Contract Number 518238

EURASNET

European Alternative Splicing Network of Excellence

LSHG-CT-2005-518238

Activity Report for the period

Jan 1, 2007 to December 31, 2007

Start date of project: January 1, 2006 Coordinator Prof. Dr. Reinhard Lührmann

Max Planck Society, Germany

completed and submitted March 17, 2008 revised September 18, 2008

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TABLE OF CONTENTS A. PERIODIC ACTIVITY REPORT 1. Publishable Executive Summary 4 2. Overview of work package activities at month 24 7 3. Project objectives and major achievements during the reporting period 14 4. Milestones and Performance Indicators 4.1 Table of Milestones 16 4.2 Table of Performance Indicators 17 5. Work package reports 5.1 Work packages 1-21 23 5.2 Consortium Management (WP22) 121 6. Deliverables for JPA month 13-30 127 7. Plan for using and disseminating the knowledge (PUDK) 134 B. PERIODIC MANAGEMENT REPORT 8. Tabular overview of all resources employed on the project 137 9. Tabular overview of major cost items 138 10. Justification of major cost items and resources

10.1 Max Planck Gesellschaft zur Förderung der Wissenschaften e.V. 144 10.2 Centre de Regulació Genòmica 149 10.3 Friedrich-Alexander-Universität Erlangen-Nürnberg 151 10.4 Uppsala University 153 10.5 European Molecular Biology Laboratory 155 10.6 Tel Aviv University 158 10.7 International Centre for Genetic Engineering and Biotechnology 160 10.8 Medical University Vienna 163 10.9 The University of Edinburgh 165 10.10 Consiglio Nazionale delle Ricerche 167

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10.11 Justus Liebig Universität Giessen 172 10.12 Centre National de Recherche Scientifique 173 10.13 University of Berne 181 10.14 Scottish Crop Research Institute 184 10.15 Medical Research Council 186 10.16 Instituto de Medicina Molecular 188 10.17 University of Leicester 191 10.18 Adam Mickiewicz University in Poznan 194 10.19 University of Aarhus 195 10.20 Universidad de Buenos Aires 197 10.21 Université de Genève 199 10.22 University of Dundee 201 10.23 The Chancellors, Masters and Scholars of the Univ. of Cambridge 203 10.24 The Hebrew University of Jerusalem 205 10.25 Centre Européen de Recherche en Biologie et Médicine - 207 Groupement d'Intérêt Economique 10.26 Institut National de la Santé et de la Recherche Médicale 209 10.27 Fondazione Centro San Raffaele del Monte Tabor 211 10.28 Swiss Institute of Bioinformatics 213

11. Person-month analysis for the Reporting Period 2007 214 12. Summary Financial Report 215 13. Report on the distribution of the community's contribution 219 14. Summary explanation of the impact of major deviations from cost budget

and from person-month budget. 222 15. Joint program of activities (month 25-42; January 2008-June 2009) 222

15.1 The future of EURASNET research: Goals and objectives after two years 15.2 Planning and timetable 241

15.3 Graphical presentation of work packages 243 15.4 Table of milestones (month 25-42) 245 15.5 Table of deliverables (month 25-42) 246 15.6 Work package descriptions 263 15.7 Work package list 290 15.8 PM Table for month 25-42 291 16. Project management level description of resources and grant for the next 18 month 293

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1 PUBLISHABLE EXECUTIVE SUMMARY EURASNET

http://www.eurasnet.info/index.shtml

The EURANET Network of Excellence set out to pursue the following project objectives: To carry out an ambitious program of joint research that will elucidate the regulatory mechanisms and significance of alternative splicing, at the molecular, cellular and organismal level. To create a communication structure that will facilitate the exchange of information, procedures, reagents and personnel, as well as sharing resources and establishing fluent interdisciplinary dialogue among the members of the network. To implement a competitive, externally-reviewed “Young Investigator” Program that will facilitate the establishment of new research groups throughout Europe. To explore and exploit the applicability of the activities of the network, particularly in the area of biomedical applications. Also, to include efforts that increase the profile and awareness of the RNA field within European society, bringing understanding to the public, policy makers and other stakeholders. Parties to the EURASNET contract: (1) Max Planck Gesellschaft zur Förderung der Wissenschaften e.V. EURASNET COORDINATOR Prof. Reinhard Lührmann, Research Director Phone: +49-551-2011407 FAX: +49-551-2011197 Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Department of Cellular Biochemistry Am Fassberg 11, 37077 Göttingen Dr. Karla Neugebauer, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden Dr. Henning Urlaub, Max Planck Institute for Biophysical Chemistry, Göttingen (2) Centre de Regulació Genòmica, Barcelona, Spain Dr. Juan Valcárcel (3) Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Prof. Stefan Stamm (4) Uppsala University, Uppsala, Sweden Prof. Göran Akusjärvi (5) European Molecular Biology Laboratory, Heidelberg, Germany Dr. Peer Bork, Associate Programme Coordinator EMBL Heidelberg, Germany Dr. Rolf Apweiler, EBI Sequence Database Group, Hinxton, UK (6) Tel Aviv University Medical School, Tel Aviv, Israel Gil Ast, PhD

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(7) International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Prof. Francisco E. Baralle (8) Medical University Vienna, Vienna, Austria Prof. Andrea Barta (9) The University of Edinburgh, Edinburgh, UK Prof. Jean Beggs (10) Consiglio Nazionale delle Ricerche, Rome Italy Dr. Giuseppe Biamonti, Istituto di Genetica Molecolare - CNR, Pavia Prof. Glauco Tocchini-Valentini, Istituto di Biologia Cellulare - CNR, Monterotondo Scalo (Roma) (11) Justus Liebig Universität Giessen, Giessen, Germany Prof. Albrecht Bindereif (12) Centre National de Recherche Scientifique, Paris, France Prof. Jamal Tazi, , Institut de Genetique Moleculaire (JRU 5535 CNRS-UMII) Montpellier Dr. Bertrand Séraphin, Centre de Genetique Moleculaire (CGM), UPR2167 CNRS Gif-sur-Yvette, France Dr. Christiane Branlant, Maturation des ARN et Enzymologie Moléculaire MAEM UMR 7567 CNRS-UHP, Vandoeuvre les Nancy Dr. Edouard Bertrand, Institut de Genetique Moleculaire (JRU 5535 CNRS-UMII) Montpellier (13) University of Berne, Bern, Switzerland Prof. Daniel Schümperli (14) Scottish Crop Research Institute, Dundee, UK Prof. John W.S. Brown (15) Medical Research Council, MRC Human Genetics Unit, Edinburgh, UK Dr. Javier Fernando Cáceres (16) Instituto de Medicina Molecular, Lisbon, Portugal Prof. Maria Carmo-Fonseca (17) University of Leicester, Leicester UK Prof. Ian Eperon (18) Adam Mickiewicz University in Poznan, Poznan, Poland Dr. Artur Jarmolowski (19) University of Aarhus, Aarhus, Denmark Prof. Jorgen Kjems (20) Universidad de Buenos Aires, Buenos Aires, Argentina Prof. Alberto R. Kornblihtt Dr. Anabella Srebrow (21) Université de Genève, Geneva 4, Switzerland Prof. Angela Krämer (22) University of Dundee, Dundee, UK Prof. Angus Lamond (23) The Chancellors, Masters and Scholars of the University of Cambridge, Cambridge UK Dr. Christopher Smith (24) The Hebrew University of Jerusalem, Jerusalem, Israel Prof. Hermona Soreq (25) Centre Européen de Recherche en Biologie et Médicine - Groupement d'Intérêt Economique, Illkirch, France Dr. James Stévenin (26) Institut National de la Santé et de la Recherche Médicale, Paris, FRANCE

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Dr. Didier Auboeuf

(27) Fondazione Centro San Raffaele del Monte Tabor, Milano Italy

Dr. Davide Gabellini

(28) Swiss Institute of Bioinformatics, Biozentrum, University of Basel, Switzerland

Dr. Mihaela Zavolan

(29) Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland

Prof. Frédéric Allain

(30) International Institute of Molecular and Cell Biology

Prof. Janusz Bujnicki

(31) Universitat Pompeu Fabra

Dr. Eduardo Eyras

(32) University of Southampton

Dr. Diana Baralle

EURASNET came into being on January 1st, 2006. Now, after two years, the second annual activity report gives testimony to the remarkable progress EURASNET made in establishing a true networked research community in the field of alternative splicing. Scientifically, EURASNET is doing well, which is perhaps not surprising, considering that it now brings together 40 of the leading labs in alternative splicing from Europe, Israel and Argentina. The publication record for 2007 provides ample evidence how EURASNET labs made substantial contributions to further our understanding of the process of alternative splicing. The biochemical purification and characterization of single components or entire native complexes of the splicing machinery, the identification and analysis of regulatory features, the structural characterization of splicing substrates and spliceosomal proteins, the disease models under investigation and the development of potentially powerful therapeutics all made substantial progress as described in the respective detailed reports. The microarray initiative continues to be one of the real success stories of EURASNET which would not have been possible without the supporting financial and intellectual infrastructure of this NoE. After the initial evaluation of commercially available solutions, the network is now in a position to finalize a contract with one of the leading vendors of microarray formats. This Network-approved and –adapted format best suits the applications and needs of Network members and their research interests. A similar set-up was started in the field of small-molecule-screening, where the first small chemical compound screens are now expected for 2008 after the necessary assays for splicing specific read-outs were established. It is in the latter field where eventually the first patentable knowledge of EURASNET may emerge. Another remarkable fact is the high level of integration achieved by the groups working on plant splicing. The commercial potential of RNA research is now clearly recognized by pharmaceutical industries. In 2007 for the first time a company contributed substantial funding to sponsor a EURASNET led meeting on Cancer & RNA. The network has stepped up efforts in establishing contacts with the medical community. Special provisions were made to bring EURASNET into the consciousness for example of genetic reference labs. EURASNET members continue to spread word about EURASNET in numerous national and regional RNA-networks. Increasingly, truly collaborative research is now executed, a strong program of meetings and workshops has improved integration, dissemination and outreach, and staff exchange between laboratories is gaining momentum. EURASNET has again enlisted five new promising young investigators in 2007 who will now pursue their research goals with the help and support of the Network and who at the same time will add to the Network's ability to achieve an ambitious research program.

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2 OVERVIEW OF WORK PACKAGE ACTIVITIES AT MONTH 24 WP1 The EURASNET Web site Many EURASNET groups have contributed material (presentations, tools and resources, protocols, publications, scientific descriptions of alternative splicing etc) to the website. Group 5 (Stamm) have taken the responsibility for uploading this information and organising the website. This has also included posting information of meetings and workshops on a regular basis, organising database content and linking content. In addition, the general maintenance and troubleshooting for the website has been carried out throughout the period by the Stamm group. During 2007, the idea of changing the website and, in particular, establishing a team to gather and input information by co-ordinating WP1, WP19 and WP21 was discussed with the management of EURASNET and Group 14 (Brown) and Group 8 Barta) as leaders of WP19 and WP21. At month 24, work on the new website has begun. WP2 Sharing resources, technology and reliable protocols Group 3 (Stamm) and group 14 (Brown) implemented a protocol collection system on the EURASNET web page. All groups participated in this collection system by adding lab-specific, tested protocols. All protocols were converted into a pdf format that will be hosted in the new EURASNET web page design. A special expertise overview has been created on the EURASNET site, which is located in the password-protected area of the web site. All groups added reagents to the EURASNET reagent collection site, which describes currently 123 antibodies/sera, 439 plasmids/DNA constructs and 36 cell lines. More than an estimated 380 plasmids and 62 antisera were exchanged among group members. Group 3 (Stamm), group 26 (Tazi) and group 7 (Baralle) have now assembled a small collection of 12 minigenes that can be distributed to clinicians that want to study alternative splicing. WP3 The Young investigator program Members 1a, 2, 3, 9, 15, 16, 21 and 22 (the YIP Committee) prepared the short list for the second YIP recruitment. Winners were selected by the SAB from the EC approved short list during the Annual Meeting in France. WP4 The alternative splicing database Group 5 (Apweiler) has integrated various databases on alternative splicing (ASD, ASTD) with the ENSEMBL database that is hosted at the European bioinformatics Institute. Each transcript was annotated with eVOC and MESH data, as well as comprehensive splicing data. Group 28 (Zavolan) brought expertise of miRNA prediction to alternative splice site selection and bioinformatically predicted alternative exons that are regulated by snoRNAs, which are experimentally tested by group 3 (Stamm). Group 6 (Ast) used several databases to create a user-friendly interface that allows users to search for splicing regulatory motifs in RNA sequences. Group 26 (Auboeuf) collected input from all groups to make the FastDB database more user-friendly for experimentalists. Easier to use visualization tools were added to this database. Together with group 23 (Smith) they implemented an algorithm in FastDB that allows visualization of affymetrix splicing data in FastDB, which allows for an easier and faster analysis of array data generated in the consortium. WP5 Ensuring durability Several EURASNET members have contributed to “ensure a durable, functional alternative splicing network” via application to regional and international funding programs. Group 1a (Lührmann), Group 9 (Beggs) and Group 2 (Valcárcel) have maintained close contact with the EU with the goal of promoting the field of RNA Biology as a strategic priority in FP7. Group 16 (Carmo-Fonseca) has informed all EURASNET members on available initiatives that may support further trans-European cooperative RNA research. WP6 In silico approaches to alternativ splicing

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Group 3 (Stamm) have identified a snoRNA regulating the processing of an mRNA expressed from a gene located on a different chromosome. Group 28 (Zavolan) implemented a snoRNA-mRNA binding model that reflects the constraints the snoRNA-rRNA interaction. Group 5A (Bork) developed a framework for the prediction of conserved alternative transcripts and identified some in as distant organisms as human and fly. Group 6 (Ast) compiled a unified dataset of splicing regulatory sequences. WP7 Molecular characterization of splicing substrates Group 4 (G Akusjarvi) characterized the properties of the adenovirus splicing factor L4-33K, Group 7 (F Baralle) studied the RNA structure of ATM and CFTR pseudoexons, identified SR and hnRNP proteins that bind to the CERES splicing regulatory elements and showed in collaboration with group 10a, that hnRNP A1 and H bind to the silencer element of exon 12 in the Ron pre-mRNA, Group 10a (G Biamonti) has studied the mechanism of silencing of exon 12 in the Ron pre-mRNA, produced and characterized a new variant of SatIII RNAs and developed a method for exhaustive identification of their protein partners Group 12c (C Branlant) characterized a structural RNA motif that plays a key role in the conformation of the HIV-1 RNA regulatory region acting at site A3, showed that it binds protein with DRBD domains, made a complete analysis of proteins associated with various HIV-1 and RSV splicing regulatory regions and identified new regulatory mechanisms at the HIV-1 acceptor sites A3 and A7. In collaboration with J Tazi, group 12c showed by RNA-structure probing, that the C to U mutation in pre-lamin A pre-mRNA, increases the accessibility of the 5’ cryptic site whose utilization leads to the progeria syndrome. Group 12c made a deep investigation on the MBNL1 RNA binding properties. Group 17 (I Eperon) has cloned several splicing regulatory proteins as fusion to fluorescent proteins and started to monitor their binding to pre-mRNAs, testing the possibility to watch protein association in real time, Group 19 (J Kjems) showed that the C362T substitution in medium-chain acyl-CoA dehydrogenase pre-mRNA generates an inhibitory hnRNP A1 site, and discovered a new regulation by tra2β of the HIV-1 site A7 , group 19 also produced a chemically modified piece of the hnRNP A1 pre-mRNA in order to test for the binding of hnRNP A1 on both sides of the alternatively spliced exon by FRET experiments, Group 23 (C Smith) determined how the PTB, Raver and MBNL proteins interact on both sides of the α-tropomyosin exon 3 and regulate its utilization, Group 29 (F Allain) was recently incorporated in WP7 as a YIP, he determined the 3D structure of the RRMs 3 and 4 of hnRNP L and made the interesting finding that the hnRNP L and PTB RRMs 2 and 3 have similar modes of interaction with RNAs. Group 29 also established the 3D structure of the 3 RRMs of hnRNP F and of the complex formed between an AGGGAU oligo-ribonucleotide and the RRM2. Surprisingly, RNA binding is not mediated by the β-sheet surface. WP8 Genome-wide analyses of splicing regulation Neugebauer 1B, Stamm 3, Apweiler 5B, Ast 6, Barta 8, Beggs 9, Biamonti 10A, Tocchini-V 10B, Bindereif 11, Brown 14, Cáceres 15, Carmo-F 16, Jarmolowski 18, Kjems 19, Krämer 21, Smith 23, Soreq 24, Auboeuf 26, Zavolan 28, [Valcarcel 2] Group 1B (Neugebauer) in collaboration with Group 2 (Valcárcel) investigated the effects of Cap Binding Complex on alternative splicing by RNAi of CBC subunits and analysis by splice-sensitive microarray. No AS events were validated suggesting that CBC does not influence AS events. Group 3 (Stamm) used ExonHit SpliceArrays and identified AS events that were influenced by Tra2β overexpression. Group 6 (Ast) analyzed groups of PTB-regulated exons provided by Group 23 (Smith) for motif enrichment. Groups 8, 14 and 18 (Barta, Brown, Jarmolowski) carried out a number of collaborative projects including; expansion from 96 to 384 AS events in the Arabidopsis RT-PCR panel; use of the RT-PCR panel to identify AS events affected by CBC knockdown, identifying defects in splicing in first introns; found that ~30% of AS events are influenced by mutations in two separate NMD components. Group 10A (Biamonti) in collaboration with Group 2 (Valcárcel) used splice sensitive arrays to profile changes in AS during epithelial-mesenchymal transition using SW480 cells grown at high (epithelial) or low (mesenchymal) density. Group 11 (Bindereif) used Affymetrix Human ExonArrays to analyze the effects of hnRNPL knockdown in HeLa cells and identified and validated a large number of novel hnRNPL target AS events. Group 19 (Kjems) carried out genomic SELEX with HIV-1 Rev and found 6-8 cellular RNAs that bound to Rev with higher affinity than HIV-1 RRE. Most targets were endogenous retroviruses or retroelements. Group 21 (Krämer) tested the effects of SF1 RNAi on AS of exons associated with SF1 CLIP tags. In 9/17 cases, splicing was affected. CLIP has been carried out for SF1 and U2AF65 in both nuclear and cytoplasmic fractions. The general distribution of U2AF65 tags was similar to those of SF1. Group 23 (Smith) identified and validated 25 PTB/nPTB targets by using quantitative gel-baed proteomic analysis of control and PTB+nPTB knockdown HeLa cells. They also tested AS predictions arising from analysis of the PTB+nPTB

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knockdown experiment using ExonHit SpliceArrays and Affymetrix ExonArrays. The ExonArrays were found to be an effective global platform for analysis of AS events. Group 24 (Soreq) used ExonArrays to characterize AS events that occur in thymic tumours associated with Myasthenia Gravis. A number of genes with multiple AS events were identified, and in parallel the levels of SF2/ASF and SC35 were found to be reduced. Group 26 (Auboeuf) used ExonArrays to analyze effects of estradiol on gene expression. A number of novel estradiol activated genes were identified, but very few AS changes were found, possibly due to the bioinformatics challenges of identifying AS changes within genes where expression levels are also varying. They have also developed methods for analysis of ExonArray data and have applied these methods to data supplied by Groups 3 and 23 (Stamm and Smith). Group 28 (Zavolan) applied computational methods to datasets of human full-length cDNA and 5’ ESTs to identify exons whose inclusion is dependent upon promoter choice. A large fraction of human exons appear to be included in a promoter dependent manner. WP9 Complexity of spliceosomal proteomes Group 1a (Lührmann) has studied the proteome on a cis-acting SRp55 ESE in a 5’ exon that is flanked by an upstream U2 snRNP interaction site (U2 anchoring site, polypyrimidine and branch site) and a downstream 5’ splice site. This group showed that the proteome of the exon complex contains the tri-snRNP stably bound but differs otherwise from previously characterized splicesomal cross-intron B complexes. The group was also able to isolate spliceosomes at different stags of assembly/function using splicing extracts from S. cerevisiae yeast and D. melanogaster cells. Group 1b (B. Seraphin) and Group 1a characterized the pre-mRNA retention and splicing (RES) complex and crystallized one of its components Pml1p, respectively. Group 12c (Branlant) group investigated the proteome on several pre-mRNA regions involved in the regulation of splicing, i.e. HIV-1 RNA regions containing the A3 and A7 acceptor sites and their regulatory elements, the NRS splicing silencer of the Rous sarcoma virus, the CUG-repeated sequences in the DMPK gene, and the lamine A pre-mRNA region containing the 5’-cryptic site. Proteomes for the most of the RNP complexes have been analysed by mass spectrometry. Group 12a (Tazi) focussed on alternative splicing events that lead to Hutchinson–Gilford progeria syndrome (HGPS) by the proteome analysis of in vitro assembled RNP complexes on wildtype and mutated LMNA mRNAs. Group 19 (Kjems) is investigating spliceosomes by MS that are formed on wildtype and mutant exon 5 sequences of the medium-chain acyl CoA dehydrogenase. Group 4 (Akusjärvi) has generatedMS2-IIIa substrates that are spliced efficiently and tested them in the first set of affinity-purification schemes. Alternatively, stable cell lines expressing L4-33K will be established in order obtain nuclear extract that should simplify purification of spliceosomal complexes. Group 9 (Beggs) is investigating the interaction of Cwc21 protein in complex with other proteins. Cwc21 shares high similarity with the human SR300 protein that is involved in splicing regulation. WP10 Post-translational modification and dynamic regulation Group 4 (Akusjarvi) is studying the role of the protein kinase PKA in pre-mRNA splicing. Group 1A (Luhrmann) has studied the function of the hPrp4 kinase. This group in collaboration with Group 1C (Urlaub) has demonstrated that the tri-snRNP protein hPrp28 is a target for the SR protein kinase, SRPK2. In a genetic screen, Group 12A (Tazi) has identified another SR protein kinase, DNA topoisomerase I as a suppressor of the developmental defects induced by overexpression of the SR protein, B52. Group 3 (Stamm) has concentrated on the role of the phosphatase PP1 in alternative splicing regulation. Group 10A (Biamonti) has continued the studies of the alternative splicing of the c-Ron proto-oncogene. Groups 15, 16 and 22 (Caceres, Carmo-Fonseca and Lamond) have used FRET/FLIM microscopy to analyse interactions among different spliceosome components. Use of FLIM has allowed them to spatially map these interactions to specific sites in the nucleus. Srebrow (a new YIP investigator) is studying the link between signaling and the alternative splicing of the Rac-1 pre-mRNA, which is regulated by hnRNP A1 and hnRNP A2. Group 26 (Auboeuf) has focused on the study of how post-translational modifications of the p68 helicase affect different cellular functions of this protein (splicing, transcription). WP11 Function of splicing factor isoforms Groups 2 (Valcarcel), 16 (Carmo-Fonseca) and 23 (Smith) have joined forces for the design and evaluation of splicing microarrays as well as the development of analytical tools to detect changes in alternative splicing of splicing factor genes. Several groups have already benefited from these technical platforms and 5 collaborations have been established involving the following groups: 2 (Valcarcel), 1B (Neugebauer), 3 (Stamm), 10A (Biamonti), 16 (Carmo-Fonseca) and 20 (Kornblihtt). Splicing factors isoform expression has been analyzed in

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Drosophila and human cells in processes ranging from sex determination to muscle differentiation or tumor progression (Groups 2 and 16, Valcarcel and Carmo-Fonseca). Analysis of isoform-specific knockdown of PTB isoforms by Group 23 (Smith, see also WP8) has rendered hundreds of novel alternative splicing events targets of regulation by these factors. Functional analysis of additional splicing factor isoforms include: SC35 autoregulation (Group 25, Stevenin); distinct effects of knocking down U2AF35 isoforms in cell proliferation, apoptosis and alternative splicing of cell cycle regulatory factors (Group 16, Carmo-Fonseca); Group 21 (Krämer) analyzed the expression of SF1 isoforms in mammalian cells and studied their intracellular localization and isoform-specific siRNAs have been generated and tested; Group 2 (Valcárcel) reported differential activities of two isoforms of the apoptosis regulatory protein TIA-1 controlled by the related protein TIAR. Group 10B (Tocchini-Valentini) has adapted original tRNA splicing-based technology for the selective inactivation or modification of splicing factor isoforms. Group 12A (Tazi) collected microarray data from Drosophila over-expressing splicing factors. Groups 8, 14 and 18 (Barta, Brown and Jarmolowski) established an alternative splicing RT-PCR panel to examine multiple AS events simultaneously. At the end of the first reporting period (months 1-12), initial analysis of three over-expression lines of Arabidopsis SR proteins (RSZ33, RSp31, SRp30) had been carried out using the pilot AS RT-PCR panel of 96 events. The data from biological reps has been analysed and 1) confirmed the auto-regulation of alternative splicing of RSZ33, 2) that over-expression of particular SR proteins affects the alternative splicing of other SR proteins, and 3) that different SR protein isoforms affect different, but over-lapping subsets of AS events in other genes. In addition, RSZ32 and RSZ33 are paralogues of the same SR protein gene. Mutants in each have been obtained and characterised for the two gene family members. Over-expression constructs of GFP-fusions of PTB-like protein genes, PTBL1 and PTBL3 have been prepared and are currently being transformed into Arabidopsis. In addition, putative knock-out mutants have been identified and are being characterized genetically. An underlying principle emerging from these studies is that splicing factors establish intricate gene networks to buffer their activity levels and possibly respond to varying cellular conditions, involving alternative splicing events in their own genes under their own control or the control of other splicing factors and RNA binding proteins. WP12 Mis-splicing and disease Group 4 (Akusjarvi) has characterized two alternatively spliced mRNAs encoded by the adenovirus L4 transcription unit, L4-33K, and L4-22K. Group 7 (Baralle F.) has continued to study several regulatory elements in the genes of interest (CFTR, NF-1, HERG and ATM) from the point of view of their RNA-protein and RNA-RNA interactions. In parallel, they are investigating the effect of knocking down interesting proteins such as TDP-43 a splicing factor involved in several neurodegenerative diseases. Group 10A (Biamonti) has continued the studies on the Ron proto-oncogene AS and found that two growth conditions are characterized by different splicing profile of Ron: skipping of exon 11 mainly occurs at low-density cells and is accompanied by unproductive splicing of the SF2/ASF gene transcripts which, in turn, directs SF2/ASF mRNA to degradation through the NMD pathway Group 11 (Bindereif) developed a combination of RNAi-mediated knockdown of a specific splicing regulator (hnRNP L) and splice-sensitive microarray analysis to identify on a genome wide level alternative splicing processes affected by this factor. Group 12A (Tazi) identified and characterized splicing factors implicated in the deregulated control of splicing of mutated LMNA mRNA in the case of Hutchinson-Gilford progeria syndrome (HGPS). Group 19 (Kjems) has used synthetic bi-functional LNA-containing oligonucleotides and a U7smOPT snRNA to counteract exon skipping in the presence of disease-causing mutation (362C>T) in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD). Group 25 (Stevenin) used specific whole cell extracts (WCE) or nuclear extracts (NE) isolated from human cells overexpressing various SR or SR-like proteins to analyse splicing of wild-type LMNA transcripts vs transcripts that are mutated in the HG Progeria syndrome. Group 27 (Gabellini) have found that FRG1 over-expression inhibits muscle differentiation of C2C12 in tissue culture. This cell culture model of Faciscapulohumeral Muscular Distrophy (FSHD) is currently used to identify the molecular pathways affected by FRG1 over-expression. Group 32 (Baralle D.) has developed a set of minigene splicing assays to study the influence of mutations in neurofibromatosis type 1 exon/intron 29 splicing and in the regulation of pathological pseudoexon inclusion in NF-1 intron 30. WP13 Co-transcriptional mechanisms of alternative splicing Group 1b (Neugebauer) has established splicing factor ChIP in yeast and mammalian cells; this includes the entirely novel strategy of ChIP of alternative splicing factors, which are expressed as tagged versions from stably transfected BACs. This has turned out to be an improvement over the use of antibodies and has therefore led to a number of new collaborations within the workpackage. Collaborative project with Group 26b (Seraphin) on the role of Pol II in U1 snRNP recruitment to nascent RNA is complete, with a manuscript in progress. Group 9 (Beggs)

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showed that the splicing factor Prp45p regulates transcriptional elongation rather than initiation rates in yeast. Group 20 (Kornblihtt) has compiled an extensive list of treatments that alter RNA polymerase II elongation rate and correlated these with effects on alternative splicing. Together with Group 12d (Bertrand), in vivo measurements of Pol II elongation rates have been undertaken and extended in new collaborative projects on alternative splicing, showing that the physiological stimulus of UV light leads to alteration of Pol II elongation rate, which brings about changes in AS of reporter genes. ARK and Group 6 (Ast) demonstrated AS of IKBKAP can be due to modulation of elongation rates. The YIP Group 26 (Aubeouf) has extended the questions regarding transcriptional regulation and splicing to an estrogen-regulated system in MCF-7 breast cancer cells, in which they showed that co-transcriptional splicing of cyclin D1 and PS2 pre-mRNAs is dependent on Ser5 phosphorylation of the CTD. Group 10A (Biamonti) found that several AS factors are recruited co-transcriptionally to SAT II loci; the RNA is retained at these sites longer than the splicing factors, suggesting a nuclear role for the SAT III RNAs. Group 19 (Kjems) has used integrated versions of HIV or globin-based intron-containing transcription units to show that the first 5’ splice site enhances transcription initiation of each gene via U1 snRNP recruitment and then enhanced accumulation of basal TFs. Group 16 (Carmo-Fonseca) showed that a specific set of repeats in the CTD is required for mRNA release from the transcription site. WP14 Chemical biology and therapeutics Partner 1a (LÜHRMANN) has identified five inhibitors of spliceosome assembly from a small collection of known bio-active compounds. These inhibitors are valuable tools to identify, isolate and characterize novel intermediates of the splicing cycle that were previously not accessible. Partner 3 (STAMM) showed that reducing PP1 activity by either the cell permeable inhibitor tautomycin or by its nuclear inhibitor NIPP1 promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 (SMN2) exon 7 in a mouse model for spinal muscular atrophy, suggesting a new principle for the treatment of diseases caused by missplicing events. Through screening of a chemical library of 6500 compounds Partner 12a (TAZI) has identified 100 indole derivatives that selectively inhibit the activity of individual SR proteins. Partner 12a in collaboration with 12c (BRANLANT) have discovered a novel inhibitor of HIV-1 replication, IDC16, among the indole derivatives. Several libraries are now available based on the structure of the active molecules. Active molecules have, also, been, found to inhibit metastatic cell motility, to correct aberrant splicing of LMNA in the case of HGPS and to induce exon skipping of mutated exons of Dystrophin gene. Partner 12a has, also, engineered a set of different luciferase reporter minigene constructs for medium and high throughput screening. Luciferase based reporters have been generated to harbour ESEs recognized by different SR proteins or mutated exonic sequences from LMNA gene responsible for premature aging (Progeria syndrome) or exonic sequences from Dystrophin gene whose skipping is important for the correction of Duschenne Muscular Dystrophy (DMD). These plasmids were included in EURASNET database (pFlucASF/SF2, pFlucSRp55 and pFlucSC35, pFlucDMD51, pFlucLMNAwt and pFlucLMNAmut). Partner 13 (SCHÜMPERLI) developed U7-based method to induce exon skipping in HIV-1, thereby inhibiting HIV-1 replication. Partner 13 developed, also, U7-based methods to induce exon inclusion in survival of motoneurons 2 gene (SMN2), which could be used in a gene therapy for spinal muscular atrophy. Partner 17 (EPERON) further investigated and optimized strategy for using TOES (targeted oligonucleotide enhancers of splicing) to rescue refractory exons as a basis for related endeavors by other members of the WP. Partner 19 (KJEMS) identified ESE and ESS in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) and showed that exon skipping in the presence of disease-causing mutation (362C>T) can be counteracted by recruitment of antisense oligonucleotide containing an ESE. Partner 19 has also demonstrated that LNAs targeted to experimentally selected binding sites can function as very potent inhibitors of HIV-1 expression in cell culture and may potentially be developed as antiviral drug in patients. Through an extensive screen, Partner 6 (AST) had identified two small molecules (potential drugs) that raised the inclusion level of exon 20 in the endogenous IKAP mRNA in FD cells. Partner 22 (LAMOND) has also screened over 2000 chemical compounds from different libraries using both HeLa in vitro splicing assays AND a Biacore assay to examine protein-protein interaction between spliceosome factors. This partner has identified 8 distinct compounds that reproducibly inhibit splicing and these are currently being evaluated by medicinal chemists for their suitability for further development, either as research tools and/or therapeutic agents. Partner 10 (BIAMONTI) used morpholino antisense oligonucleotides to change the Ron proto-oncogene alternative splicing. By masking an ESE-binding site for SF2/ASF with Mo-SF2, Partner 10 inhibited the expression of deltaRon isoform, an inducer of metastic phenotype. Partner 25 (STEVENIN) has developed new tools for screening chemicals targeting specifically SR proteins. They prepared nuclear extracts from cells overexpressing each of the SR proteins to analyse (SRp20, SC35, 9G8, ASF/SF2, SRp30c, SRp40, SRp55, as well as the SR-related Tra2β1 protein).

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WP15 Development of Enabling Technologies The recruitment of five new YIPs added to the broad spectrum of techniques available in the network. Members 1b, 3 and 10b tested ne cell lines that stably express a tagged pre-mRNA. Member 1c successfully lowered the amount of starting material in feasibility studies with U1 snRNP and 15.5K-61K-U4atac snRNP l for MS-detection of UV-induced cross-links of proteins to RNA. Members 1a and 1c developed new strategies for the detection of protein-RNA crosslinks WP16 Conferences and meetings 12A organised the Second Annual Meeting in Ile de Bendor, France, and also the IFM “RNA and Cancer” in La Grande Motte, France. 15, 20 and 22 organized an IFM on "Cell Biology, Signaling and Alternative Splicing" in Bariloche, Argentina. 1A organised the IFM, “Biophysical Methods to Study Alternative Splicing”, in Berlin. workshops (formally part of WP17). 8, 14 and 18 organised a workshop on Alternative Splicing in Plants in Carry-le-Rouet, France. 8 organised a workshop on Alternative Splicing and Bioinformatics in Vienna. 9 organised a workshop on High Throughput Approaches in Biology and oversaw the organisation of the meetings programme generally. WP17 Staff exchange and training 1a trained two colleagues from UU, Uppsala, Sweden and MPG, Dresden, Germany in methods of spliceosome assembly and purification. 2 provided training in microarrays and data analysis for students and postdocs from four laboratories (IMM, Lisbon, Portugal; MPG, Dresden, Germany; FCEN-UBA, Buenos Aires, Argentina; CNR, Pavia, Italy). 9 trained two colleagues from CNRS (Nancy, France) in yeast microarrays. 14 hosted three students and post-docs from AMU (Poznan, Poland) for use of the AS RT-PCR panel and organized the Plant Intra-workpackage meeting at the SCRI (Dundee, UK) with participation of eight colleagues from AMU (Poznan, Poland) and MUW (Vienna, Austria). 21 coordinated staff exchange and training. Ten students and post-docs from five groups (LUG, Giessen, Germany; UNIGE, Geneva, Switzerland; UCAM-DBIOC, Cambridge, UK; INSERM, Paris, France; TAU, Tel Aviv, Israel) participated in the ExonArray Workshop organized by 23 at the UCAM-DBIOC in Cambridge, UK. WP18 Career development Partner 19 (Kjems) gave a lecture at the Ile Bendor meeting about career development including aspects of women in science and obligations for mentors and mentees. Partner 19 Kjems posted the seminar and an assembled list of career development resources available at the web on the EURASNET homepage. Partner 1B (Neugebauer) has posted her seminar on ”Database of Expert Women in the Molecular Life Sciences” on the EURASNET web page. Partner 1B (Neugebauer) and partner 19 (Kjems) have initiated the planning of the Krakow meeting in May 2008. Partner 11 (Binderief) participated in a Ph.D. committee for a student in partner 1A (Lührmann’s) laboratory March 19, 2007. Partner 12 (Tazi) and partner 1B (Neugebauer) participated in Ph.D. committees in connection with 2 PhD thesis in partner 21 (Krämer) laboratory. Partner 17 (Eperon) is official supervisor for a student working in partner 7 (Baralle’s) laboratory. Most of the partners have reported to be involved in career and mentor activities and have given seminars on this topic at their local institutions. WP19 Public understanding of RNA biology The webpage has been replenished; information material about splicing and alternative splicing as well as alternative splicing in disease has been added. Collection of teaching material about RNA biology for the webpage is ongoing. A media training workshop is currently organised; it will take place at the Fist International EURASNET Conference (Krakow, Poland) in May 2008. Jørgen Kjems and Guiseppe Biamonti were involved in local press events, press releases were made by Albrecht Bindereif and Jørgen Kjems. The EURASNET was presented in a magazine called “The Parliament” in a full page advertisement.

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Jean Beggs, Jørgen Kjems and John Brown as well as Mihaela Zavolan organised or took part in open house events and visits of laymen at their institutions. Artur Jarmolowski gave a talk at the Festival of Science and Art in Poznan. Several talks were given to a lay-audience (undergraduate students, journalists and/or general public) by Reinhard Lührmann, Hermona Soreq, Davide Gabellini, Albrecht Bindereif, Chris Smith, Francisco Baralle, Karla Neugebauer and Andrea Barta. Daniel Schümperli had several visits of SMA patients and meetings with SMA helper-associations as well as patients. On a number of occasions the EURASNET leaflet was displayed. Claudia Panuschka is in the process of generating a comprehensive brochure about the EURASNET, its aims and research topics. The brochure will act as a dissemination tool for specific target groups, namely medical doctors, teachers and pupils. She is in direct contact with the webpage master in Dundee to organise the rearrangement of the webpage domain meant for the general public. WP20 SMEs and technology transfer Group 22 (Lamond) has developed in vitro RNA splicing assay formats for use in high throughput chemical compound screening in collaboration with Group 1A (Lührmann). Group ? (Tazi) has identified a small molecule inhibitor of SR protein splicing factors and is characterizing its effect on cells in vivo and its mechanism of action in vitro. Group 13 (Schümperli) is collaborating with the groups of Prof. Christian Leumann, Department of Chemistry and Biochemistry, University of Bern and that of Dr. Luis Garcia, Institut de Myologie Paris, France on tricyclo-DNA applications to exon skipping in Duchenne Muscular Dystrophy (DMD). Group 19 (Kjems) has developed a novel transfection reagent for introducing RNA into cells and is negotiating with Biotechnology companies to commercialise it. Group 22 (Lamond) has expanded the Commercialisation Factfile to include detailed advice and information on strategic planning issues involved in forming a spinout venture. Group 22 (Lamond) has formed a spin-out company, Dundee Cell Products, to commercialise RNA processing reagents and cell fractions that can be used for in vitro splicing assays. Group 16 (Carmo-Fonseca) has transferred to Dundee Cell Products hybridoma cell lines expressing monoclonal antibodies to splicing factors for commercialisation. WP21 Reachout to the broader RNA community All 40 EURASNET members have contributed to the “Broader outreach to the wider RNA community” via a significant number of publications, workshops, presentations and consultancies at national and international meetings, to industry, clinicians and medical scientists and to students. Towards the end of the reporting period, Group 14 (Brown), Group 11 (Barta), Group 5 (Stamm) and Group 1 (Lührmann) have re-organised the management of the website (WP1) and established co-ordination between the Dissemination work-packages dealing with the website (WP1), Public Understanding of Science (WP19) and Broader outreach to the wider RNA community (WP21). This will benefit the network in the future in terms of capturing and disseminating information. In particular, Group 14 (Brown) and Group 11 (Barta) have now created a structure to regularly gather and collate information from the 40 groups in the network as suggested in the first year report. WP22 Management Participant 1A (R. Lührmann) coordinated the Network's scientific, financial and administrative actions. In particular he lead the reporting sessions during the Annual Meeting 2007 at Ile de Bendor, was in permanent contact with SAB, Network members (in particular the Steering Committee) and the EC. During the preparation phase of the Annual Meeting 2007 and during the meeting he coordinated the selection of five new YIP members. He provided logistic support for various workshops, meetings and IFMs. The coordinator prepared the Annual Report 2007 and organized an IFM in Berlin which included a PI-only session to prepare the network for the reporting period. Participant 2 (J. Valcárcel) as one of the Deputy Coordinators and as member of the Steering Committee supervised major parts of the Network's activities in the field of micro-arrays. He also provided secretarial and logistic support for various meetings and workshops. Participants 1A and 2 acted as members of the YIP committee. Participant 3 (S. Stamm) as one of the Deputy Coordinators and as member of the Steering Committee was primarily involved in integrating activities related to the web-site and decisions of the Committee. Participants 9 (J. Beggs), 15 (J. Cáceres), 16 (M. Carmo-Fonseca), 21 (A. Krämer) and 22 (A. Lamond) acted as members of the Steering Committee.

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3 PROJECT OBJECTIVES AND MAJOR ACHIEVEMENTS DURING THE REPORTING PERIOD RESEARCH: To carry out an ambitious programme of joint research that will elucidate the regulatory mechanisms and significance of alternative splicing, at the molecular, cellular and organismal level. Workpackages 4, 6 and 11 (The Alternative Splicing Database, In silico Approaches to Alternative Splicing, and Function of splicing factor isoforms) comprise the network's main activities towards establishing joint research between computer biologists and experimentalists. Data have now been merged into the unified ASTD database. Other user-friendly databases have been created as well. The EURASNET microarray initiative (WP8) finished a two year exploratory study of all commercially available experimental platforms, collected proposals for interesting genes to be analyzed among network members and then proceeded to perform experiments under controlled unified conditions. Many network labs participated in this exploratory phase. The operations were funded from the HTE fund. During the Berlin IFM the results were discussed and a decision for the most suitable platform was made. The network is now finalizing a contract to secure exclusive access to this most advanced microarray. Suitable microarray platforms are also of critical importance for e.g. the WP11 (splicing factor isoforms) due to the ever increasing numbers of alternative splicing event targets discovered by network members during 2007. The HTE funded Arabidopsis RT-PCR panel enabled the plant groups of EURASNET to collect a tremendous amount of new data on alternative splice events in plants. Work on this subject proceeded in a highly integrated fashion. Small molecule screening is the other enabling technology supported through the HTE fund (WP15 Development of enabling technologies). A cooperation with the Dundee screening facility was established and first screens will be performed in 2008. Considerable progress has been made in the isolation and characterization of spliceosomes and other RNP complexes assembled on constitutively and alternatively spliced pre-mRNAs using mass-spectrometric analysis techniques. These studies revealed that the protein composition of the spliceosome is both highly complex and highly dynamic, but at the same time shows high evolutionary conservation. The molecular characterization of splicing substrates (WP7) was able to map a host of new regulatory sites. Proteomic studies of complexes assembled on several mutated pre-mRNA substrates as well as viral RNAs which are causative agents for diseases are steadily advancing (WP9, 12). The role of alternative splicing in disease processes is the main topic of WP12. Studies on various model systems continue. Work performed in WP14 identified several new inhibitors of disease related splicing events or have investigated regulatory sites with a potential for therapeutic use. Within WP10 (Post-translational modification and dynamic regulation) among other subjects research on the role of protein kinases and phosphatases in splicing made substantial progress. Several groups use fluorescence microscopy techniques to study protein-protein interactions. INTEGRATION: To create a communication structure that will facilitate the exchange of information, procedures, reagents and personnel, as well as sharing resources and establishing fluent interdisciplinary dialogue among the members of the network. The EURASNET website is currently undergoing a major redesign jointly organized by members 8 and 14. The number of resources, protocols, available reagents like plasmids and antibodies increased dramatically (WP1, 2). Webmaster, Integration manager and PSO develop new efficient tools for data collection from members. The number of cutting edge technologies within the network has again been increased with the incorporation of five Young Investigators in 2007. To further increase the use of the staff exchange and training program (WP17) this is now also available for students and postdocs to attend intra-workpackage meetings where work package participants can intensify their discussions. A comprehensive list of RNA researchers was compiled and is used for further reach-out activities. The official EURASNET flyer is currently extended into a full-blown brochure about EURASNET. EURASNET members continued to give presentations about the EURASNET NoE during various national and international meetings. A EURASNET advertisement was placed in "The Parliament".

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YOUNG INVESTIGATOR PROGRAM: To implement a competitive, externally-reviewed “Young Investigator” Programme that will facilitate the establishment of new research groups throughout Europe. The second round of YIP recruiting was completed during the reporting period (contract start is Jan 1, 2008). The winners of this competitive call substantially expand the existing spectrum of technologies and expertise available in the network. The YIP winners integrated in the first recruiting round (contract start was July 1, 2006) are all well integrated, that is participating in scientific cooperative projects and in the meeting program. TRANSLATION AND DISSEMINATION: To explore and exploit the applicability of the activities of the network, particularly in the area of biomedical applications. Also, to include efforts that increase the profile and awareness of the RNA field within European society, bringing understanding to the public, policy makers and other stakeholders. Many EURASNET members are also involved in national and regional European RNA networks using these connections to further propagate and disseminate knowledge about the goals of EURASNET. EURASNET has become an important reference point for commercially available microarray formats. In 2007 the RNA and Cancer in France was generously supported by industries. As a result of the microarray initiative the network is now considering the employment of a bioinformatician experienced in data analysis of this particular high-throughput approach. This IFM like the other IFMs in 2007 provided excellent platforms for discussions among PIs not only about currently pursued research goals but also about the type of research beyond the funding period of this network. As an important precondition for the future development of business relations in the field of alternative splicing, within WP 20 a constantly updated commercialization fact file is maintained under the leadership of EURASNET member A. Lamond. He recently also provided the Network with a Strategic Planning for a New Biotech Spinout or Startup Venture file. MANAGEMENT: To organize a management scheme that will ensure the implementation of the other four objectives. Network operations for the scientific aspects of EURASNET are efficient and smooth. Also, the financial operations are efficiently organized and controlled from the Coordinator's location. Annual reporting activities in a network of now 40 members is increasingly complex and time consuming due the large number of highly diverse member administrations involved.

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4.1 TABLE OF MILESTONES JPA MONTH 13-30

Milestones for months 13 - 30

No Title Month 11 First European Conference on Alternative

Splicing 14, achieved (was regular reporting meeting)

12 Up-to-date database of splicing factors, protein and RNA regulators and potential novel members of these categories

18, achieved with delays due to EBI restructuring

13 2nd round of YIP recruiting starts 18, achieved 14 Selection and integration of YIP award

recipients (2nd round) 24, achieved

15 Four interdisciplinary focus meetings 24, achieved 3/4 16 Following the initial two year proof of concept

phase of the HTE-fund, EURASNET makes decision which assay formats and screening targets will be pursued in the future for microarray- and small molecule screening approaches.

24, achieved (see WPs 8 and 20)

17 Second annual workshop programme completed

24, achieved

18 Annual NoE Meeting 28, achieved month 29, will be International Meeting

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4.2 TABLE OF PERFORMANCE INDICATORS JOINT RESEARCH • Number and quality of publications resulting from EURASNET • Number of publications in peer reviewed journals • Number of joint posters and meeting abstracts • Number of collaborative publications

2007 saw a substantial increase in collaborative publications.

Publications acknowledging EURASNET - 2007

Activity Number Refereed publications from one EURASNET lab 60 Refereed publications from interactions between more than one EURASNET lab

9

Conference proceedings involving one EURASNET lab 59 Conference proceedings from interactions between more than one EURASNET lab

15

• Number of presentations at international meetings

EURASNET members presented their work during the Annual EURASNET conference at Ile de Bendor, France, the Interdisciplinary Focus Meetings 2007 in La Grande Motte (France), Bariloche (Argentina) and Berlin (Germany), during Eurasnet workshops and countless other international international meetings. Detailed reports of all EURASNET financed events are included in the WP16 report.

Broader outreach to the wider RNA community - 2007

Activity Number Presentations at workshops 40 Presentations at national RNA groups or meetings 14 Organisation of national/international meetings 15 Presentations at national/international meetings 31 Invited seminars 35 Presentations/consultancy to industry/SMEs 57 Consultancy to therapeutic groups 5 Education of RNA focused students 32

• Number of shared resources newly established The network-wide database of antibodies, cell lines, plasmids and protocols continued to grow during 2007. The EURASNET microarray initiative (initiated by J. Valcárcel and continued by C. Smith) with much input from many EURASNET members

18

continued to evaluate the available commercial formats. At the end of this explorative phase EURASNET is currently finalizing a contract with Affymetrix providing EURASNET exclusive access to the best available technology on the market. The setup of small molecule screening experiments at the University of Dundee facility has now advanced to the stage where first screens are planned for 2008.

• Number of methodologies developed within the NoE New methodologies unique to research and application in alternative splicing like bioinformatics approaches, microarrays, biochemical purifications, electron microscopy techniques and analytical mass spectrometry continue to be developed in the network.

• Number of identified new regulatory factors in alternative splicing Due to stepped up efforts in array experiments the number of potential regulatory factors has grown far beyond the number of 240 factors in 2006.

• Number of identified regulatory sequences 285 ESR candidates (Gil Ast lab, http://ast.bioinfo.tau.ac.il/ESR.htm), 77 high-quality, experimentally verified splicing regulators Hiller, M., Zhang, Z., Backofen, R., and Stamm, S. (2007). pre-mRNA secondary structure and splice site selection. PLOS Genetics 3, 2147-2155). Quantifications are also provided in the WP12 report.

• Number of disease models under investigation Retinitis pigmentosa, Ron oncogene, spinal muscular atrophy SMA, AIDS, FSHD, CFTR, β-thalassemias, contribution of alternative splicing to various nervous system diseases and to cancers

• Number of organisms under investigation human cells, yeast, plants, viral systems, Drosophila melanogaster, C. elegans, mouse

• Press coverage of the network's output The IFM in La Grande Motte, France was covered by national TV, the IFM in Bariloche by national newspapers. A EURASNET info page appeared in THE PARLIAMENT.

• Attendance of workshop programme Detailed quantification of each workshop attendance is provided in the WP16 report. The three workshops in 2007 were attended by 70, 81 and 22 participants, respectively,

• Number of exchanged reagents The exact number of plasmids, constructs, extracts, antibodies, cell lines etc distributed throughout the network is unknown, but dramatically increased over the 2006 numbers due to increased collaborative efforts (estimates assume a number of more than 120 antibodies and about 380 plasmids).

SHARING RESOURCES, RELIABLE PROTOCOLS • Use of joint network facilities

Set-up of joint micro-array experiments under uniform and controlled conditions will now result in a contract providing all EURASNET members access to the best technology on the market. Small molecule screening facilities in Dundee does first EURASNET screens in 2008.

• Research manager to maintain updated catalogue of shared technologies,

resources and protocols

19

The Integrating Manager in Erlangen (participant 3, Stamm) (from July 2007 on in Dundee, participant 14 Brown) surveys the collections, an updated list of available technologies was included in the amended Annex I after the second YIP integration.

• Number of protocols available online 51 protocols available. YI PROGRAM • Number of applications in each open call (2nd call) 19 applications were received for the 2007 open call. • Recruitment of 5 new YIs to the network by month 24

Accomplished! The addition of five new labs substantially extended the experimental and technical potential of the network.

• Start second recruiting round by month 18 accomplished

• Number of publications of YIs and success in future career Three publications.

• Integration of YIs into the network Integration is excellent, all YIPs are involved in collaborative projects. EURASNET WEB SITE • Progress with setting up and using the website in all its aspects

The EURASNET website is functional in all its important modules awaiting further input from member contributions. The reorganisation and redesign of the site will further enhance functionality. Website organizer and Integration Manager (participant 14) and PSO (participant 8) are closely cooperating.

• Number of "site-hits" on the average 40 hits/hour • User feedback and user satisfaction evaluation No statistical information available. STAFF MOBILITY AND TRAINING • Number of labs participating in exchange program

Twelve labs participated in the exchange program. • Number of short- and long-term visits between labs and their outcome A total of twelve exchanges was recorded. • Annual total of offered PMT

A total of 5.2 person months training was achieved. This number will increase after it was decided in 2007 that travel bursaries will become available also for intra-work package meetings between collaborating network members and workshop attendance of students and postdocs.

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CAREER DEVELOPMENT • Number of people moving between EURASNET laboratories

Twelve short term visits (see report for WP18). • Number of joint PhD students and postdocs Four cases of joint PhD committees in 2007. • Attendance at workshops/seminars on career development About 40 students at the Ile de Bendor Annual meeting. CONFERENCES AND MEETINGS • Number of participants during Annual Conference 2007

Of the 35 research groups in the network, 33 were present with their respective principal investigator or an authorized legal representative. 38 additional lab members attended the conference. Four of the seven Scientific Advisory Board members were present. For the YIP decision the SAB members not attending were contacted by telephone. The required quorum for the YIP decision was thus achieved. The European Commission was represented by the external reviewer Prof. Benoit Chabot, Sherbrooke, Canada.

• Number of non-NoE participants during European Conferences No European Conference yet. • Feedback from participants and SAB members

The Annual Conference, as well as the IFMs and workshops all successfully brought together participants and SAB members in the most fruitful way. EURASNET is increasingly recognized in the scientific community and industry as a serious player in AS-related research. The intellectual exchange within the network is considered very constructive and mutually beneficial.

• Meeting reports in journals such as EMBO reports An official report on the Bariloche IFM will appear in EMBO Reports 2008. • Attendance of Interdisciplinary Focus Meetings

Bariloche Meeting (Cell Biology, Signaling and Alternative Splicing): 52 scientists attending

Berlin IFM (Biophysical Methods) was attended by 40 EURASNET scientists. • Attendance of Interdisciplinary Focus Meeting dedicated to splicing and disease

The RNA and Cancer meeting (La Grande Motte, France) was attended by 91 scientists.

SMEs AND TECHNOLOGY TRANSFER • Number of EURASNET links to industry

As a precondition for such links a commercialization fact-file was updated under the leadership of EURASNET member Angus Lamond, Dundee. He also prepared a document " EURASNET Strategic Planning for the New Venture Biotechs" available for participants. Comments from legal departments of EURASNET members continue to be evaluated and eventually incorporated into a new, updated version of

21

the fact-file. The Dundee Cell Products Ltd (DCP) will discuss the potential for collaboration between EURASNET and DCP. A contract between EURASNET and Affimetrix is being finalized.

• Number of patent applications None • Establishment of partnerships with a drug discovery company None. PUBLIC SCIENCE ACTIVITIES • EURASNET brochure

Has been updated upon YIP integration and extended formats are under development (see Report WP 19).

• Attendance at workshops on PSU training Workshop on PSU is part of Annual Meeting Krakow 2008 (month 29) • Webpage for Science and Society "site-hits" still under construction • Number of lay articles published None DURABILITY • Number of additional funds used to perpetuate the network

EURASNET funding provides only a minute amount of the funds required to carry out the agreed upon Joint Program of Activities. For each participating lab the major part of funding therefore relies on other grants and/or institutional funding.

• Number of successful joint grant applications Currently statistics on such grant applications are not available, although we know that such successful applications exist.

• Integration of the broader European RNA community Workshops and IFMs served as suitable tools to bring EURASNET and the broader RNA community into close contact. Participant 8 and 14 (Barta and Brown) have considerably stepped up efforts to reach the European RNA community with mailing lists.

• Number of NoE activities sponsored by industry The Cancer & RNA Workshop in France, 2007 received sponsoring from industry.

GENERAL MANAGEMENT • Reports by the managers for approval

Contract amendments have been approved for YIP integration (1st round) and underspending in 2006. Amendment for YIP integration (2nd round) was submitted Dec 2007.

• Efficient information flow within NoE

22

was achieved through e-mails and telephone/video conferences. • Efficient execution of committee decisions

Major decisions executed in swift time related to YIP integration, meetings, workshops and funding for high-throughput enabling technologies.

• Attendance of board meetings Four of seven board members attended Annual meeting in France in person, the others by telephone.

• Smooth and timely flow of resources according to budget Research grant money could not be distributed due to late payment of 2007 advance payment.

• Steering committee meeting minutes None • Timely reporting to the EC

Annual Report 2007 submission was only moderately delayed, primarily due to slow processing of financial report forms within members' financial departments.

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5 WORK PACKAGE REPORTS Work Package 1 The EURASNET Web site Lead Participant: StefanStamm/John Brown

month planned achieved deliverables: 61 Optimization of database content, in particular through

linking of already available content (month 30) 30 24

62 Improve content intended for the general public (month 30) 30 24 63 Software improvements, bug-fixes and more user-friendly

interface (month 30) 30 24

Objectives of the work package • To establish, curate and maintain a EURASNET website as a tool to promote European research on alternative splicing by concentrating all available information, facilitating the exchange of information between NoE members and by disseminating the activities of the NoE to the wider scientific community and public interest groups Short description of activities 61 Optimization of database content. Additional, new content has been posted on the website. Available content and the new content has been linked as appropriate. Database content in particular has been added to and the form of presentatioin and retrieval is being revised. 62 Improve content intended for the general public Information on some splicing-related and alternative splicing-related diseases as well as information on alternative splicing for lay audiences has been put on the website. In addition, educational presentations for students from various EURASNET members is available. Before the end of the reporting period the website will be modified to have clickable access for information specifically prepared and targetted to the general public, clinicians and students. 63 Software improvements, bug-fixes and more user-friendly interface. A number of bugs have been removed and improvements made. A new user-friendly website is under development. Problems and explanations for delays, postponements etc. None

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Work Package 2 Sharing Resources, Technology and Reliable Protocols Lead Participant: Stefan Stamm

month planned achieved deliverables: 130 Web pages containing protocols and useful information

regarding RNAi, shared databases and analysis programs (continuation of del.2, month 30)

30 24

131 Updated list available to all participants describing the network's access to services, shared facilities and technical expertise via collaborative arrangements (continuation of del.3, month30)

30 24

Objectives of the work package • To create a seamless, integrated network that allows each lab to draw upon the knowledge and resources available in other labs. • To promote and encourage shared use of already existing facilities belonging to individual institutes whenever this is compatible with existing on site work loads and work flows. • To actively ensure transfer of a technology fully developed in one laboratory to another participating laboratory still lacking this technology. Short description of activities 130 Web pages containing protocols and useful information. All labs contributed protocols to the EURASNET webpage that contains now more than 51 entries. Currently, the protocols are only visible for members and are being tested and discussed, before they will be released to the general public. Protocols were already used in the workshop on alternative splicing and disease in Montellier in Feburary 2008. A second set of protocols will be made available to the public at a RNA protein interaction workshop that is planned to take place in Barcelona. Based on the feedback of users, the current protocol entry forms will be optimized in a new entry form. With the release of the new EURASNET web site, all protocols will be in pdf format and will be open to the public. All protocols will be published in book format in mid 2009. 131 Updated list available to all participants describing the network's access to services, shared facilities and technical expertise via collaborative arrangements. User profiles were regularly updated on the EURASNET web site. Based on these profiles, more than 28 collaborations between EURASNET members were initiated. Problems and explanations for delays, postponements etc. All deliverables were achieved

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Work Package 3 The Young Investigator Programme (YIP) Lead Participant: Reinhard Lührmann

month planned achieved deliverables: 5 Start selection of the second group of five YIP awards by

month14 (Annual Meeting, France). Integrate successful candidates by month 25.

25 25

Objectives of the work package • To actively promote and support the most-talented, young investigators in the field of alternative splicing in Europe.

• To integrate young and talented group leaders into the EURASNET and thereby ensure the vigour, durability and dynamic expansion of the NoE. Short description of activities 5 Second YIP selection. The second round of YIP recruitment was initiated with an open call early in 2007. Winners were selected during the Annual EURASNET meeting at Ile de Bendor, France 2007, where short-listed candidates presented their projects. Full documentation of the call and selection process was send to the EC. After approval of the selected candidates, financial and other documents were collected from the five new members and Annex I and Consortium agreement were amended. Starting date of new members is Jan 01, 2008. Problems and explanations for delays, postponements etc. None

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Work Package 4 The Alternative Splicing and Transcript Diversity (ASTD) Database Lead Participant: Rolf Apweiler

month planned achieved deliverables: 8 First incorporations of curated information from

databases generated by individual laboratories into the ASD structure and report of discussions between ASD staff and computational groups on implementation of data access for large-scale computational studies and assembly of combined databases. (month 19)

19 20

64 Using the exon index generated by the ASTD pipeline for human, mouse and rat, we aim to annotate exons with experimental properties to capture the microarray data that is available from individual laboratories within the Network (month 30).

30 --

132 Improved tools for combined searches and user-friendly access to the ASD database; Integration of additional species to ASTD (continuation of del.7, month 30).

30 30

Objectives of the work package • To maintain, update and expand the alternative splicing database (ASD) hosted at the EBI. Short description of activities 8 First incorporations of curated information from databases generated by individual laboratories into the ASD structure. Prediction of miRNA target sites is being studied by the Zavolan group and is publically available at:

http://www.mirz.unibas.ch/Computational_prediction_of_microRNA_targets.shtml

The latest EIMMo human and mouse predictions from January 2008 (Gaidatzis et al, 2007) have been incorporated into the ASTD schema to provide additional annotations to the genomic DNA. Computationally-predicted miRNA target sites are selected on that basis that perfect base pairing between the 5' end (nucleotides 1-8) of the miRNA and its target are essential for miRNA function. As evolutionary conservation is an indicator of the functionality of miRNA target sites, the predicted sites in the EIMMo model also take into account the evolution of orthologous target sites in a set of related species.The mappings will be available with the next release of the ASTD data. The Ensembl Funcgen database (eFG) database and API stores functional genomics experiments submitted to Ensembl from their collaborators. This includes miRNA targets predicted by the miRanda programme, published in miRBase (Griffiths-Jones et al., 2006). miRBase Targets is a web resource (http://microrna.sanger.ac.uk/) developed by the Enright Lab at the Wellcome Trust Sanger Institute containing computationally predicted targets for microRNAs across many species. For each published miRNA, miRBase provides the genomic coordinates of the predicted precursor sequence, the nucleotide sequences of both the precursor and mature miRNA sequences and predicted targets of the mature miRNA. When ASTD and Ensembl are fully integrated the 3’ UTRs will be annotated for miRNA target predictions from both EIMMo and miRanda. Gaidatzis D, van Nimwegen E, Hausser J, Zavolan M. Inference of miRNA targets using evolutionary conservation and pathway analysis. BMC Bioinformatics. (2007) 8:69 Griffiths-Jones S., Grocock R.J., van Dongen S., Bateman A., Enright A.J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 2006;34:D140–D144 64 Exon index generated by the ASTD pipeline for human, mouse and rat. Within ASTD, a pipeline defines exons and introns by blasting a recent database of EST’s and mRNA’s against the Ensembl known gene set. The resulting data is then analysed looking for high scoring pairs (HSPs). The Ensembl gene sets are created using an automated analysis pipeline that has been significantly optimized based on the completeness of the genome sequence and the availability of species-specific supporting data.

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Work is currently underway to compare and unify these two pipelines, to optimize the parameters achieving a consensus exon index for Ensembl that reflects the best representation of each the genome. 132 Improved tools for combined searches and user-friendly access to the ASD database. The current ASTD website and tools (http://www.ebi.ac.uk/astd) will be maintained until approximately Feb 2009. The pages will be updated and maintained for all new releases and developments until the date of its removal. The Ensembl genome browser available at http://www.ensembl.org provides visualization for its own and collaborators genome annotations, alignments, variation and functional genomics data and supporting additional data integration through the DAS protocol. The BioMart data mining tool, which supports sophisticated Ensembl-specific queries, provides functions to retrieve gene annotation, GO, SNP, sequence and other information directly from the BioMart databases. With the integration, all functionality on the ASTD web pages will be transferred to Ensembl to ensure a smooth transfer for our users. In addition to the site at the EBI, a user-friendly interface was implemented into FASTdb, which can be seen at http://www.fast-db.com/fastdb2/frame.html FAST DB was developed by EURASNET member Didier Auboeuf and Pierre deLa Grange who were leading the discussions with the scientists of EURASNET and implemented their suggestions into FAST-DB. The screenshot shows the integration of databases that predict splicing regulatory motifs with FAST-DB. (de la Grange, P., Dutertre, M., Correa, M., and Auboeuf, D. (2007). A new advance in alternative splicing databases: from catalogue to detailed analysis of regulation of expression and function of human alternative splicing variants. BMC Bioinformatics 8, 180)

Screenshot of FAST-DB. Clicking of each sub-database creates a hyperlink that allows analysis of alternative exons with this databases Problems and explanations for delays, postponements etc. 64 Specific aim 64 could not be achieved due to a restructuring of the databases. As an alternative, microarray data will be integrated into the FASTDB database.

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Work Package 5 Ensuring Durability Lead Participant: Maria Carmo-Fonseca

month planned achieved deliverables: 9 Generation of a webpage dedicated to disseminating

information for NoE members about future funding (month 18).

18 14

Objectives of the work package • To ensure a durable, functional alternative splicing network that will serve as a model for cooperative research and innovation in the field of RNA splicing and RNA biology in Europe long beyond the NoE funding period. Short description of activities 9 Webpage dedicated to disseminating information for NoE members about future funding.

1. Fostering applications to international funding programs. The EURASNET website has been launched and is regularly updated (see WP1) with information on funding opportunities. A forum has been created at EURASNET meetings to inform participants of available funding possibilities. Particular emphasis is given to international programs and other initiatives that may support further European cooperative research and innovation in the field of RNA biology. As a result of these activities, several members of EURASNET have submitted applications to regional and international funding programs.

2. Liaison with relevant scientific and other bodies throughout Europe. The Network´s leading participants have maintained close contact with the EU with the goal of maintaining a sensitive response to perceptions of future need, and influencing this perception where necessary. Problems and explanations for delays, postponements etc. NIL

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Work Package 6 In silico approaches to alternative splicing Lead Participant: Gil Ast

month planned achieved deliverables: 65 The Stamm lab will concentrate on verifying the

bioinformatically predicted targets for snoRNAs. This will be done by two approaches: a) direct verification of ‘promising-looking’ hits and b) construction of a custom-made DNA array that detect all the predicted changes in splice site usage (month 30).

30 25

66 The Zavolan lab in collaboration with the Stamm lab will validate some initial predictions of snoRNA HBII-52 targets, based on which the model will be updated to predict targets for other orphan snoRNAs (month 30). The Zavolan lab will predict miRNA target sites in 3’ UTRs of human and mouse transcripts, including transcripts which use alternative polyadenylation sites.

30 25

67 The Bork lab will identify conserved and therefore functionally constrained alternative transcripts in divergent Metazoa. We will also use information on conservation to identify associated regulatory sequences (month 30).

30 25

68 The Ast lab will compile a unified dataset of splicing regulatory elements and examine the minimum/maximum potential regulatory sequences. Also, we will examine the level of alternative splicing among metazoan (month 30).

30 25

N6 N6 Computational structural model of splicing regulators hnRNP-L and hnRNP-L-like in complex with RNA (month 30)

30 30

N7 N7 Computational modeling and experimental testing of novel RNA binding domains (month 30) 30 30

Objectives of the work package

• Improved identification of known regulatory sequence motifs • Computational prediction of novel regulatory motifs • Computational prediction of regulatory proteins and RNAs • Investigation of correlations between promoter structure and alternative splicing • Investigations on the evolution of alternative splicing

Short description of activities 65 Verifying the bioinformatically predicted targets for snoRNAs. The Stamm lab have identified a snoRNA regulates the processing of an mRNA expressed from a gene located on a different chromosome. In collaboration with the Zavolan group who predicted snoRNA targets, they experimentally validate the accuracy of these predictions. Additional hits came from an Affymetrix gene chip experiment that was performed together with Chris Smith and Didier Aubouef. We constructed a custom made array, however, this array failed to indicate even known changes in snoRNA-dependent alternative splicing and in the detection of new events . 66 Refinement of snoRNA target prediction models to take into account new information from validated snoRNA-mRNA interactions. The Zavolan group implemented a snoRNA-mRNA binding model that reflects the constraints the snoRNA-rRNA interaction and predicted several targets for snoRNA HBII-52. In collaboration with the Stamm group they experimentally validate the accuracy of these predictions. The same model was applied to snoRNAs from Arabidopsis to generate predictions of mRNA targets that will be tested by the group of John Brown. Predictions of miRNA sites. Objective achieved, including the incorporation of miRNA target sites in the ASTD. Predictions are updated on a regular basis, in accommodate new genome sequence data and new reference transcripts.

30

67 Identify conserved and therefore functionally constrained alternative transcripts in divergent Metazoa. The Bork group developed a framework for the prediction of conserved alternative transcripts and identified some in as distant organisms as human and fly. This work needs to be extended to more organisms for proper quantification. The group further identified as planned conserved regulatory sequences associated with alternative splicing. 68 Unified dataset of splicing regulatory elements. The Ast lab compiled a unified dataset of splicing regulatory sequences and generated a web server: http://ast.bioinfo.tau.ac.il/ESR.htm. They are currently examine the minimum/maximum potential regulatory sequences. Problems and explanations for delays, postponements etc. None

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Work Package 7 Molecular Characterization of Splicing Substrates Lead Participant: Christiane Branlant

month planned achieved deliverables: 33 Agreement on pre-mRNA substrates, nuclear

extracts and cell lines; computer analysis of secondary structures and distributions of known preferred binding sites for SR and hnRNP proteins (month 18).

18

69 Further analysis of local RNA secondary structures and effects on splicing (month 30)

30 24

70 Complete identification of components in complexes and assessment of roles in the functions of influential sequences (month 30)

30 24

71 Expansion of work on detailed analysis of RNA-protein complexes formed with recombinant protein and repeat sequences or regulatory sequences, including structural analysis. (month 30)

30 24

72 Identification of more in vivo targets of the functional activity of splicing regulatory proteins (to be subsumed in WP8). (month 30)

30 Planned in connection with WP9, delayed

73 Development of single molecule methods to follow the kinetics of splicing and factor association. (month 30)

30 24

N10 Determine the structure of hnRNP F, hnRNP L and Etr3 in free state and in complex with RNA using NMR spectroscopy

30 24

Objectives of the work package • To develop strategies with selected pre-mRNA substrates for the description of what spliceosomal components encounter upon assembly on pre-mRNAs.

• RNA 2D structure, • Nuclear proteins bound to RNA transcript

The developed strategies include biochemical, structural and molecular methods and the use of single molecule approaches. The selected substrate sequences are viral pre-mRNAs sequences, repeated cellular RNA sequences or pre-mRNA sequences with deregulated alternative splicing in genetic diseases. Short description of activities 33 Agreement on pre-mRNA substrates, nuclear extracts and cell lines. 69 Further analysis of local RNA secondary structures and effects on splicing. (Participants 7, 12c in collaboration with 12d WP9 and WP14)

• HIV-1 The effect of the RNA 2D structure on protein binding at the HIV-1 splicing sites A3 (needed for tat mRNA production) and A7 (needed for both tat and Rev mRNA production) was further investigated. We found that the highly conserved apical part of the HIV-1 SLS3 stem-loop structure, that contains the regulatory splicing elements acting on site A3, is needed to preserve the specific folding of these regulatory elements. The 3D structure of this apical part was established by NMR. It forms an RNA double-stranded motif that was expected to be recognized by proteins containing DRBD (double stranded RNA binding domain) domains. The proteomic analysis of nuclear proteins associated with this HIV-1 region (WP9) confirmed this hypothesis.

• Human Mutations altering alternative splicing A C to U point mutation in exon 11 of the lamin A pre-mRNA is responsible for the progeria syndrome. This mutation strongly reinforces the utilization of a cryptic 5’ splice site. We analyzed the 2D structure of a 200-nt long fragment of the

32

lamin A pre-mRNA that contains this cryptic site, and showed that this site is located in a highly structured region. However, its accessibility is markedly increased, after the C to U mutation responsible for progeria. The consequence of this structural alteration on protein binding is studied by a proteomic approach (WP9). An increased inclusion of Exon 5 of human troponin T pre-mRNA is frequently observed in DM1 patients. A study of the RNA secondary structure of the troponin T exon 5 and its flanking intronic sequences has been started. The RNA secondary structure of the ATM and CFTR pseudoexon, whose inclusions lead to pathologies, was studied by the use of antisense oligonucleotides. This strategy is expected to be useful for future therapeutic applications.

• RNA secondary structure of RNA that may regulate the availability of splicing factors - SC35 Partner PID26 has identified RNA segments in the 3’ UTR of the SC35 pre-mRNA that are responsible for a negative retro-control of SC35 production (WP12). They identified the repressor binding proteins (hnRNP H and TDP43) and Partner PID 12c will now study the 2D structure of the interacting RNA segments for further analysis of the RNA protein interaction at this regulatory site. - ASF/SF2 Partner 10a showed that various stresses activate different transcription factors resulting in the transcription of large amounts of satellite III RNAs, which are already known to bind protein SRp30c. partner 10a developed a proteomic approach for the identification of the other proteins bound to these RNAs (WP9). The idea is that the SatIII RNAs modulates the concentration of ASF/SF2 protein available for splicing depending on the physiologic conditions. Partner PID12c will now study the 2D structure of SatIII RNAs in collaboration with partner 10a. 70 Components in complexes and assessment of roles. (Participant 12c in collaboration with12d WP9 and WP14, Participant19) This work is done in close connection with the work done in WP9. As soon as the 2D structure of a viral or cellular pre-mRNA region containing a splicing site and its regulatory elements or an RNA sequence responsible for deregulation of alternative splicing is established, delimited domains of this RNA are fused to three binding sites for the phage MS2 coat protein and the MS2-based affinity chromatography method is applied to purify the RNP formed by the RNA domains in HeLa cell nuclear extracts. The associated proteins are identified by mass spectrometry and those interacting directly with the RNA are defined by UV cross-linking experiments and by gel-shift assays using recombinant proteins. During this work period such analyses were done on the HIV-1 A3 and A7 regions, the NRS inhibitory element of Rous sarcoma virus, RNA containing the repeated CUG sequences known to be responsible for type 1 muscular dystrophy. Finally, work is currently done on WT and mutated exon 11 of the lamin A pre-mRNA and on WT and mutated exon 5 of the medium chain acyl-CoA dehydrogenase. It should be pointed out that information on proteins that may bind to splicing regulatory sites are also obtained by purification of complete splicing complexes by partner PID1c (WP9). The probing studies of early spliceosomal complexes by this partner, using hydroxyl radical is also informative on how regulatory proteins interact with the RNA in the early step of spliceosome assembly (WP9). Therefore, altogether the effort made in WP7 and WP9 should strongly contribute to answer the question: what is the identity and characteristic features of the RNP substrates of splicing complexes. 71 Expansion of work on detailed analysis of RNA-protein complexes. (Participants 4, 7, 10a, 12c,19, 23).

• HIV-1 Further analyses were performed on the mechanisms of regulations acting at the HIV-1 acceptor sites A3 and A7. A new mode of activation of site A3 by the combined action of 9G8 and ASF/SF2 at a previously unidentified activator site was discovered. The interference of the 9G8 and ASF/SF2 associations with the binding of other nuclear proteins, in particular hnRNP A1, is investigated. The proteomic analysis of RNP complexes formed at site A7 identified nucleolin, hnRNP H and hnRNP K as potential new partners of the A7 regulatory regions, their binding sites were identified by various methods. Interestingly, we showed that hnRNP K and hnRNP A1 cooperate together for binding to the A7 region and as expected hnRNP K reinforces the inhibitory effect of hnRNP A1 at this site. New activation factors were also detected at site A7.

• Adenovirus The adenoviral L4-33 protein was shown to be a virus encoded alternative splicing factor. It activates splicing by stimulating pre-spliceosome formation. UV crosslinking experiments demonstrated that, as well alone as in nuclear extract, L4-33 does not bind directly to the pre-mRNA. Current efforts are made to try to identify which of its partner protein is able to bind the RNA directly. A surprising finding in the course of these experiments was the observation that L4-33K stimulates splicing of its own pre-mRNA through an auto-regulation feed forward mechanism. The identification of the targeted L4-33 pre-mRNA is underway.

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• Splicing deregulations linked to genetic diseases

Type 1 Muscle Dystrophy (DM1) is due to the amplification of CTG repeats in the DMPK gene. CUG repeats form peculiar long helical structures that sequester the MBNL splicing factor. Work is done in the view to establish the 3D structure of the RNA binding domain of protein MBNL1. The RNA specificity of MBNL1 was studied by using both the SELEX and the triple hybrid approach. Nine isoforms of MBNL1 are produced by alternative splicing, the difference of RNA specificities of these isoforms was studied by the triple hybrid approach. The binding sites of MBNL1 and CUG-BP were identified on the intronic sequences that border exon 5 of human troponin T (the inclusion of exon 5 is increased in DM1 patients). MBNL1 binding sites were detected on both sides of exon 5. The fine tuning of the mechanism of regulation by MBNL1 and CUG-BP is investigated. In parallel, a deep study of the roles of PTB, Raver and MBNL in the regulation of the alternative splicing of exon 3 from human α-tropomyosin was achieved (exon 3 is excluded in DM1 patient). Both Raver and PTB bind on both sides of this exon. Binding sites for MBNL1 were detected also on both sides of exon 3 and the knock down of MBNL1 by siRNA limits the in cellulo utilization of exon 3. Hence, while binding sites of MBNL1 are found on both sides of the troponin T exon 5 and a-tropomyosin exon 3, depletion of MBNL1 has opposite effect on these exons. This illustrates the importance of the context of the binding sites of splicing factors on the action of these factors. The mechanism by which the C362T substitution in the medium-chain acyl-CoA dehydrogenase pre-mRNA induces exon 5 skipping was elucidated. The C to T point mutation generates an hnRNP A1 binding site that abolishes an activator site. CERES elements are short RNA splicing regulatory elements. When mutated, they induce either enhancer or silencer activities depending on the nucleotide altered. They were first identified in the CFTR exon 12 because clinical studies identified within their sequences several mutations capable of affecting the splicing of exon 12. By pull down assays with WT and mutated CERES elements, followed by western blot assays against common RNA binding proteins, ASF/SF2, SRp50, hnRNPA1/A2 and C2 were found to be protein partners of the CERES elements. Functional validations are underway.

• Ron oncogene The proteins bound to the silencer element in exon 12 of the Ron pre-mRNA were studied. Both hnRNP A1 and hnRNP H bind to this element. 72 Identification of more in vivo targets of the functional activity of splicing regulatory proteins. Not yet started, will be done in studies concerning both WP7 and WM9. 73 Development of single molecule methods to follow the kinetics of splicing and factor association. (Participants17 and19) Participant PID19 has established an in vitro assay for the detection by FRET experiments of the association of hnRNP A1 with the two binding sites that flank an alternative exon of its own pre-mRNA. The capability of hnRNP A1 to inhibit inclusion of its targeted exon within the chemically modified pre-mRNA has been verified by in vitro splicing assays. In addition, single molecule FRET experiments have recently been initiated. Partner PID17 has cloned about 20 splicing regulator proteins as fusions to GFP, Cherry and other fluorescent proteins and is monitoring their binding on pre-mRNA. He will now be able to collaborate with other partners who are involved in the biochemical study of the binding of these proteins to their favourite pre-mRNAs. Single molecule analysis of assembly of complexes in crude extracts has not been described previously. Methods have been developed to detect the binding of proteins to pre-mRNA in nuclear extracts, which permits the stoichiometry and heterogeneity of complexes to be measured for the first time. Simultaneous occupancy by proteins with different fluorophores can be determined. The binding of PTB to tropomyosin pre-mRNA has been assayed (PID 17 and PID 23). Evaluation of the possibility of watching protein association in real time is under way. The new YIP partner 12d E Bertrand also produced a HeLa cell line with a stably inserted DNA sequence derived from HIV-1 cDNA. It contains the D1 5’ss and the A7 3’ss, 24 binding sites for the phage MS2 coat protein are inserted downstream from site A7. This construct is now used to study protein binding at transcription sites by in live HeLa cells fluorescence studies. ASF/SF2 was found to be bound to RNA at the transcription site. N10 Determine the structure of hnRNP F, hnRNP L and Etr3 in free state and in complex with RNA using NMR spectroscopy. The new YIP partner (PID 29) F Allain recently determined the structure of two of the 4 RRMs of hnRNP L (RRM3 and 4). Protein hnRNP L recognizes CA-repeats. Interestingly, its RRM 3 and 4 domains interact with the RNA in a way very similar to how the RRM 3 and 4 domains of PTB are interacting. This suggests that hnRNP L and PTB might have similar modes of action, with different sequence specificities. Partner PID29 also established the 3D structure of the 3 RRMs of hnRNP F in their free form. Surprisingly, this reveals the presence of a C-terminal α-helix that covers the β-sheet surface of the RRM. To get an explanation, for this unexpected

34

observation, the structure of the complex formed by an AGGGAU oligoribonucleotide and RRM2 was determined. It reveals a new mode of binding for an RRM to RNA, since the β-sheet surface is not used for RNA binding as is usually the case. These data explain how G-tracts are recognized by hnRNP F. Problems and explanations for delays, postponements etc. 72 delayed

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Work Package 8 Genome-wide Analyses of Splicing Regulation Lead Participant: Chris Smith

month planned achieved 74 Validation by Q-RT-PCR of 40-50 AS changes predicted

by ExonHit SpliceArrays and Affymetrix ExonArrays (month 16)

16 16

75 Comparison of different data analysis procedures for extracting AS information from ExonArrays (month 30)

30 21

76 Exon junction microarray analysis of effects of CBC knockdown upon alternative splicing (month 30)

30 24

77 Validation of SF1 CLIP targets to test whether SF1 is an alternative splicing factor. CLIP of U2AF65 to compare binding sites with SF1 and application of CLIP to hnRNP L (month 30).

30 24

78 The expanded AS RT-PCR panel will be used to address a) the effects of individual factors in mRNA biogenesis, b) the effects of stress on AS events and levels of trans-acting factors, c) the link between AS and nonsense-mediated decay (NMD), and d) combinatorial control of alternative splicing of subsets of transcripts. (month 30)

30 24

79 The ability of new Arabidopsis genome tiling arrays from Affymetrix to detect and report on AS will be examined (month 30).

30 24

80 Genome-wide identification and analysis of promoter-dependent exons in human (month 30).

30 24

133 A web-based resource of validated techniques for global analyses of splicing (continuation of del.36, month 30).

30 24

134 Feasibility testing of key experimental approaches (continuation of del.37, month 30).

30 24

135 An agreed coordinated strategy for subsequent analyses of global splicing factor activity (continuation of del.38, month 30).

30 16

Objectives of the work package • To develop and share relevant expertise, techniques and reagents.

• To test the feasibility of and to optimize key techniques e.g. splicing factor ChIP, CLIP, iTRAQ analysis of alternative splicing. Where possible, to coordinate such tests within common model systems.

• To develop a co-ordinated strategy for genome-wide investigation of splicing factor activity, aiming to maximize complementarity and minimize overlap between participants, particularly in the area of mammalian splicing factor activity, where the majority of participants are active. To identify and eliminate major “gaps” (e.g. important splicing factors not currently being analyzed). Short description of activities 74 Validation of ExonHit SpliceArrays and Affymetrix ExonArrays. Biological triplicate RNA samples from HeLa cells treated with control or PTB+nPTB siRNAs were provided to ExonHit and Affymetrix. The companies were not told the identity of the splicing factors that had been knocked down. Hybridizations to arrays were carried out by the companies. Affymetrix supplied three lists of candidate events produced via different data analysis pipelines using Partek Genomics Suite, Biotique XRay, and an “Internal Analysis”. Data from ExonHit was supplied in the form of an Excel spreadsheet (the Splice Array Tool) with no prioritization of targets. Upon request, a small number of higher priority predictions were supplied. Validation was carried out by RT-qPCR of triplicate samples. Only 7/15 of the ExonHit predictions were validated. Among the 32 “high confidence”predictions from the Affymetrix Internal Analysis the validation rate was 25/27 (~93%). An independent analysis of hnRNPL knockdown in HeLa cells (Bindereif) also used ExonArrays to successfully identify a variety of hnRNPL regulated AS events in HeLa cells (Hung, et al. (2008). Diverse roles of hnRNP L in mammalian mRNA processing: a combined microarray and RNAi

36

analysis. RNA 14, 284-296). 75 Comparison of different data analysis procedures. RT-qPCR validation of the three sets of predictions of PTB/nPTB-regulated events from the ExonArray gave the following results: Partek, 17/24 (71%), Xray 12/21 (57%), Affymetrix Internal Analysis, “High Confidence” 25/27 (93%), “Medium Confidence” 5/12 (42%). A workshop was held in Cambridge in September 2007 to discuss ExonArray data analysis. Tyson Clark explained in full the Affymetrix analysis procedure to WP8 members. Independent analysis procedures have also been developed by the groups of Bindereif and Auboeuf. 76 Exon junction microarray analysis of effects of CBC knockdown. In a collaboration between the Neugebauer and Valcarcel labs, the cap binding complex (CBC) has been depleted from HeLa cells by RNAi. Because CBC plays a role in exon definition, by recruiting the U1 snRNP to the cap-proximal 5' splice site, we wanted to test for the possibility that CBC is important in alternative splicing. A predoctoral student (Marta Pabis) from the Neugebauer lab in Dresden travelled to Barcelona with the RNA samples resulting from three biological replicates in which CBC proteins were depleted by at least 80%. There she produced labeled probes, hybridized them to the custom-designed splice junction arrays developed by the Valcarcel lab, hybridized and scanned them, and finally extracted the data. Together with Claudia Ben-Dov of the Valcarcel lab, she determined that only two candidate alternative splicing events could be identified. Marta was unable to validate these AS events by RT-PCR, so we conclude that at least under the conditions of this experiment, we were unable to detect a role for the CBC in AS. This result will serve as an important control in a distinct set of experiments addressing the role of CBC in U snRNA export. 77 Validation of SF1 CLIP targets. 17 pre-mRNAs identified as potential SF1 targets by CLIP were tested for splicing changes after RNAi-mediated depletion of SF1. In nine cases splicing was either reduced or the AS pattern was changed, in five cases no changes were observed and three cases were non-conclusive. Together these data indicate that SF1 is only required for the splicing of certain introns and acts as an alternative splicing factor. The CLIP method was also applied to identify targets of U2AF65, which cooperates with SF1 in 3’ splice site recognition. The general distribution of CLIP tags in introns and exons was similar to the distribution observed for SF1. CLIP was also applied to hnRNPL in HeLa cells. Of 100 sequenced tags, none contained the clusters of CA motifs that are typical of known hnRNPL binding targets, possibly reflecting insufficient stringency of the immunprecipitation step. 78 Expanded AS RT-PCR panel. The Arabidopsis alternative splicing RT-PCR panel has been expanded from the initial 96 AS events to 384. The AS events have been selected from databases and focused on genes encoding RNA-interacting proteins, transcription factors and genes involved in signalling and stress responses. To date, the full 384 AS RT-PCR panel has been used to analyse mutants of the cap-binding complex proteins (factors in mRNA biogenesis – 78a). Transgenic over-expression lines and mutant lines of transacting factors such as SR proteins and PTB are being prepared and checked genetically before being analysed (78b). An initial experiment with 96 AS events using two NMD mutants indicates that around one third of plant AS transcripts may be regulated by NMD (78c). In some treatments, similar patterns of changes in AS among subsets of genes have been identified and may indicate combinatorial control of AS (78d). 79 Arabidopsis genome tiling arrays. Arabidopsis genome tiling arrays have been obtained in order to assess their utility in detecting and quantifying alternative splicing. The difficulties in extracting the data on a single primer basis however are not trivial and we are currently developing contacts with labs who have this capability. 80 Genome-wide identification and analysis of promoter-dependent exons in human. We developed a method to identify cassette exons whose inclusion in the mature mRNA is dependent upon the promoter used to initiate the transcript. We applied this method to human full-length cDNA data, and to 5’-end ESTs. We found that the results of our initial analysis performed on mouse full-length cDNA data hold, and that a large fraction of the human exons appear to be included in the mature mRNA in a promoter-dependent manner. This fraction is even higher when one analyzes presumably unbiased libraries of 5’-end ESTs. We found a weak correlation between the probability of promoter-dependence of human and mouse orthologous exons, but otherwise we did not find features that distinguish the promoter-dependent exons from those that appear to be promoter-independent or constitutive. The mechanisms underlying this dependency remain to be determined. 133 A web-based resource of validated techniques for global analyses of splicing. The analysis tool for Splicearrays and a visualization was implemented in FASTDB. Addition of validated protocols (from deliverable 134) to the website is an ongoing process that is envisaged to last for the lifetime of EURASNET. 134 Feasibility testing of key experimental approaches The Kramer lab has used the CLIP method with nuclear and cytoplasmic fractions of cross-linked HeLa cells to distinguish between functions of SF1 and U2AF65 in the two cellular compartments. A very high reproducibility between different experiments was obtained, having optimized the preparation of extracts to minimize cross-contamination. The CLIP method

37

has been improved following protocols provided by Jernej Ule and is currently being adapted for deep sequencing. The Smith lab carried out a quantitative gel-based proteomic analysis of PTB/nPTB knockdown in HeLa cells. An improved experimental design with 6 biological repeats and a 3-dye system with a Cy2 labeled internal pooled standard was adopted, allowing reliable identification of 45 spots that had changed by > |1.15| fold after a short RNAi treatment (to reduce the number of secondary events identified). 41 proteins were identified by MS sequencing and so far 25 of 55 identified AS events have been validated. Only three of these events were also identified by microarray analysis. The Stamm lab tested ExonHit SpliceArrays and compared them by RT-PCR, resulting in a collaborative paper with Claudia Ben dov fromValcarcel’s group: (Novoyatleva, et al. . (2008) Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing. Hum Mol Genet, 52-70. 135 Agreed coordinated strategy for subsequent analyses of global splicing factor activity. WP participants discussed future strategy at the 2007 Ile de Bendor reporting meeting. The increasing interactions and integration of WP members was considered to be sufficient to avoid unintended duplication. The plant groups (Barta, Brown, Jarmolowski) have established a productive closely coordinated joint programme. It was agreed that such a closely coordinated programme would not be appropriate for the larger number of groups using diverse animal systems, but that collaborative activities would develop by a series of smaller scale future collaborations. Problems and explanations for delays, postponements etc. nil

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Work Package 9 Complexity of Spliceosomal Proteomes Lead Participant: Reinhard Lührmann

Month planned achieved deliverables: 81 Complete the analyses of the protein composition of RNP

complexes formed on the wildtype/mutant exon 11 of the LMNA pre-mRNA, the wildtype/mutant exon 5 of the MCAD pre-mRNA, the SRp55 ESE, the DMPK CUG repeats, and the HIV-1 A7 site and its regulatory region (including an upstream RRE) (month 30).

30 24

82 Perform MS2 affinity selections with complexes formed on the second important and highly regulated acceptor site of HIV-1 RNA, site A3 (month 30).

30 24

83 Mass spectrometry analyses of spliceosomal complexes from adenovirus infected cells, RNP complexes formed in the presence and absence of the rev protein on RRE-containing model constructs (month 30).

30 in progress

84 Determine whether different types of cells can be used for the preparation of splicing active nuclear extracts (month 30).

30 24

85 Investigate the role of the RES complex, and in particular, the human factors homologous to the yeast RES subunits and of the EJC and associated factors in splicing regulation (month 30).

30 24

86 Mass-spectrometric analysis of Prp8p protein-protein crosslinks, nuclear Brr2-containing complexes, and other yeast protein complexes involved in splicing with the aim of identifying new, possibly post-translationally modified, factors involved in this process (month 30).

30 24

Objectives of the work package • Determine the composition of enhanceosomes assembled on different pre-mRNA substrates containing defined cis-acting regulatory elements.

• Elucidate qualitative and quantitative changes in the composition of spliceosomes formed under different physiological conditions (i.e., after viral infection or heat shock), in different cell types, and at different stages of the cell cycle. Short description of activities 81 and 82 Analyses of the protein composition of RNP complexes Partner 12a (J. Tazi) is focussing on alternative splicing events that lead to Hutchinson–Gilford progeria syndrome (HGPS), which is caused by a single mutation in exon 11. Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. It is clinically characterized by postnatal growth retardation, midface hypoplasia, micrognathia, premature atherosclerosis, absence of subcutaneous fat, alopecia, and generalized osteodysplasia. One of the main aims of this project is to understand the mechanism(s) underlying the selection of alternative splice sites in the case of mutations leading to such diseases. The majority of HGPS cases are due to a single nucleotide mutation (GGC>GGT) in exon 11 of the LMNA gene. It has been shown that this mutation alters the splicing profile by creating a cryptic splice site, therefore leading to the production of a truncated protein. In a collaboration between Partner 12a and Partner 1a (R. Lührmann) it was shown by both in vitro and ex vivo experiments that the single point mutation (GGC>GGT) in exon 11 was sufficient to induce utilisation of a cryptic 5' splice site in exon 11. Cross-linking studies have revealed that SF2/ASF and SRp55 members of the SR proteins confirmed their involvement ex-vivo. SRp55 activate the authentic splice site, whereas SF2/ASF activates the cryptic splice site. The activity of SRp55 is dependent on its phosphorylation status. Hyperphosphorylation of SRp55 leads to selective usage of the authentic splice site. A manuscript is in preparation to describe all these findings. Collaboration between Partner 12a and Partner 25 (J. Stévenin) using in vitro assays in HeLa extract demonstrated that the cryptic splice site can be used in the absence of the mutation. Different extracts from cells overproducing SR proteins are currently used to dissect the mechanism by which SR proteins activate an exonic cryptic splice site. In parallel, collaboration between Partner 12a and Partner 12c (C. Branlant) used purification of RNP complexes assembled around the wild type and mutated sequences using MS2 tag affinity purification procedure developed by partner 1a and have compared the proteomic profiles. The characterisation of pertinent proteins is in progress.

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Partner 12c (C. Branlant) applied proteomic approaches to identify nuclear proteins associated with pre-mRNA regions that are responsible for the regulation of alternative splicing. They investigated several pre-mRNA regions involved in the regulation of splicing, namely HIV-1 RNA regions containing the A3 and A7 acceptor sites and their regulatory elements, the NRS splicing silencer of the Rous sarcoma virus, the CUG-repeated sequences corresponding to the CTG expansions in the DMPK gene, which are responsible for type 1 myotonic dystrophy (DM1), and the lamine A pre-mRNA region containing the 5’-cryptic site, which is abnormally used in progeria patients. The binding sites of some of the associated proteins that were found to interact directly with the HIV-1 RNA region containing site A7 and its regulatory ESE3 element were identified (i.e. nucleolin, hnRNP K and hnRNP H). While nucleolin has a positive effect on splicing in vitro, hnRNP K reinforces the negative effect of hnRNP A1. In addition, several proteins known to modulate the Rev activity were found to be associated with this stem-loop structure (NFAR 90, RNA helicases A and DDX1, PSF). NFAR 90 and NFAR 41 and RNA helicase A, which are known to form a heterotrimer, were also found to associate with the HIV-1 RNA region containing the regulatory elements of site A3. In the Rous sarcoma virus (RSV) system, alternative splicing of the virus' genomic RNA is negatively controlled by a 150-nt long silencer (Negative Regulatory Element, NRS) which is known to bind the U1 and U11 snRNPs, several SR proteins and hnRNP H. On the basis of the 2D structure that we established for this silencer element, we have identified which nuclear proteins bind to each of its stem-loop structures. Reporter constructs have been built to test for their functional roles. When long CUG repeats are present in the pre-mRNA of the DMPK kinase, these pre-mRNAs are found in nuclei foci. In these foci, they are associated with at least two proteins: the splicing regulatory factor MBNL and hnRNP H. The idea is that MBNL is titrated in these foci and cannot play its normal role in the regulation of the alternative splicing of several pre-mRNAs, the splicing of which is found to be deregulated in DM1. By MS2-based affinity chromatography of RNP particles, we purified complexes formed by CUG repeats in nuclear extracts. Among the numerous proteins detected one was found in large amounts, nucleophosmin, and its direct binding to CUG repeats was demonstrated. The biological significance of this binding is currently being investigated. For proteome studies on lamin A pseudo 5’ splice site the MS2-based chromatography approach is currently being used to identify the nuclear proteins bound to the lamin A pre-mRNA region containing the point mutation responsible for progeria. Both the normal and the mutated RNA regions are being studied. How splicing factors are recruited to nascent transcripts in the nucleus in order to assemble spliceosomes on newly synthesised pre-mRNAs is unknown. The group pf Partner 16 (Carmo Fonseca) in collaboration with Partner 1a (R. Lührmann), compared the intranuclear trafficking kinetics of snRNP and non-snRNP proteins in the presence and absence of splicing activity. Photobleaching experiments showed that spliceosomal proteins move continuously throughout the entire nucleus independently of ongoing transcription or splicing. Using quantitative experimental data, a mathematical model was applied for spliceosome assembly and recycling in the nucleus. The model assumes that splicing proteins move by Brownian diffusion and interact stochastically with binding sites located at different sub-nuclear compartments. Inhibition of splicing, which reduces the number of pre-mRNA binding sites available for spliceosome assembly, was modeled as a decrease in the on-rate binding constant in the nucleoplasm. Simulation of microscopy experiments before and after splicing inhibition yielded results consistent with the experimental observations. Taken together their data argue against the view that spliceosomal components are stored in nuclear speckles until a signal triggers their recruitment to nascent transcripts. Rather, the results suggest that splicing proteins are constantly diffusing throughout the entire nucleus and collide randomly and transiently with pre-mRNAs. To study exon recognition via ESEs, Partner 1a (R. Lührmann) investigated complexes that assemble in vitro on intron-exon-intron constructs. One construct is derived from the second exon of the MINX pre-mRNA, and contains an anchoring sequence, branchpoint, polyrimidine tract, 3’ splice site, exon 2 and a downstream 5’ splice site plus three MS2 stem loops at its 3’ end for affinity purification. The second construct is based on the second β -globin exon, and contains - besides all aforementioned sequence features- three consecutive SRp55 ESEs. Interestingly, Partner 1a found that cross-exon complexes affinity-purified under mild conditions contained not only U1 and U2, but also the U4/U6.U5 tri-snRNP, with the downstream 5’SS playing only a minor role in formation of these complexes. Moreover, psoralen crosslinking revealed that while U4 and U6 RNA are still basepaired, U6 is also basepaired to U2 RNA in purified cross-exon complexes. These data indicate that similar to the situation in the B complex spliceosome, which forms across an intron, tri-snRNP recruitment to spliceosomal complexes formed across an exon also involves U2-U6 RNA helix II. Mass spectrometric analyses carried out together with partner 1c (H. Urlaub), identified all known snRNP proteins and U2-related proteins such as SPF45, U2AF65/35, SPF30, in purified cross-exon complexes Moreover, with few exceptions, most other proteins typically present in cross-intron A complexes, were also found, including SR and hnRNP proteins, p68, BUB3 and general mRNA binding proteins. The most striking difference in protein composition between cross-exon complexes and cross-intron B complexes (both of which contain the tri-snRNP), is the significant underrepresentation of hPrp19/CDC5 complex proteins - as demonstrated by very low peptide numbers and independent immunoblotting studies - and the complete absence of so-called Prp19-related proteins, such as Syf1, Syf3, hIsy1 etc., and also proteins of the RES complex, in the cross-exon complex. It was previously shown by others that, in the presence of nuclear extract, addition of a short 5’ SS oligonucleotide, which binds the ACAGA box of the U6 RNA, to a 3’ SS RNA substrate (similar to those used in this work), leads to a low level of trans-splicing. Interestingly, addition of such a 5’ SS oligonucleotide to the cross-exon complex, stabilized the association of

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the tri-snRNP. Proteomic analysis of purified exon complexes stabilized in the presence of the 5’ SS oligo, revealed the presence of increased amounts of hPrp19/CDC5 complex proteins and ca. 10 additional proteins that are typically found in cross-intron B complexes. RES complex proteins and some Prp19-related proteins were nonetheless still absent or not bound stably enough to withstand affinity purification. Based on these results Partner 1a has proposed a model where the transition from cross-exon to cross-intron recognition is initiated by the recruitment of the tri-snRNP to the U2 snRNP, which is stably bound to the 3’ SS by cross-exon interactions involving ESEs and the downstream 5’SS. This cross-exon bound U2/U4/U6/U5 tetra-snRNP may then serve as a docking site for the interaction with the upstream 5’ SS/U1 snRNP complex, and subsequently allow stable recruitment of the Prp19/CDC5 complex and additional cross-intron B complex proteins prior to catalytic activation. Partner 1a is currently investigating the RNA structural requirements for stable cross-intron complex formation with separate 5’ SS and 3’ SS pre-mRNA constructs. Partner 19 (J. Kjems) is focussing on Exon 5 of the medium-chain acyl CoA dehydrogenase. Exon 5 contains a disease-causing mutation (362C>T) that causes skipping of Exon 5. The group has purified proteins that bind to wild-type Exon 5 of the pre-mRNA and to Exon 5 containing the disease-causing mutant. The MS analysis of the bound proteins is currently being performed. 83 Mass spectrometry analyses of spliceosomal complexes from adenovirus infected cells The work on the affinity selection of spliceosomes formed in adenovirus-infected cells has been initiated. Partner 4 (Akusjärvi) has generatedMS2-IIIa substrates that are spliced efficiently and tested them in the first set of affinity-purification schemes. The efficiency of spliceosome formation has to be improved on this particular substrate. A stable cell line expressing L4-33K will be established in order to characterise cellular and viral protein–protein complexes containing L4-33K. The stable cell line will also be used to make nuclear extract that should simplify purification of spliceosomal complexes. The subcellular localisation of L4-33K will also be studied. 84 Splicing extracts from diffeent cell types Proteomic studies of purified spliceosomes assembled in vitro at defined stages of function have in recent years primarily been carried out with splicing extracts from human cells. Partner 1a (Lührmann) in collaboration with partner 1c (Urlaub) have now expanded these studies to other organisms in order to gain deeper insight into the evolutionary conservation and/or diversity of spliceosomal proteomes. The yeast S. cerevisiae was chosen, as there is no alternative splicing in yeast cells and thus regulatory splicing factors such as SR and hnRNP proteins are not abundant, if present at all. Nonetheless, the basic (constitutive) spliceosomal machinery appears to be conserved between yeast and human. Therefore, a comprehensive proteomic analysis of yeast spliceosomes should reveal the minimum set of proteins required for driving spliceosomal RNP dynamics and for catalysing the removal of a pre-mRNA intron. We initially focussed on the purification of the pre-catalytic B complex (containing all snRNPs), the catalytically activated B* complex (lacking U1 and U4 RNPs) and the catalytic step I spliceosome (C complex), to learn more about the dynamics of protein recruitment and release during catalytic activation and step 1 of splicing. The yeast system offers unique opportunities to stall complexes at defined functional stages and thus allowed for the first time the purification of the B* complex under native conditions. MS analysis of the purified B* complex revealed a homogenous protein composition with only ca. 40 distinct proteins, compared to more than 120 in human B or C complexes, making the yeast B* complex also very attractive for structural studies. Indeed, preliminary EM studies revealed that the B* complex is particularly homogenous, with defined outlines and structural features. Comparisons of the proteomes of purified B, B* and C complexes reveal defined sets of proteins which are specifically recruited during the transition from B to B* or B* to C. The limited number of the proteins involved in these dynamics raise the exciting possibility of recapitulating individual functional steps of the spliceosome cycle from purified components using partial complementation studies As a second system we have started to investigate the proteome of major spliceosomes from Drosophila. Despite the fact that text book models on alternative splicing regulation such as the sex determing pathway are derived from studies in Drosophila, little is known about the overall similarity and/or differences between the splicing machineries of Drosophila and human. For this prupose, spliceosomal complexes were assembled on an MS2-tagged Fushi tarazu (Ftz) pre-mRNA substrate or (for C complex purification) a truncated Ftz construct that lacks a 3’ss and 3’ exon but has an extended pyrimidine tract. MS analyses of the spliceosomal B and C complexes revealed remarkable similarities in protein composition compared to their human counterparts, including, among others, Drosophila homologues of Prp19 and CDC5 and their associated proteins, which are implicated in the catalytic activation of the human and yeast spliceosomes. The same group also analysed B complexes formed on a second construct, Zeste, containing a short intron of only 62 nt, which is too small to be spliced in vertebrates. Despite the fact that the mechanism of splice site recognition for short Drosophila introns differs from that of long introns (i.e., intron versus exon recognition, respectively), the protein composition of B complexes formed on Zeste appears to be almost identical to that of complexes formed on Ftz pre-mRNA. Finally, the protein composition of spliced and unspliced Ftz mRNPs. Initial MS analyses indicate that, like in humans, an exon junction complex is deposited on Drosophila mRNAs in a splicing dependent manner. 85 RES complexes The yeast pre-mRNA retention and splicing (RES) complex counteracts the escape of unspliced pre-mRNA from the nucleus and activates splicing of a subset of Mer1p-dependent genes (Dziembowski et al. EMBO J. 23, 4847-4856 (2004)) MS

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analysis showed that a homologous complex is present in activated human spliceosomes, but not in pre-spliceosomes. Partner 1b (B. Seraphin) in collaboration with the group of group of H. van Tilbeurg (Orsay) and partner 1a (R. Lührmann in collaboration with M. Wahl) performed biochemical analyosis of the architecture of the yeast RES complex and solved the crystal structure of the Pml1p subunit. In the RES complex an atypical RRM of the Snu17p subunit acts as a scaffold that organises the other two constituents, Bud13p and Pml1p. As shown by crystal-structure analysis performed by both groups, the C-terminal portion of Pml1p harbours a forkhead-associated (FHA) domain, which could be involved in phosphopeptide-binding. In this respect it is interesting to note that partner 1c (H. Urlaub) in collaboration with R. Lührmann demonstrated a high degree of phosphorylation of human Pml1p in purified human splicesomal B complexes. The combined biochemical and structural studies of the yeast RES complex proteins indicate further that the non-canonical Snu17p RRM binds several ligand proteins through short peptide motifs and, thereby, acts as a platform that displays functional modules of the ligands, such as the FHA domain of Pml1p and a conserved polylysine motif of Bud13p. The Seraphin group also performed functional assays by analyzing splicing of reporter RNAs in yeast mutated for various subunits of the RES complex. This did not reveal any implication of the FHA phosphothreonine binding pocket in splicing but demonstrated that the RES complex may affect alternative splice site choices in a model system. Currently the group of Reinhard Lührmann is establishing stable human cell lines expressing tagged human orthologues of the RES complex with the goal to purify native complexes and identify their interaction partners and and to study their role in alternative splicing. 86 Prp8 protein–protein cross-links (Partner 9, Jean Beggs) Yeast two-hybrid screens with different regions of Prp8p showed interactions of the N terminus of Prp8p with Cwc21p and with Snu114p (van Nues & Beggs, 2001 and unpublished results). Snu114p is a GTPase and a component of U5 snRNPs, with similarity to the ribosomal translocation factor EF-2. It has been proposed that, through GTP hydrolysis, the protein Snu114p regulates the ATPase/helicase activity of protein Brr2p, possibly by inducing a conformational change involving Prp8p, and thereby activates the spliceosome for catalysis (Brenner & Guthrie, 2005; Small et al 2006). Cwc21p is a protein of unknown function that was found as a component of the Cef1 complex (CWC). Using recombinant proteins, we have demonstrated that Prp8p, Snu114p and Cwc21p undergo direct physical interactions. Using chemical cross-linking combined with MS, we pinpointed specific amino-acid residues in Prp8p and Cwc21p that are in contact with one another. These contacts occur in regions of Cwc21p and Prp8p that are highly conserved between yeast and human. This information will be the basis for future mutagenesis studies. We have shown that human SRm300, an SR protein that is implicated in splicing regulation in humans, is a functional homologue of Cwc21p. Cwc21p (135 aa) is a much smaller protein than SRm300 and related proteins from other organisms, but it has strong similarity throughout its length to the N-terminal regions of these other proteins, which are longer mainly because of SR repeats that are lacking in the budding yeast protein. The N-terminal region of human SRm300 also interacts with yeast Prp8p in an in vitro pull-down assay. This similarity with SRm300 raises the interesting possibility that Cwc21p may play a role in modulating splicing in yeast through its interaction with Prp8p and/or Snu114p. Boon, K.L., Grainger, R.J., Ehsani, P., Barrass, J.D., Auchynnikava, T., Inglehearne, C. and Beggs, J.D. (2007) prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nature Struc. Mol. Biol. 14:1077–83. Problems and explanations for delays, postponements etc. Deliverable 81 Various attempts have been made to purify spliceosomes assembled on pre-mRNA introns containing a Rev-response element (RRE) in the presence of recombinantly expressed Rev protein with the goal of identifying trans-acting cellular factors which would interfere with splicing of such an intron and/or nuclear export of unspliced pre-mRNAs (collaboration between partners 1a (Lührmann) and 12c (Branlant)). Problems occurred at various stages of the project, in particular in obtaining soluble native Rev protein. But even with soluble Rev protein spliceosome formation was effected in a non-reproducible way even in the absence of an intronic RRE. Therefore, it was decided to no longer pursue this goal.

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Work Package 10 Post-translational Modification and DynamicRegulation Lead Participant: Javier Cáceres

month planned achieved deliverables: 87 Measure the elongation rate of RNA polymerase II in vivo

by imaging techniques using FRAP (fluorescence recovery after photobleaching) (month 30).

30 24 (now part of WP 13)

88 The functional significance of adenoviral L4-33K phosphorylation and stability will be investigated by mutational analysis. Establish the effect of PKA on alternative splicing and a potential subcellular colocalization between PKA and splicing regulatory proteins (month 30).

30 24

89 Develop FRET microscopy techniques to compare protein-protein interactions in the spliceosome and in nuclear speckles. Analyze by FRET-FLIM the possible interactions of U1 with SF2/ASF, at the HIV-1transcription site (month 30).

30 24

90 Use the tap-tagged SRp30 protein to select and MS-analyze ribonucleoprotein complexes in nSBs (month 30).

30 delayed

91 Investigate the function of hPrp4 kinase in splicing employing a combined cell biological and biochemical approach (month 30).

30 24

92 Investigate the effect of the RNAi-mediated silencing of SRPK1 and SRPK2 on the subnuclear distribution of snRNPs and other splicing factors using fluorescence microscopy methods (month 30).

30 24

93 Analyse the post-translational modifications of spliceosomal proteins at defined functional stages of the spliceosome, with particular emphasis on phosphorylation (month 30).

30 On target

94 Continue experiments to elucidate relationships between post-translational modifications of p68 and specific functions in different steps of the gene expression process (e.g. transcription, splicing, or export) (month 30).

30 24

Objectives of the work package • The aim of this work package is to characterize molecular interactions that play a function role in linking signal transduction pathways and the regulation of alternative splicing.

• Study how post-translational modifications of protein splicing factors affect their activity and/or subcellular localization.

• Assess using mutational studies the functional role of specific modifications in regulating alternative splicing pathways. The ultimate goal is to understand how extracellular signals modulate gene expression through mechanisms involving changes in alternative pre-mRNA splicing. Short description of activities 87 Measure the elongation rate of RNA polymerase II A collaboration between Groups 12D and 20 (Bertrand and Kornblihtt) used FRAP technology to measure Pol II elongation using HIV-1 reporters. This experiments are discussed more extensively in WP13 (Boireau et al., 2007) 88 Adenoviral L4-33K phosphorylation and stability The Adenoviral L4-33K protein is translated from an mRNA containing an intron that interrupts the protein reading frame. The unspliced L4 mRNA is translated into a

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protein called L4-22K (same amino-terminus as L4-33K), whereas the spliced mRNA generates the L4-33K protein. Results from Group 4 (Akusjarvi) have suggested that L4-22K regulates transcription of the L4 region by stimulating transcription from the Major Late Promoter whereas L4-33K functions a splicing factor stimulating Major Late alternative splicing. The interplay between these two viral proteins is currently studied in detail. This group has also shown that the protein kinase PKA regulates adenovirus E1A alternative splicing in vivo. The catalytic subunit of PKA phosphorylates SF2/ASF and colocalizes with nuclear splicing factor compartments (Kvissel et al., 2007). Current efforts are aimed at establishing cell lines overexpressing the C subunit of PKA and developing an in vitro splicing system responding to PKA C subunit expression. 89 FRET microscopy techniques Splicing factors SF1 and U2AF associate cooperatively with the pre-mRNA and play a crucial role in the recognition of the 3’ splice site during early steps of spliceosome assembly. SF1 is subsequently displaced at a later stage during formation of the active spliceosome, and little is known on how this remodeling process occurs in vivo. Group 16 (Carmo-Fonseca) used Fluorescence recovery after photobleaching (FRAP) analysis in live cells to determine that SF1 and the two subunits of U2AF (U2AF65 and U2AF35) are significantly less mobile than dextran molecules of similar size and established that this was due to the fact that they interact with each other in the nucleus. Use of Fluorescence resonance energy transfer (FRET) methods in fixed and living cells, both in the nucleoplasm and in the nuclear speckles, allowed them to demonstrate that SF1 interacts with U2AF in a splicing-independent manner, forming extra-spliceosomal complexes localized at nuclear speckles. This implies that SF1 associates with U2AF before and after splicing, and that this interaction is transiently disrupted to allow assembly of the catalytic spliceosome (Rino et al., 2008). A collaboration between Groups 15 and 22 (Javier Caceres and Angus Lamond) has focused on the study of the interaction between SR proteins and splicing components that are bound at the 5’ or 3’ splice site. Previously, these interactions have been extensively characterized biochemically and shown to be critical for both intron and exon definition. Use of fluorescence resonance energy transfer (FRET) microscopy allowed the identification of interactions of individual SR proteins with the U1 snRNP-associated 70 kDa protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live cell nuclei. These interactions were shown to occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively co-transcriptional. FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM) allowed the mapping of these interactions to specific sites in the nucleus. Thus, spatial mapping using FLIM-FRET has revealed differences in splicing factors interactions within complexes located in separate sub-nuclear domains (Ellis et al., 2008). Furthermore, Group 12D (Bertrand), a member of the YIP program, pursued an in vivo analysis of splicing by FRAP. As previously reported, this group developed cell lines containing an integrated array of a MS2-tagged, HIV-1 reporter gene that carried an intron from HIV-1 (SD1 and A7), and showed poor co-transcriptional splicing. At its transcription site, U1 snRNP, but not the other snRNPs, were detected. FRAP curves with GFP-U1A and GFP-U170K show fast recoveries (few seconds). This indicates that either the proteins come on and off the snRNA rapidly, or that the snRNA itself is turning-over quickly. Experiments to discriminate this are underway. Experiments testing the feasibility of analyzing interaction by FRET-FLIM at the HIV-1 transcription site have also been performed in collaboration with groups 15 and 22 (Caceres and Lamond) and showed positive interaction between U1 70K and ASF/SF2. Bertrand's group has now derived new reporters and cell lines containing the artificial Ad2 derived MINX intron. They see co-transcriptional splicing and recruitment of many splicing factors, and are characterizing a particular cell line in more details. They have also made mutants in the splice donor and splice acceptor sites that show strong nuclear retention, and may allow the enrichment of a particular spliceosomal state (i.e. complex C with the 3'ss mutant), then suitable for in vivo analysis with imaging techniques. 90 Ribonucleoprotein complexes in nSBs Group 10 A (Biamonti) devoted considerable effort to set up a cell line that expresses the Tap-tag version of SRp30c. Unfortunately, the version with the Tap at the C-terminus is highly unstable and does not show a distribution consistent with that expected for splicing factors. Following many problems, they have very recently obtained a cell line in which the Tap-tag is at the N-terminus of the protein. In this case, the ectopic SRp30c protein is nuclear and displays a distribution similar to that of SF2/ASF. Moreover the protein associates with nSBs after heat shock. Thus, the conditions have now been set up to be able to procede with this deliverable. 91 The function of hPrp4 kinase An interaction between hPrp4 kinase and the U5 snRNP component hPrp6 has been previously shown. Now,

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Group 1A (Luhrmann) found that siRNA-mediated depletion of hPrp4 in HeLa cells resulted in splicing defects. Furthermore, hPrp4 has been identified in MS2 affinity purified B and Exon complexes, whereas it is absent in the A as well as in the C complex. It was observed that two tri-snRNP proteins become heavily phosphorylated in the B complex prior to the catalytic activation of the B complex, i. e. before U4 RNA dissociates from the spliceosome. Phosphorylation of the same tri-snRNP proteins was also observed in a purified cross-exon complex (containing tri-snRNPs) upon base-pair interaction of a 5’ splice site RNA with U6 RNA. This group is now investigating in more detail the possible role of the phosphorylation of the two tri-snRNP proteins in early steps of catalytic activation of the spliceosome and whether hPrp4 kinase is responsible for these phosphorylation events. 92 Silencing of SRPK1 and SRPK2 Group 1A (Luhrmann) in collaboration with group 1C (Urlaub)has investigated the role of phosphorylations of the tri-snRNP protein, hPrp28. They showed that this phosphorylation is important for the interaction of hPrp28 with the tri-snRNP and also for the stable integration of the tri-snRNP into B complexes. They also found that the SR protein kinase, SRPK2, but not the related kinase SRPK1, appears to be the major kinase responsible for phosphorylating hPrp28 and yeast spliceosomes in purified spliceosomes at different stages of assembly (in collaboration with Group 1C (Urlaub) (a member of the YIP program). 93 Post-translational modifications of spliceosomal proteins Group 1A (Lührmann) and 1C (Urlaub) have started to characterize the phosphoproteome of purified human spliceosomes stalled at defined stages of assembly and/or function. At this point, phoshopeptides of spliceosomal B complex proteins have already been characterized. They have also started to raise phosphopeptide-specific antibodies with the goal to use these antibodies for visualizing spliceosomes in intact cells by immune fluorescence microscoy methods Group 3 (Stamm) has shown that the phosphatase PP1 binds to the RRM of several splicing factors and regulates alternative pre-mRNA processing. PP1 activity in the cell is regulated by PKA, as PKA phosphorylates PP1 inhibitors, such as DARPP32 and Inhibitor 1. Recent results from this lab showed that cAMP-mediated activation of PKA changes splice site selection of PP1-dependent exons. Group 10A (Biamonti) has continued the study of the alternative splicing of the c-ron protooncogene in a model of epithelial-mesenchymal transition (EMT). SW480 cells have an epithelial phenotype when grown at high-density, but they display a mesenchymal morphology and express mesenchymal markers (down-regulation of E-cadherin, nuclear beta-catenin, and presence of stress fibers) at low density, constituting a good system to study EMT in vitro. They have now shown that skipping of exon 11 of the Ron gene, which is regulated by the SR protein SF2/ASF, occurs at low density and this is accompanied by reduction of unproductive splicing of SF2/ASF. During EMT, the ERK and JNK are activated in mesenchymal cells. Pharmacological inhibition of the ERK pathway results in inclusion of exon 11 and increases unproductive splicing of SF2/ASF. Group 12 a (Tazi) had previously shown that overexpression of the Drosophila SR protein B52 in vivo leads to developmental defects. Use of genetic screen based on random overexpression of endogenous Drosophila genes resulted in the identification of 7 lines that rescue the B52-induced phenotypes in two different tissues. These factors include RNA binding proteins, transcription factors and chromatin-associated factors. Interestingly, among these proteins they identified the Drosophila DNA topoisomerase I (TopoI), which is known to phosphorylate SR proteins in mammals and in Drosophila. Furthermore, a TopoI mutant larvae display hypo-phosphorylation of B52 whereas overexpression of TopoI increases B52 phosphorylation in vivo. Moreover, TopoI overexpression can rescue the phenotypes induced by B52 overexpression in several tissues, suggesting that TopoI phosphorylation negatively regulates B52 activity. This group recently observed that B52 overexpression in the developing eye imaginal disc strongly impairs eye development. They demonstrated that B52 overexpression increases the inclusion of an alternative exon of the eyeless (PAX6) gene, leading to the production of isoforms with impaired DNA binding activity (see WP 11). In this context, co-overexpression of TopoI restores a correct splicing of eyeless and restores a normal eye phenotype. These results demonstrate for the first time in a whole organism the implication of TopoI in the regulation of an alternative splicing event regulated by the B52/SRp55 SR protein. Finally, Anabella Srebrow, a recent incorporation to our YIP program, is focusing on a regulated splicing

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event, the alternative splicing of the Rac1 pre-mRNA by matrix metalloproteases, in particular MMP3 and the involvement of hnRNPA1 and hnRNP A2 in this regulation. 94 Post-translational modifications of p68 and specific functions In addition to play a role in transcription and splicing, the RNA helicase p68 is involved in mRNA export. Supporting a model in which the ability of p68 to be engaged in different steps of the gene expression process depends on post-translational modifications (PTMS), ten different PTMs of p68 have been reported. Group 26 (Auboeuf/YIP program) is testing the role of these different isoforms of p68 in different cellular functions. Their immediate goals are identify p68 target regulated transcripts using p68-depleted cells and Affymetrix ExonArrays. Problems and explanations for delays, postponements etc.

88 The studies on the significance of SR protein kinases on L4-33K phosphorylation has turned out negative. Current work is aimed at using a mutational approach to look at protein stability and subcellular localization and put that in relation to the major phosphorylated epitopes of the protein. 90 As described above, what has been limiting for this deliverable was to obtain a cell line expressing a Tap-tag version of SRp30c. This has been problematic, but fortunately the problem has now been circumvented.

PUBLICATIONS

Boireau,S., Maiuri,P., Basyuk,E., de la Mata,M., Knezevich,A., Pradet-Balade,B., Bäcker,V., Kornblihtt,A., Marcello,A. and Bertrand,E. (2007) The transcriptional cycle of HIV-1 in real-time and live cells. J. Cell Biol., 179, 291-304. Kvissel,A.K., Ørstavik,S., Eikvar,S., Brede,G., Jahnsen,T., Collas,P., Akusjärvi,G. and Skålhegg,B.S. (2007) Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing. Exp. Cell Res., 313, 2795-2809. Mathew,R., Hartmuth,K., Mohlmann,S., Urlaub,H. Ficner,R. and Luhrmann,R. (2008) Phosphorylation of PRP28 by SRPK2 is required for integration of the U4/U6.U5 tri-snRNP into the spliceosome. Nat. Struct. Mol. Biol., in press. Michlewski,G., Sanford,J.R. and Cáceres,J.F. (2008) The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1. Mol Cell, in press. Fic,W., Juge,F., Soret,J. and Tazi,J.(2007) Eye development under the control of SRp55/B52-mediated alternative splicing of eyeless.PLoS ONE, 2(2):e253. Gabut,M., Dejardin,J., Tazi,J. and Soret,J. (2007) The SR family proteins B52 and dASF/SF2 modulate development of the Drosophila visual system by regulating specific RNA targets. Mol.Cell Biol.,27, 3087-3097. Novoyatleva,T., Heinrich,B., Tang,Y., Benderska,N., Butchbach,M.E., Lorson,C.L., Lorson,M.A., Ben-Dov,C., Fehlbaum,P., Bracco,L., Burghes,A.H., Bollen,M. and Stamm,S. (2008) Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing. Hum. Mol. Genet., 17,52-70. Rino,J., Desterro, J.M., Pacheco,T.R., Gadella,T.W. and Carmo-Fonseca,M.A. (2008) Splicing factors SF1 and U2AF associate in extra-spliceosomal complexes. Mol.Cell.Biol., in press. MANUSCRIPTS

Ellis,J.D., Lleres,D., Denegri,M., Lamond,A.I. and Cáceres,J.F. Spatial mapping of splicing factor complexes involved in exon and intron definition. under second revision in J.Cell.Biol. Juge,F., Fic,W, Fernando,C. and Tazi,J. The splicing factor B52 influences DNA topoisomerase I localization and function in Drosophila. submitted for publication.

46

Work Package 11 Function of Splicing Factor Isoforms Lead Participant: Juan Valcárcel

month planned achieved deliverables: 95 Further optimization of microarray data analysis (month

30). 30 20

96 Analysis of the effects of knocking down specific isoforms in cellular function (month 30).

30 24

97 Application of microarrays to the study of effects of knocking down splicing factor isoforms (month 30).

30 24

98 Complete the assessment of AS in plant splicing factors and prepare genetic material for analysis of AS isoforms in plants and analysis using the AS RT-PCR panel (month 30).

30 24

Objectives of the work package • To analyse functional differences between alternatively spliced isoforms of splicing factors and regulators. Short description of activities 95 Optimization of microarray data analysis Progress has been made in comparing available data analysis packages (see also WP8) including X-ray Exon Array Analysis (Biotique systems), Partek Genomics Suite (Partek Inc) and Affymetrix proprietary analysis methods for Affymetrix Exon arrays and ExonHit SpliceArray Analysis Tool for custom Affymentrix and Agilent platform-based ExonHit array designs. This has been carried out within the context of a EURASNET-based contest experiment to compare alternative splicing changes in samples where two isoforms of the splicing factor PTB have been knocked out. Additional optimization of data analysis has been performed through a collaboration between the Valcarcel and Carmo-Fonseca laboratories involving the development of statistical and visualization tools (SAPO) aimed at evaluating alternative splicing changes in splicing factor isoforms. Validation rates as high as 90% for microarray predictions of alternative splicing changes have been achieved through some of these analysis packages, making this high-throughput technology accessible for the community. 96 Effects of knocking down specific isoforms Functional analysis of splicing factor isoforms have been carried out for the following factors: a) PTB / nPTB (Smith): evidence for crossregulation between the two isoforms has been found, together with the identification of tens of specific targets; b) SC35 autoregulation (Stevenin); evidence for autoregulation has been found, implicating alternatively spliced transcripts leading to productive or non-productive mRNAs, SC35 regulating this alternative splicing event to buffer changes in the levels or activity of the factors; c) U2AF35 (Carmo-Fonseca): distinct effects of knocking downU2AF35 isoforms in cell proliferation, apoprtosis and alternative splicing of cell cycle regulatory factors have been documented; c) SF1 (Krämer): a full catalogue of isoforms has been generated and isoform-specific siRNAs have been generated and tested. d) TIA-1 (Valcarcel): differential activities of two isoforms of the apoptosis regulatory protein TIA-1, generated by alternative splicing have been found; the balance between these isoforms is controlled by the related protein TIAR. An overriding principle emerging from these studies is that splicing factors establish intricate gene networks involving other splicing factors and RNA binding proteins to buffer their activity levels and possibly respond to varying cellular conditions. Progress has also been made in the functional analysis of plant splicing factor isoforms. T-DNA insertion mutants of the RSp and RSZ protein families of Arabidopsis have been isolated and characterized on the molecular level. Double mutants of the RSZ family have been created and are currently characterized. 97 Application of microarrays to the study of effects of knocking down splicing factor isoforms Efforts have concentrated in setting up analytic tools to interpret microarray results (see above), in the full design of microarrays able to distinguish between alternatively spliced versions of all know splicing factors and in establishing physiological changes in splicing factor isoforms which will report variations that can guide experiments aimed to study the effects of knocking down specific isoforms. Isoform expression has been analyzed in a) Drosophila sex determination, b) muscle cell differentiation in vitro and comparing healthy vs myotonic dystrophy tissue, c) comparing mammalian tissues, and d) changes occurring in a cell system that model Hodgkin lymphoma tumor progression. Insights obtained in these studies include the identification of RNA binding proteins as a major category of genes suffereing changes in alternative splicing in a variety of physiological conditions and systems. A second conclusion is the frequent changes in alternative splicing affecting 3’ untranslated regions, with the potential to generate distinct transcripts subject to regulation by a variety

47

of RNA binding proteins and microRNAs. In addition, analysis of isoform-specific knockdown of PTB isoforms (see also WP8) has rendered tens to hundreds of novel alternative splicing events which are targets of regulation by these factors. 98 Assessment of AS in plant splicing factors Using the pilot AS RT-PCR panel we have demonstrated the utility of approach to investigate changes in AS due to over-expression of SR proteins. Initial analysis of three over-expression lines of Arabidopsis SR proteins (RSZ33, RSp31, SRp30) had been carried out using the pilot AS RT-PCR panel of 96 events. The data from biological reps has been analysed and 1) confirmed the auto-regulation of alternative splicing of RSZ33, 2) that over-expression of particular SR proteins affects the alternative splicing of other SR proteins, and 3) that different SR protein isoforms affect different, but over-lapping subsets of AS events in other genes.Further over-expression lines and mutants are being prepared for the next phase of analysis using the complete AS RT-PCR panel. Problems and explanations for delays, postponements etc. NONE Significant progress has been made to fulfill the objectives of this Workpackage. Advances have been made in all Deliverables, which can be considered achieved for the purpose of setting up proof-of-principle technologies and studies to address the variety of activities of splicing factor isoforms in various experimental systems. Technologies and data analysis software are now available for consortium members and beyond and will have an enduring effect for the work of many groups.

48

Work Package 12 Mis-splicing and Disease Lead Participant: Francisco Baralle

month planned achieved deliverables: 136 Generation of diagnostic and bioinformatics tools useful to

identify mutations in humans that affect splicing (continuation of del.48, month 30).

30 24

137 Databases (genes vulnerable to inactivating mutations due to the presence of distant branch points, splicing factors and related proteins) and update of motif database at the EBI with mutations seen in diseases studied by the network (continuation of del.49, month 30).

30 24

138 RNAi of trans-acting factors to modulate aberrant splicing (continuation of del.50, month 30).

30 24

Objectives of the work package • Further analysis of splicing-affecting mutations in human disease genes. • Identification of the trans-acting factors binding to newly identified regulatory sequences and assessment of their roles in the regulation of the splicing progress. • Development of new database/database entries useful to identify and predict mutations in humans that affect splicing. • Identification of more potential siRNA targets to modify/rescue aberrant splicing profiles. • Participant 13 will continue to work on the inhibition of HIV-1 replecation, in particular by looking at viral replication in primary human T-lymphocytes and on SMN2 splicing by using a transgenic mouse model (knockout for mouse SMN, knock-in for human SMN2). Short description of activities 136 Generation of diagnostic and bioinformatics tools useful to identify mutations in humans that affect splicing. Studies were carried out focusing on specific mutations that produce aberrant splicing and functional activity of Alternative Splicing isoforms. The data collected by WP12 will be useful to assess simplified diagnostic and therapeutic procedures. The deliverable outcome is summarized in table 1below:

Pathologic mutation mechanism.

Identification of elements involved

Diagnostic procedure

development

Biological activity of AS/

aberrant isoforms

Alternative/ Aberrant splicing system

WP12 participant

Cis-acting

Trans-acting

2ry structure

AdL4+ 4 Akusjärvi - - - - Function identification CFTR exon 9 7 Baralle F Yes Yes Yes Yes - CFTR exon 12 7 Baralle F Yes Yes Yes - CFTR pseudoexon intron 11

7 Baralle F Yes Yes Yes Yes -

Herg 7/KCNH 2 7 Baralle F Yes Yes Yes Yes - ATM pseudoexon intron 20

7 Baralle F Yes Yes Yes Yes -

NF1 exon 29 32 Baralle D Yes Yes Yes - NF1 exon 31 32 Baralle D Yes Yes Yes - NF1 exon 37 7 Baralle F Yes Yes Yes Yes -

49

LMNA 12a Tazi 25 Stévenin

Yes Yes Function modulation

MCAD 19 Kjems Yes Yes Therapeutic approach SMN 13 Schümperli Yes Yes Therapeutic approach Ron 10a Biamonti Yes Yes Function identification FRGI 27 Gabellini Function identification various hnRNPL targets

11 Bindereif Yes Yes Function identification

Bioinformatics tools In collaboration with the group of Igor Vorechovsky at the University of Southampton, group 7 has recently developed a database of aberrant splice sites in human disease genes, which is currently freely available at http://www.dbass.org.uk (Vorechovsky, 2006; Buratti et al., 2007; Kralovicova and Vorechovsky, 2007). The updated DBASS3 and DBASS5 now contains 625 records of aberrant 3ʼ and 5ʼ splice sites generated by 682 different mutations in 235 genes. Each database can be searched by phenotype, gene, mutation, location of aberrant splice sites in introns and exons, their distance from authentic counterparts, and by bibliographic references. The deliverable outcomes are summarised in tables 2 and 3 below. Table 2. Summary of aberrant 3ʼ splice sites in DBASS3

Location of cryptic or de novo 3' splice sites Exon Intron Both

Mutation in the 3ʼss consensus

(cryptic)

elsewhere ('de

novo')a

in the 3ʼss consensus

(cryptic)

elsewhere ('de

novo')a

All mutations

Number of genes 61 29 31 63 148 Number of phenotypes 66 31 31 66 156 Number of cryptic and de novo 3ʼss (percent) 99 (36.0) 41 (14.9) 38 (13.8) 97 (35.3) 275 (100) Number of unique 3'ss (percent) 94 (36.4) 36 (14.0) 36 (14.0) 92 (35.6) 258 (100) Number of aberrant 3'ss affecting terminal exons (percent)

11 (11) 4 (10) 9 (24) 4 (4) 28 (10.9)

Median distance (nt) between authentic and aberrant 3' splice sites

13 49 -44 -13 1

Legend: a, the number of pseudoexons (cryptic exons) was 13 (4.7% of all reported aberrant 3ʼss and 5.0% of unique aberrant 3ʼss). The 3ʼss consensus is YAG/G, where Y is a pyrimidine and slash is the intron-exon boundary. Table 3. Summary of aberrant 5ʼ splice sites in DBASS5

Location of cryptic or de novo 5' splice sites

Exon Intron Both

Mutation in the 5'ss consensus

(cryptic) elsewhere ('de novo')a

in the 5'ss consensus

(cryptic) elsewhere ('de novo')a

All mutations

Number of genes 91 41 81 43 194 Number of phenotypes 91 46 88 44 206 Number of cryptic and 138 (33.9) 62 (15.2) 150 (36.9) 57 14.0) 407 (100)

50

de novo 5ʼ ss (percent) Number of unique 5ʼss (percent) 132 (36.1) 62 (16.9) 118 (32.2) 54 (14.8) 367 (100)

Number of aberrant 5'ss affecting terminal introns (percent)

5 (42) 1 (8) 6 (50) 0 (0) 12 (3.3)

Median distance (nt) between authentic and aberrant 5' splice sites

-46 -56 48 36 -8

Legend: a, the number of pseudoexons was 39 (9.6% of all reported aberrant 5ʼss and 10.6% of unique aberrant 5ʼss). The 5ʼss consensus is MAG/GURAGU, where M is A or C, R is purine and slash is the exon-intron junction. 137 Databases and update of motif database at the EBI. The bioinformatics tools described in deliverable 136 and available at http://www.dbass.org.uk are being submitted to WP4 to verify the possible integration in the AS Database managed by EBI. 138 RNAi of trans-acting factors to modulate aberrant splicing The (GU)n binding protein TDP43 is a negative splicing factor that has also been shown to downregulate gene transcription in a couple of cases and has been described as having a prominent role in the occurrence of several neurodegenerative diseases such as FTD/ALS. In order to better define its functions the Group of Prof. Baralle has performed RNAi depletion of TDP43 in human cells followed by microarray analysis to search for eventually misregulated transcripts. Among the transcripts analyzed, the results have highlighted significant increases of cyclin dependent kinase 6 (CDK6) at the protein and transcript levels. Most likely as a consequence of CDK6 upregulation, TDP43– cells also show an increase in retinoblastoma suppressor (pRb) phosphorylation. This data is supported by observed alteration of the cell cycle distribution and changes in the expression levels of several factors involved in cell proliferation, when comparing RNAi treated cells vs. control cells. To systematically identify hnRNP L target genes on a genome-wide level the Bindereif group has combined splice-sensitive microarray analysis and an RNAi-knockdown approach. As a result, they have recently described 11 target genes of hnRNP L that represent several new modes of hnRNP L-dependent splicing regulation, involving both activator and repressor functions: first, intron retention; second, inclusion or skipping of cassette-type exons; third, suppression of multiple exons; and fourth, alternative poly(A) site selection. In sum, this approach revealed a surprising diversity of splicing-regulatory processes as well as poly(A) site selection in which hnRNP L is involved. The deliverable outcomes are summarised in the tables 4-5 below: Table 4

Available Tools Trans-acting factors

WP 12 participant siRNA Overexpres

sion Mutants

Identified target genes

Biological activity of Trans-acting factors

TDP-43

7 Baralle F Yes Yes Yes CFTR exon 9, BRCA1 exon 12, Apo AII exon 3

Splicing inhibitory factor Activates RB pathway and apoptosis Affects processing of selected microRNAs

hnRNP H

7 Baralle F Yes Yes No NF-1 exon 3 -

PTB 32 Baralle D Yes Yes No NF-1 IVS 30 hnRNP L

11 Bindereif Yes Transcriptome wide analysis

L4-33K 4 Akusjärvi Modulation of function L4-22K 4 Akusjärvi Modulation of function FRG1 27 Gabellini Yes Modulation of function

51

Table 5

The deliverables seen in Table 5 are relevant to the study of the function of splicing regulatory elements and can be considered as the basis for WP14 therapeutic approaches. Problems and explanations for delays, postponements etc. remark related to deliverable 136: WP12 (specifically F. Baralle together with non-EURASNET scientist I. Vorechovsky) developed a database containing 625 records of aberrant splice sites generated in 235 human disease genes. Since the groups specialized in bioinformatics are concentrated in WP4, the bioinformatics tools developed as part of deliverable 136 were submitted to WP4 and are now fully merged into the alternative splicing database managed by EBI. The collaboration with I. Vorechovsky continues and he now liaises directly with EBI to keep the database updated. Since development of advanced database formats and bioinformatics tools do not belong to the core expertise of WP12, further activities to these two aspects are now within the responsibility of WP4. This will also help to reduce the redundancy of multiple requests for submission of the same data sets. WP Restructuring The deliverables of Daniel Schümperli (participant 13) and Ian Eperon (participant 17) are more relevant to WP14 (see there) and for this reason the report on their activities is summarized separately below although they are included in the tables. Group 13 (Schümperli): has developed a U7-based methodology to induce exon skipping in HIV-1, thereby inhibiting HIV-1 replication. Moreover the group has developed U7-based methods to induce exon inclusion in survival of motoneuron 2 gene (SMN2), which could be used in gene therapy for spinal muscular atrophy. Regarding this second issue they now have very preliminary evidence that this approach works in vivo in a very severe mouse model for SMA where symptoms are clearly reduced and survival of the animals is prolonged. Moreover, they have made significant progress towards engineering inducible U7 snRNA cassettes. Group 17 (Eperon): further investigated and optimized strategy for using TOES (targeted oligonucleotide enhancers of splicing) to rescue refractory exons as a basis for related endeavours by other members of the WP. Regarding this issue, the site of annealing of the oligonucleotides, the tail sequence and the chemical composition for rescuing the expression of SMN2 exon 7 have been varied. The outcome has been assessed in splicing in vitro, and in fibroblasts from patients with spinal muscular atrophy. In the latter case splicing has been assessed by real time PCR, protein expression by western blotting, cell viability by standard methods, and oligo uptake and lifetime in the cell by standard fluorescence and also by FRET. With the best combination, increased levels of protein were seen at up to 1 week after transfection. Oligos that increased the level of protein 3-fold also showed high toxicity, whereas those that increased it about 2-fold showed no toxicity. FRET confirmed that the oligos remain intact in the nucleus even at the longest time tested post-transfection (15 hr). COLLABORATIONS AND INTERACTIONS WITH CLINICIANS

Akusjärvi R. Lührmann Baralle F. Clinicians/non-NoE:

B. Bembi, IRCCS Burlo Garofolo, Trieste (Italy) P.Schwartz, Department of Cardiology, University of Pavia and IRCCS Policlinico

Therapeutic approaches WP12 participant

target genes Biological activity obtained

Antisense oligonucleotides 7 Baralle F ATM and CFTR pseudoexons

Inhibition of aberrant pseudo exon inclusion

LNA 19 Kjems MCAD Increase mutated MCAD exon5 inclusion TOES 17 Eperon CFTR Extend increased exon inclusion

observed in other models to exon 9 CFTR mutants

U7-based 13 Schümperli HIV/SMN Inhibition of HIV gowth/ Increase SMN exon 7 inclusion

52

S. Matteo, Pavia (Italy) G. Woods, Dept. of Medical Genetics, Addenbrookes Hospital, Cambridge (UK)

C. Shaw, Kings College, London (UK) NoE: D. Baralle, Princess Anne Hospital, Southampton (UK) R. Lührmann J. Valcarcel G. Biamonti I. Eperon R. Apweiler

Biamonti Collaboration with Juan Valcarcel (11) for the screening of a splicing sensitive array

to search for splicing differences between High and Low density cell cultures. Data validation through RT-PCR analysis in progress.

Bindereif Clinicians/non-NoE: K. Stangl, Cardiologist at Charite’ Berlin. NoE:

Collaboration with Reinhard Lührmann on the functional role of recycling factors (joint publication in prep.); collaborations with the new YIPs Henning Urlaub (mass-spectrometric protein identification over the last two years; joint publication being planned); with Janusz Bujnicki (modelling of hnRNP L-RNA interaction; site-specific mutagenesis of hnRNP L to study RNA-protein interaction; since 2007; we had a lab visit of Janusz Bujnicki and one of his students in 2007, which could not be funded yet through EURASNET). Received antibodies from Juan Valcarcel (11).

Schümperli Prof. Didier Trono, School of Life Sciences, Ecole Polytechnique Fédérale de

Lausanne, Lausanne, Switzerland (lentiviral vectors, mouse transgenesis, HIV multiplication assays and reagents) Dr. Martine Barkats, Généthon-CNRS FRE 3018, Evry, France (AAV vectors, somatic gene transfer)

Dr. Barbara Rothen-Rutishauser, Institute of Anatomy, University of Bern, Switzerland

(Confocal microscopy)

Prof. Christian Leumann, Department of Chemistry and Biochemistry, University of Bern, Switzerland

(Oligonucleotide chemistry) Eperon Collaboration with F. Baralle on CFTR; supervision of an Open University PhD

student; exchange of plasmids. Kjems D. Schümperli Stévenin Collaboration with the Jamal Tazi' group (12A), on the splicing of the LMNA gene

in patients with the HG Progeria syndrome.

53

Work Package 13 Co-transcriptional Mechanisms of Alternative Splicing Lead Participant: Karla Neugebauer

month planned achieved deliverables: 139 Initial description of co-transcriptional spliceosome

assembly in vivo in yeast and design of systems/mutants in yeast for analysis of co-transcriptional spliceosome assembly mechanisms (continuation of del.51, month 30).

30 24

140 Feasibility study of splicing factor ChIP in mammalian cells (continuation of del. 52, month 30).

30 12

141 Examination of the kinetics of RNA synthesis and co-transcriptional association of GFP-tagged versions of splicing regulators (e.g. SR proteins) to the SAT III locus, including investigation of the effect of SAT III RNA synthesis on alternative splicing of heterologous gene transcripts (continuation of del. 53, month 30).

30 24

142 Investigation of relationship between transcription elongation rates and alternative splicing, through coordinated development of reporter systems and compilation of transcriptional regulators that effect alternative splicing changes (continuation of del.54, month 30).

30 24

99 Examination of the kinetics of cyclin D1 and PS2 RNA synthesis and RNA splicing in response to a transcriptional stimulus. (continuation of del. N5 , month 30)

30 24

100 Use pol II elongation inhibitors and Pol II mutants to study how elongation impacts alternative splicing. (month 30)

30 24

101 Determine the specific step at which transcription is affected by Prp45p mutants. (month 30)

30 24

102 Investigate the requirement of the RNA Pol II CTD for mRNA release from the site of transcription and alternative splicing of human IKBKAP exon 20. (month 30)

30 24

103 Determine the recruitment patterns of distinct GFP-tagged SR proteins to a variety of selected genes (intronless, constitutively spliced intron-containing, and alternatively spliced) (ChIP) and to the SATIII locus (fluorescence microscopy). (month 30)

30 24

104 Comparison of c-fos and cyclin D1 genes with respect to: splicing factor recruitment (ChIP), spliced levels on chromatin (ChRIP), and spliced levels attached to RNA pol II. (month 30)

30 24

105 Feasibility study of application of yeast 3-hybrid system to identify RNA binding proteins interacting with SATIII transcripts. (month 30)

30 24

106 Investigation of feedback from splicing to transcription. (month 30)

30 24

Objectives of the work package • Analysis of the coupling between transcriptional and splicing regulation in order to ultimately explain how alternative mRNAs are expressed in vivo. The overall aim is to optimize and extend key techniques. A particular focus of this period will be exchanging expertise and reagents, such as Pol II mutants, antibodies, and newly established cell lines, for application among the experimental systems.

54

Short description of activities 139 Initial description of co-transcriptional spliceosome assembly in vivo in yeast. The initial description and mutant study carried out in collaboration between the Seraphin and Neugebauer labs is done, with a joint manuscript in preparation. 140 Feasibility study of splicing factor ChIP in mammalian cells This study is done and was published by the Neugebauer lab in 2006. Now being extended to other cell lines in collaboration with Aubeouf (see new deliverable). 141 Examination of the kinetics of RNA synthesis and co-transcriptional association of GFP-tagged versions of splicing regulators to the SAT III locus. Biamonti has found that splicing factors (SF2, Clk/Sty) are co-transcriptionally recruited to sites of transcription of SatIII non-coding RNAs. However, SatIII RNAs are detectable at sites of transcription for more than 48 h while splicing factors return to their original distribution within 10 h from heat shock. Neugebauer and Biamonti also screened for additional SR proteins recruited to SAT III with FP-tagged versions; no other SR protein was recruited to SAT III. 142 Investigation of relationship between transcription elongation rates and alternative splicing. A detailed list of inhibitors and their effects of alternative splicing outcome has been compiled, based on the experiments of members of this workpackage, principally Kornblihtt and Aubeouf (see Table). Neugbauer and Aubeouf furthermore used ChIP to show how Pol II density and spliceosome assembly is influenced by inhibition of elongation with camptothecin. 99 Examination of the kinetics of cyclin D1 and PS2 RNA synthesis DA’s lab demonstrates that splicing of the first intron of cyclin D1 but not of PS2 occurred during transcription. The mechanism involved in co-transcriptional splicing depends on the level of the serine 5 phosphorylation of RNA polymerase II at the gene 5’-end that we show to be selectively stimulated by estradiol in a gene-specific manner. Our data indicate that the mRNA production from a subset of estradiol-stimulated genes such as cyclin D1 could occur in a very efficient “assembly line”, while in the case of the PS2 gene, the production of mRNA is not optimized owing to inefficient co-transcriptional splicing (in revision at Mol Cell Biol.). Therefore, coupling between transcription and splicing may permit the efficient production of a subset of mRNAs in response to transcriptional stimuli. DONE 100 Use pol II elongation inhibitors and Pol II mutants A detailed list of inhibitors and their effects of alternative splicing outcome has been compiled, based on the experiments of members of this workpackage, principally Kornblihtt and Aubeouf (see Table). Kornblihtt and Bertrand labs have collaborated extensively to measure elongation rates in vivo. All of this work has led to several publications, of which some are co-authored by multiple labs within the workpackage. 101 Determine the specific step at which transcription is affected by Prp45p mutants. Prp45 shown to influence elongation rather than initiation. 102 Investigate the requirement of the RNA Pol II CTD for mRNA release and alternative splicing of human IKBKAP exon 20. Initial observation that the CTD is required for release was published (Custódio N, Vivo M, Antoniou M, Carmo-Fonseca M. 2007. Splicing- and cleavage-independent requirement of RNA polymerase II CTD for mRNA release from the transcription site. J Cell Biol. 179(2):199-207) and is currently being pursued biochemically. Kornblihtt and Ast demonstrated AS of IKBKAP can be due to modulation of elongation rates. 103 Determine the recruitment patterns of distinct GFP-tagged SR proteins. The KN lab has now accomplished this on selected genes c-fos, HSP70, histone, PGK1 and LDHA, using ChIP. In collaboration with Biamonti, have also determined which SR proteins are recruited to the SATIII repeats upon stress, using a microscopy assay. A manuscript is now in preparation. 104 Comparison of c-fos and cyclin D1 genes. KN’s and DA’s lab demonstrate that splicing of c-fos and cyclin D1, respectively occur during transcription. However, the mechanisms behind the co-transcriptional splicing of both genes might be different. While splicing efficiency of both genes decreases in response to inhibitors of pol II serine 5 phosphorylation, only c-fos splicing efficiency decreases by inhibitor of pol II serine 2 phosphorylation. 105 Feasibility study of application of yeast 3-hybrid system. The Biamonti lab established a Y3H assay and showed that SF2, a protein known to localize to the SAT III locus, interacts with SAT III RNAs. The assay permits studies of the interaction of SF2/ASF mutants with SatIII RNAs, as well as screening of a human cDNA library for additional interacting proteins. In agreement with Deliverable 141, only SF2 was identified in the screen, showing the specificity of SF2 association with the locus.

55

106 Investigation of feedback from splicing to transcription. Using HIV-1 or β-globin mRNAs as model systems UAAR has demonstrate a strong positive correlation between splicing efficiency and transcription activity and that a 5' splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced promoter-docking of transcription initiation factors TFIID, TFIIB and TFIIH on a gene containing a functional 5' splice site. and, in addition to their promoter association, the TFIID and TFIIH components, TBP and p89, are specifically recruited to the 5' splice site region. These data suggest a model in which a promoter proximal 5' splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing pre-initiation complex assembly. Published paper acknowledging EURASNET funds:

Christian Kroun Damgaard, C.K., Lykke-Andersen, S., Kahns, S., Nielsen, A.L., Jensen, T.H. and Kjems, J.: A proximal 5’ splice site stimulates basal transcriptional initiation in vivo. Mol. Cell 29(2):271-8

Problems and explanations for delays, postponements etc. none

56

Work Package 14 Chemical Biology and Therapeutics Lead Participant: Jamal Tazi

month planned achieved deliverables: 107 Analysis of chemical derivatives of newly identified

inhibitors of pre-mRNA splicing (month 30). 30 24

108 Purify SR protein associated complexes and probe SR protein/drug interaction with photo-affinity molecules (month 30)

30 24

109 Adaption of the in vitro pre-mRNA splicing assay with a fluorescence readout to additional pre-mRNA substrates (month 30).

30 24

Objectives of the work package • Characteriziation of novel inhibitors of pre-mRNA splicing regarding their mode of action and molecular target

• Final optimization and implementation of the high-throughput screening for inhibitors of in vitro pre-mRNA splicing using large libraries (up to 100,000 compounds)

• Development of indole based probes for the comprehension of their mode of interaction with SR proteins, and RNA

• Compound library synthesis using alternative scaffolds-pharmacological profiling

• Validation of the efficacy of novel synthesized compounds

• Development of clinically useful drugs that direct the splicing pathway. Short description of activities 107 Analysis of chemical derivatives of newly identified inhibitors of pre-mRNA splicing During the second year a major focus of this work Package was further characterization of novel inhibitors of pre-mRNA splicing identified by different partners. Two strategies were conducted in parallel (i) based on the analysis of the small molecules that have already been shown to affect selectively the (alternative) splicing of specific pre-mRNAs involved in different diseases (Parners 1a, 6, 12a, 12c, and 22), and (ii) on the usage of antisense oligonucleotides or derivatives thereof (e.g. TOES) to correct aberrant splicing patterns by triggering either exon skipping or exon inclusion (Partners 13, 17 and 19). Following the initiative of Partner 12a (Tazi) in collaboration with Partner 12c (Branlant) and chemist at the Curie Institute, a small-molecule (IDC16) is discovered as inhibitor of HIV pre-mRNA splicing which represents a novel antiretroviral therapy to overcome drug resistance (Bakkour et 2007). IDC16 inhibits the exonic splicing enhancer activity of SF2/ASF thereby preventing Tat and Rev synthesis and HIV-1 multiplication. IDC16 was initially developed as a DNA-intercalating cytotoxic drug because of its planar fused polycyclic indole structure (see figure), but it was not pursued because of weak activity; nevertheless, for a new anti-HIV drug administered chronically this potentially mutagenic side-effect is particularly undesirable. Therefore, we hypothesize that appropriately functionalized and conformationally mobile “ring opened analogues” of IDC16 will retain strong affinity for the anti-HIV SR protein target, but lose capacity to intercalate DNA. This constituted the main objective of the last 18 month research proposal which is now achieved. 200 new compounds were synthesized to generate new and selective SR protein inhibitors as lead compounds for HIV/AIDS drug development. The strategy was to focus on disrupting the fused polyaromatic ring system of IDC16 and generating a series of ring opened analogues that maintain some of the putative essential structural features. The generation of these libraries of analogues is now screened to identify lead compounds for optimization and for use in studies to elucidate the mechanism of SF2/ASF protein inhibition. These libraries of compounds will be tested in the primary screen to measure their ability to interfere with alternative splicing via inhibition of SF2/ASF. The more active compounds will also be tested for their cytotoxicity and the more promising compounds which have the best profile in these assays will be further examined for their ability to stop HIV replication. It is expected that toward the end of the Phase 1 primary screening it will be possible to organize the active molecules into groups depending upon their potency, structure and substituent patterns. In this operation QSAR techniques will be used to study different options (hydrophobic, polar, hydrophilic substitutions) for further optimization.

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Structure of IDC16 Partner 12a has identified a novel inhibitor of NMD (NMDI 1) among the indole derivatives (Durand et al 2007). This compound was able to inhibit nucleus-associated as well as cytoplasmic NMD and allowed accumulation of NMD factory in cytoplasmic processing bodies (P-bodies). NMDI 1 was, therefore, used to purify mRNP particles subjected to NMD. In collaboration with Partner 1a a proteomic analysis of these stalled mRNPs was performed (see WP 9). Given that one third of genetic diseases and many forms of cancer have a nonsense mutation as origin, NMDI 1 could constitute a new therapeutic pathway for some genetic diseases. As proof of principle NMDI 1 was shown to stabilize dystrophin mRNA subjected to NMD in fibroblast from DMD patients. In order to test whether the chemical molecule was capable of inhibiting NMD in vivo, mice models in which target mRNA are subjected to NMD with variable efficacy in different tissues were injected with NMDI 1. NMDI 1 induces an abrogation of NMD in vivo leading to the stabilization of NMD substrates and the synthesis of truncated proteins. Partners 1a (Lührmann) has characterized the mechanism of action of various HDAC and HAT inhibitors as novel inhibitors of pre-mRNA splicing. Purified spliceosomal complexes formed in the presence of the inhibitors were obtained and their compositions were analyzed by mass spectrometry (a manuscript summarizing these results is submitted).

Partner 22 (Lamond) following identification of 8 distinct compounds that reproducibly inhibit splicing, these compounds are currently being evaluated by medicinal chemists for their suitability for further development, either as research tools and/or therapeutic agents. Partner 6 (Ast) has screened a variety of substances regarding their effect on the splicing pattern of the IKAP mRNA in FD cells. Two small molecules (potential drugs) raised the inclusion level of exon 20 in the endogenous IKAP mRNA in FD cells. This partner will optimize parameters for the drugs found (concentration, time of action, toxicity, effect on the protein level) as well as evaluate the effect of additional drugs, some already found to affect splicing of other genes involved in neurological diseases. Partner 13 (Schümperli): has developed a U7-based methodology to induce exon skipping in HIV-1, thereby inhibiting HIV-1 replication. Moreover the group has developed U7-based methods to induce exon inclusion in survival of motoneuron 2 gene (SMN2), which could be used in gene therapy for spinal muscular atrophy. Regarding this second issue they now have very preliminary evidence that this approach works in vivo in a very severe mouse model for SMA where symptoms are clearly reduced and survival of the animals is prolonged. Moreover, they have made significant progress towards engineering inducible U7 snRNA cassettes. Partner 17 (Eperon): further investigated and optimized strategy for using TOES (targeted oligonucleotide enhancers of splicing) to rescue refractory exons as a basis for related endeavours by other members of the WP. Regarding this issue, the site of annealing of the oligonucleotides, the tail sequence and the chemical composition for rescuing the expression of SMN2 exon 7 have been varied. The outcome has been assessed in splicing in vitro, and in fibroblasts from patients with spinal muscular atrophy. In the latter case splicing has been assessed by real time PCR, protein expression by western blotting, cell viability by standard methods, and oligo uptake and lifetime in the cell by standard fluorescence and also by FRET. With the best combination, increased levels of protein were seen at up to 1 week after transfection. Oligos that increased the level of protein 3-fold also showed high toxicity, whereas those that increased it about 2-fold showed no toxicity. FRET confirmed that the oligos remain intact in the nucleus even at the longest time tested post-transfection (15 hr). Partner 10 (Biamonti) has designed a morpholino targeting the region of the Ron enhancer containing the SF2/ASF binding site (Mo-SF2) to sterically block the binding of this splicing factor and, thus, the skipping of exon 11. The efficacy of Mo-SF2 was tested using human gastric carcinoma KATOIII cells characterized by high level of SF2/ASF and DeltaRon isoform an inducer of metastic phenotype. Mo-SF2 inhibits DeltaRon splicing Partner 19 (Kjems) identified ESE and ESS in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) and showed that exon skipping in the presence of disease-causing mutation (362C>T) can be counteracted by recruitment of antisense oligonucleotide containing an ESE. Partner 19 has tested antisense DNA and LNA oligonucleotides, DNA- and LNAzymes targeted to the four most accessible sites for their abilities to block reverse transcription and dimerization of the HIV-1 RNA template in vitro, and to suppress HIV-1 production in cell culture. The neutralization of HIV-1 expression declined in the following order: antisense LNA > LNAzymes > DNAzymes and antisense DNA. The LNA modifications strongly enhanced the in vivo inhibitory activity of all the antisense constructs and some of the DNAzymes. Notably, two of the LNA modified antisense oligonucleotides inhibited HIV-1 production in cell culture very efficiently at concentration as low as 4 nM.

N

N

NCl

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108 Purify SR protein associated complexes and probe SR protein/drug interaction with photo-affinity molecules In order to identify which proteins of a nuclear extract are able to interact with indole derivatives, modified molecules were synthesized by Partner 12a to graft them onto a chromatography support. IDC16 derivative comprising a methoxyethanol chain at the N-2 and C-10 positions were generated and are currently used by Partner 12a to prepare affinity column. Several supports are tested in order to minimize non specific interactions. This type of experiment will help to determine whether IDC16 allows ASF/SF2 to interact with the RNA or on the contrary blocks its’ interaction. Also, this approach will allow testing an eventual interaction between IDC16 and RNA. If IDC16 doesn’t block the ASF/SF2-RNA interaction, we will be able to envisage purifying non functional spliceosomal complexes obtained in presence of the drug and compare their composition with active complexes obtained without the drug. The protein content in each case will be identified by mass spectrometry. These different types of analyses should allow us to identify the nuclear factors capable of interacting directly and/or indirectly with IDC16. The same approach could be envisaged with certain potentially interesting derivatives. Partner 12a has synthesized 10 and 6 azido derivatives of compound IDC16 in order to affinity label proteins directly interacting with these compounds. This labelling will be carried out both in vitro in nuclear extracts and ex vivo if the modified product can easily penetrate into the cells. The labelled proteins will be separated on a 2D gel and each spot will be analyzed by mass spectrometry. The sum of all of these results should allow us to identify precisely the target(s) of IDC16. Collaboration between Partner 12a, 12c, Partner 25 and Partner 1a. Hutchinson-Gilford progeria syndrome (HGPS) is a pathology in which mRNA splicing alterations affect the normal expression of Lamin A/C proteins encoded by the LMNA gene. We are mostly interested in the heterozygous de novo point mutation located in exon 11 of the LMNA gene, which is transcribed to lamin A, while the shorter lamin C is unaffected by the mutation. The GGC>GGT G608G single-base substitution reveals a cryptic splice site and is proposed to result in a truncated transcript encoding a protein that is 50 amino acids shorter. Most of the results are described in WP9. Both in vitro and ex vivo experiments showed that the single point mutation (GGC>GGT) in exon 11 was sufficient to induce utilisation of a cryptic 5 splice site in exon 11. Cross-linking studies have revealed that SF2/ASF and SRp55 members of the SR protein family are involved in the aberrant splicing. Transfection assays with reporter constructs in cells treated with siRNA against these SR proteins confirmed their involvement ex-vivo. SRp55 activate the authentic splice site, whereas SF2/ASF activates the cryptic splice site. The activity of SRp55 is dependent on its phosphorylation status. Hyperphosphorylation of SRp55 leads to selective usage of the authentic splice site. A manuscript is in preparation to describe all these findings. Collaboration Partner 12a and Partner 25 using in vitro assays in HeLa extract demonstrated that the cryptic splice site can be used in the absence of the mutation. Different extracts from cells overproducing SR proteins are currently used to dissect the mechanism by which SR proteins activate an exonic cryptic splice site. In parallel, collaboration between Partner 12a and Partner 12c used purification of RNP complexes assembled around the wild type and mutated sequences using MS2 tag affinity purification procedure developed by partner 1a and have compared the proteomic profiles. The characterization of pertinent proteins is in progress. 109 Adoption of the in vitro pre-mRNA splicing assay with a fluorescence readout to additional pre-mRNA substrates To develop a high-throughput system for pre-mRNA splicing in vitro, a splicing reaction that gives a fluorescence readout was required. To this end, different procedures were tested in a standard test tube format by Partner 1a. Using MINX as a model in vitro pre-mRNA substrate, it has been possible to detect spliced mRNA by hybridization of a fluorescently labeled oligonucleotide to the exon-junction, the newly formed sequence element present only in the mRNA product, but not in the pre-mRNA. Hybridization of the oligonucleotide to the mRNA can be recognized as a change in its diffusion time by fluorescence correlation spectroscopy (FCS) using the Olympus MF20 multiwell plate reader. The FCS measurement is performed in a confocal volume with a single molecule detection mode, thus, this assay requires only a very small reaction volume (theoretically below 1 µl), which makes it ideal for high-throughput procedures. Initial screening format which was based on FCS turned out not to be robust enough for high throughput screening. As an alternative to establish a more robust assay format, Partner 1a is testing the possibility to use trans-splicing assay system to see whether it can be adjusted to an ELISA based format. Partner 12a has set general conditions, ex-vivo, suitable for medium throughput screening (96 wells plates) of molecules that are able to selectively interfere with the activity of SR proteins and putatively apt to correct pathological splicing events. This partner has established stable cell lines expressing luciferase based reporters to harbour either mutated exonic sequences from LMNA gene responsible for premature aging (Progeria syndrome) or exonic sequences from Dystrophin gene whose skipping is important for the correction of Duschenne Muscular Dystrophy (DMD).

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Problems and explanations for delays, postponements etc. 109 Fluorescence correlation spectroscopy (FCS) for in vitro splicing turned out not to be robust enough for high throughput screening. We decided not to continue this development. Current work is aimed at using luciferase reporter and ex vivo screening. An in vitro trans splicing system is also tested.

Publications 1: Marquis J, Meyer K, Angehrn L, Kämpfer SS, Rothen-Rutishauser B, Schümperli D. Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence. Mol Ther. 2007 Aug;15(8):1479-86. Epub 2007 Jun 5. 2: Asparuhova MB, Marti G, Liu S, Serhan F, Trono D, Schümperli D. Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping. J Gene Med. 2007 May;9(5):323-34. 3: Bakkour N, Lin YL, Maire S, Ayadi L, Mahuteau-Betzer F, Nguyen CH, Mettling C, Portales P, Grierson D, Chabot B, Jeanteur P, Branlant C, Corbeau P, Tazi J. Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance. PLoS Pathog. 2007 Oct 26;3(10):1530-9. 4: Durand S, Cougot N, Mahuteau-Betzer F, Nguyen CH, Grierson DS, Bertrand E, Tazi J, Lejeune F. Inhibition of nonsense-mediated mRNA decay (NMD) by a new chemical molecule reveals the dynamic of NMD factors in P-bodies. J Cell Biol. 2007 Sep 24;178(7):1145-60. 5: Jakobsen MR, Haasnoot J, Wengel J, Berkhout B, Kjems J. Efficient inhibition of HIV-1 expression by LNA modified antisense oligonucleotides and DNAzymes targeted to functionally selected binding sites. Retrovirology. 2007 Apr 26;4:29. 6: Kuhn A, van Santen M, Riester D, Hildmann C, Schwienhorst A, Urlaub H, Lührmann R Small molecule inhibitors of protein acetylation and deacetylation stall spliceosome assembly at distinct stages prior to the first catalytic step manuscript in preparation

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Work Package 15 Development of Enabling Technologies Lead Participant: Bertrand Séraphin

month planned achieved deliverables: 58 Extending the network's repertoire of available

technologies through integrating suitable candidates during the 2nd YIP recruiting round (month 25).

25 24

59 Experimental setup and optimization for D2 bombardment feasibility studies (month 18).

18 18

60 Construction of plasmids for use in establishing cell lines that stably express a tagged pre-mRNA (month 18).

18 24

110 Reduce the amount of RNP starting material for successful routine cross-linking experiments to less than 10 pmol (month 30).

30 24

111 Selective identification of cross-linked sites in various protein-RNA complexes by online nanoLC-Electrospray Ionization (ESI) MS/MS and off-line 2-dimensional nanoLC with subsequent MALDI-ToF/ToF analysis (month 30).

30 24

112 Increase of the protein-RNA cross-linking yield by a factor 2 - 3 using a frequency quadrupled Nd-Yag Laser (month 30).

30 24

Objectives of the work package • To identify and develop new technologies necessary to advance research on alternative splicing. Short description of activities 58 Extending the network's repertoire of available technologies. The network repertoire of technologies was extended with the arrival of new group leaders through the YIP program. These new groups provide extended and/or new expertises in various areas including biocomputing (M. Zavolan, E. Eyras, J. Bujnicki), biomedicine and biomedical applications (D. Baralle, D. Gabellini), mass spectrometry (H. Urlaub), microscopy (E. Bertrand) or structural biology (F. Allain). 59 Experimental setup and optimization for D2 bombardment feasibility studies. Partner 1a has implemented an experimental setup for D2 bombardment experiments and tested the feasibility of such studies. These experiments demonstrated that the extent of modification was extremely low. Moreover, detection of deuterated peptides by mass spectrometry proved also extremely difficult. Overall, the results of this feasibility study demonstrate that D2 bombardment is not a method of choice to map interaction surfaces of proteins. 60 Construction of plasmids for use in establishing cell lines that stably express a tagged pre-mRNA (month 18). Various cloning procedure have been tested to prepare constructs for establishing cell lines including systems based on the commercial Gateway system, using BAC recombineering or based on the original cis- and trans- Archaea-express technology (partner 1b, 3, 10b). Some delay was experienced in making these constructs but proof of principles has been obtained and such strategies can now be used in the network to establish cell lines expressing pre-mRNAs of interest. 110 Cross-linking experiments with less than 10 pmol. In feasibility studies with U1 snRNP and 15.5K-61K-U4atac snRNP we lowered the amount of starting material. We used less than 10 pmol for UV-induced cross-linking of proteins to RNA. We followed two strategies to detect and sequence the cross-linked species after the irradiated particles were hydrolyzed with endoproteinases and ribonucleases: (i) Selective detection of the cross-linked species in the mass spectrometer (multiple reaction monitoring) due to generation of marker ions specific for cross-links (ii) enrichment of cross-linked species over the excess of non-cross-linked species by using Ti02 and/or IMAC chromatography. - Lenz C. Kühn-Hölsken E. & Urlaub H. (2007) Detection of protein–RNA cross-links by nanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring (MRM) experiments, J. Am. Soc. Mass Spec. 18, 869–881. 111 Selective identification of cross-linked sites in various protein-RNA complexes. We applied the above strategies to the U2-specific protein SF3b14a bound to an RNA oligonucleotide encompassing the branch site interaction region in U2 snRNA. Enrichment of the cross-linked species and subsequent MALDI-MS analysis

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revealed that the C-terminal helix of SF3b14a is in contact with the BSiR oligonucleotide. Snurportin-1 bound to U1 snRNP was cross-linked to U1 snRNP, and the cross-linking site on snurportin-1 and U1 snRNA was identified by a combination of precursor-ion-scanning techniques and MSMS that was triggered by multiple-reaction monitoring experiments; furthermore, MALDI-MSMS was used subsequent to enrichment of the cross-linked species. Surprisingly, we found that snurportin-1 is in contact with stem III of U1 snRNA (in addition to its previously characterized contact site - Kühn-Hölsken E, Dybkov O, Sander B, Lührmann R, Urlaub H., 2007). Improved identification of enriched peptide–RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry. Nucleic Acids Res. 35(15):e95 - Kühn-Hölsken, E., Lenz, C., Richter, F., Raabe, M., Pleßmann, U., and Urlaub, H. (2007) Combined LC-ESI-MS/MS and LC-MALDI-MS/MS analysis reveals an unusual protein–RNA contact in human [U1 snRNP-Snurportin1] particles, Proceedings of the 55rd ASMS Conference and Allied Topics, 3rd.–7th. June, Indianapolis, Indiana, USA. 112 Increase of the protein-RNA cross-linking yield. Initial experiments were performed by using LASER and UV flash lamps. U1 snRNP was used as substrate. The cross-linking yield was monitored with multiple reaction monitoring, which gives a quantitative signal. We did not observe a significant improvement of the cross-linking yield in comparison with the yield obtained by using standard UV lamps. Overall, the results of this study demonstrate that normal UV lamp provide the highest possible cross-link leveL. Problems and explanations for delays, postponements etc. Technical delays in making plasmid constructs to integrate tagged reporter RNA in cells.

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Work Package 16 Conferences and meetings Lead Participant: Jean Beggs

month planned achieved deliverables: 11 2007 Annual Meeting by month 16. 16 16 113 Four Interdisciplinary Focus Meetings in 2007:

RNA&Cancer (month 16) Cell Biology, Signaling&Alternative Splicing (month 23). Biophysical Methods to Study Alternative Splicing (m 25)

16

16 23 25

114 First International EURASNET Meeting, Krakow – Poland (month 29)

29 29

115 Three workshops on areas of strategic relevance in 2007: Alternative Splicing in Plants Alternative Splicing and Bioinformatics High Throughput Approaches in Biology

17 19 21

17 19 21

Objectives of the work package To organise three types of conferences and meetings:

• Biennial European Conference on Alternative Splicing, participation open world-wide, for the exchange of information on the latest advances in the field.

• Annual Interdisciplinary Focus Meetings, open for NoE members and invited participants, for strategic planning and the establishment of collaborations across research field boundaries. • Courses/workshops for graduate scientists and clinical researchers/clinicians

• Annual NoE meeting, open to NoE members. Short description of activities

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11 2007 Annual Meeting. The Second Annual Meeting was a EURASNET members’ meeting. It was organized by participant J. Tazi and took place in Ile de Bendor, France April 14-18, 2007. There were 71 EURASNET participants. The Steering Committee and Network Management Team and four SAB members were present. Professor Benoit Chabot (Canada), appointed by the European Commission as External Reviewer, was present throughout the meeting. During this meeting the second round of YIP selection was successfully completed. Ten YIP candidates gave presentations and five were selected. This fulfilled Deliverable 11. 113 Four Interdisciplinary Focus Meetings in 2007. 1. The IFM “RNA and Cancer” was organized by NoE member J. Tazi April 12 – 13, 2007 in La Grande Motte, France. 2. Participants A. Kornblihtt, A. Lamond, J. Caceres and organized an IFM on "Cell Biology, Signaling and Alternative Splicing" in Bariloche, Province of Río Negro, Argentina. November 29-30, 2007. This was an independent meeting but held back-to-back with the "Gene Expression and RNA Processing" meeting Nov 26 – 29, organized by the International Centre for Genetic Engineering and Biotechnology, Universidad de Buenos Aires. 3. “Biophysical Methods to Study Alternative Splicing”, organised by participant R. Lührmann was held in Berlin, on February 20-23, 2008, in association with a EURASNET Principal Investigators meeting. Information on all three events is presented at the end of this WP16 report. Deliverable 113 of WP16 was therefore 3/4 achieved. 115 Three workshops on areas of strategic relevance in 2007. 1. The “EURASNET Workshop on Alternative Splicing in Plants” in Carry-le-Rouet, France on May 11, 2007, organized by participants J. Brown, A. Barta and A. Jarmolowski as an independent meeting associated with the 6th Symposium on Post-transcriptional Regulation of Plant Gene Expression, May 10-13. 2. A workshop on “Alternative Splicing and Bioinformatics” was organized by participant A. Barta in Vienna, Austria July 19-20, 2007. 3. A Workshop entitled “High Throughput Approaches in Biology” was held in Edinburgh, Scotland, September 4-5, 2007, organized by participant J. Beggs. This was an independent workshop, held immediately after the inaugural symposium of the Edinburgh Centre for Systems Biology, September 3-4. Deliverable 115 of WP16 was thus fully achieved. Problems and explanations for delays, postponements etc. The Second Annual Meeting was originally proposed to take the form of the first European Conference on Alternative Splicing in 2007. However, as it proved to be impractical to organize a meeting of such dimensions so early in the lifetime of the NoE it was agreed last year that the European Conference would be postponed until 2008. Therefore the Second Annual Meeting was a EURASNET members’ meeting. In terms of IFMs the EURASNET network is one meeting event behind schedule for 2007, as an IFM on the topic of Combining computational and experimental approaches to study alternative splicing was not held due to there being so many meetings scheduled already in 2007 (EURASNET and non-EURASNET). There will, however, be a meeting on this topic held in Krakow in May 2008 in association with the International Conference.

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WP16: MEETING REPORTS ANNUAL MEETING 2007 Minutes of the 2007 EURASNET Annual Meeting Ile de Bendor, Marseille, France (April 14-18, 2007) Of the 35 research groups in the network, 33 were present with their respective principal investigator or an authorized legal representative. 38 additional lab members attended the conference. Four of the seven Scientific Advisory Board members were present. For the YIP decision the SAB members not attending were contacted by telephone. The required quorum for the YIP decision was thus achieved. The European Commission was represented by the external reviewer Prof. Benoit Chabot, Sherbrooke, Canada. 1. EURASNET YIP Program. Winners of 2nd round selection. The short-listed candidates were invited to attend the EURASNET Annual Meeting at Ile de Bendor to present their projects in the presence of the Scientific Advisory Board. All of them accepted this invitation. All members of the SAB, as the ultimate decision-making body on YIP awards, received the full documentation of the short-listed applications well ahead of the Annual Meeting. The quality of the short-listed candidates was very high this year, so that it was with much regret that the SAB could award YIP positions to only five candidates. In continuation of the existing Annex I the selected YIP winners will be integrated according to the following scheme: Frédéric Allain, Participant 29 Diana Baralle, Participant 23B Janusz M. Bujnicki, Participant 30 Eduardo Eyras, Participant 31 Anabella Srebrow, Participant 20B The decision was met with wide acclaim and support by YIP Committee and Consortium. The Steering Committee therefore asked the Coordinator to forward the names of the five winning candidates and the proceedings to the European Commission for approval. 2. Decisions related to meetings and workshops. The following commitments were discussed and agreed upon: • Interdisciplinary Focus Meeting RNA and Cancer (April 2007, Montpellier - La Grande Motte, France) • EURASNET Workshop Alternative splicing in plants (May 2007, Carry-le-Rouet, France) • Workshop Alternative Splicing and Bioinformatics (July 2007, Vienna, Austria) • Workshop High Throughput Approaches in Biology (September 2007, Edinburgh, UK) • Interdisciplinary Focus Meeting Cell Biology, signaling and RNA Processing (November 2007, Bariloche, Argentina) • First International EURASNET Meeting on Alternative Splicing (May 2008, Krakow, Poland), in conjunction with Career Development Workshop • Interdisciplinary Focus Meeting Alternative Splicing and Disease (June 2008, Rome, Italy) • Workshop. Hands-on-course on basic and advanced techniques in RNA – protein interactions (Fall 2008, Barcelona, Spain) • Workshop 2nd Cell Imaging Workshop (2008, Montpellier, France) • Workshop RNA Techniques for Clinical Researchers (2008) General agreement was reached that travel bursaries should become available on request for small intra-workpackage meetings whenever the complexity of projects and discussions among participants within a workpackage require such meetings.

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3. Public Understanding of RNA Biology The new Public Science Officer, Claudia Panuschka, Vienna, Austria was introduced by EURASNET member Andrea Barta. Concerning training bursaries the General Assembly decided to make these funds available on request for necessary small intra-workshop meetings in case such discussions are necessitated by complexity of networked projects and number of participants. 4. Microarray Initiative In continuation of the microarray initiative of EURASNET, the General Assembly decided to support the application "Expansion and Application of Plant Alternative Splicing RT-PCR Panel", submitted by consortium labs working in this field, with 60.000 Euro from the HTE fund. The panel is set up at the SCRI, Dundee and funds will be transferred to the EURASNET account of member John Brown.

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REPORTS ON INTERDISCIPLINARY FOCUS MEETINGS HELD IN 2007

1) “RNA and Cancer”

April 12 – 13, 2007 in La Grande Motte, France

Organised by J. Tazi The purpose of this two day EURASNET IFM was to give scientists the opportunity to present and discuss their research observations and to meet up with current or potential industrial partners. The meeting was enhanced by the participation of scientists from the Cancéropôle Centre of South-Western France and from Sigma Aldrich, at no cost to EURASNET. We placed particular emphasis on new technological advances and their potential impact on Alternative Splicing research in Cancer. The main objective was to facilitate these interdisciplinary exchanges and promote interactions between students, researchers and clinicians. EURASNET, lends itself naturally to these joint objectives. The meeting was attended by 94 participants including 50 students and post-docs, 10 industrials, 24 researchers and 10 clinicians. There were 14 seminars, one poster session and 4 round tables. The topics covered by the meeting include, non-coding RNA, RNA Aptamers and the basic mechanism of pre-mRNA splicing and its connection to cancer. The industrial participants discussed technical aspects both at plenary sessions and at the round tables. Four EURASNET members were invited to speak at this meeting. The feed back from the participants in the form of a questioner was very positive. The duration of the meeting, the documentation and the time dedicated to every presentation obtained an average score of 3.5/4. The highest scores (3.8/4) was attributed to the scientific level of the seminary.

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RNA AND CANCER MEETING ABSTRACTS

TRAFFICKING AND ASSEMBLY OF NON-CODING RNAs: THE CASE OF SnoRNPS AND TELOMERASE

Edouard Bertrand1, Séverine Boulon1, Nathalie Marmier-Gourrier2, Bérangère Pradet-Balade1,

Laurence Wurth3, Céline Verheggen1, Beata Jady4, Benjamin Rothé2, Christina Begon-Pescia1, Marie-Cécile Robert1, Tamas Kiss4, Barbara Bardoni5, Alain Krol3, Christiane Branlant2, Christine

Allmang3, Bruno Charpentier2

1-IGMM-CNRS UMR5535; 1919 route de Mende; Montpellier Cedex 5; France. 2-MAEM, UMR 7567 CNRS-UHP, Faculté des Sciences et Techniques; Vandoeuvre-les-Nancy; France. 3-Architecture et Réactivité de l'ARN, Université Louis Pasteur de Strasbourg, CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg, France 4-LBME; Université Paul Sabatier; 118, route de Narbonne; Toulouse; France 5- Université Nice-Sophia antipolis; Faculté de Médecine, Av. de Valombrose; Nice; France RNA-binding proteins of the L7ae family are at the heart of many essential RNPs, and some of them display the remarkable ability to initiate formation of distinct RNP complexes, depending on their RNA binding site. Here, we show that Nufip and its yeast homolog Rsa1 are part of a conserved machinery that assembles RNPs of the L7ae family: U3 B/C RNP; box C/D and H/ACA snoRNPs, telomerase, U4 snRNP, and mRNPs coding for selenoproteins. We demonstrate that Nufip and Rsa1 have two functions: first, to tether together core proteins in the immature RNPs; second, to physically link assembling RNPs with HSP70/90 chaperones and R2TP proteins: Pih1, Tah1/SGT, and the AAA+ ATPases Rvb1 and Rvb2. Our results suggest an assembly line, which goes from translation of core RNP proteins, loading on co-chaperones, to assembly and refolding on nascent RNAs. HnRNP A1 IS REQUIRED FOR THE PROCESSING OF AN ONCOGENIC MIRNA

CLUSTER

Javier F. Caceres

MRC Human Genetics Unit, Edinburgh, EH4 2XU, UK The SR and hnRNP A/B family proteins have antagonistic roles in alternative splicing regulation. A subset of SR proteins, as well as hnRNP A1 shuttle from the nucleus to the cytoplasm, suggesting that their activities may not be limited to nuclear pre-mRNA processing events. Indeed, we have recently described a novel cytoplasmic role for SF2/ASF in mRNA translation (1). We are using the CLIP (ultraviolet cross linking and immunoprecipitation) method in order to identify mRNA targets of SF2/ASF and hnRNP A1. This approach is allowing us to identify and classify nuclear and cytosolic mRNA targets for these proteins that could operate at the level of pre-mRNA splicing (nuclear fraction), mRNA stability and/or translation (cytoplasmic fractions). Interestingly, we found that hnRNP A1 binds to and is necessary for the processing of a microRNA precursor, pre-miR-18a. This microRNA is expressed as a cluster of intronic RNAs, the miR-17~18a~19a~20a~19b-1~92 microRNA polycistron, which has been proposed to act as a potential human oncogene. We found that hnRNP A1 binds specifically to pri-miR-18a, but not to any other of the miRNAs present in the same cluster, prior to Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity of this pri-miRNA cluster and also display reduced levels of endogenous pre-miR-18a. In addition, we demonstrate that hnRNP A1 is required for

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the processing of miR-18a in a context-dependent manner. Furthermore, we show that hnRNP A1 is required for miR-18a-mediated repression of a target reporter in vivo. Thus, a general RNA binding protein that has been implicated in various roles in RNA metabolism has a function in miRNA biogenesis. These results underscore a novel role for RNA binding proteins as auxiliary factors that facilitate the processing of specific miRNAs. 1. Sanford,J.R., Gray,N.K., Beckmann,K. and Cáceres,J.F. (2004) A novel role for shuttling SR proteins in mRNA translation. Genes Dev.,18, 755-768. 2. Guil,S. and Cáceres,J.F. (2007) hnRNP A1 is required for the processing of an oncogenic miRNA cluster. Submitted for publication.

IMPACT OF ANTI-CANCER DRUGS ON THE ALTERNATIVE SPLICING OF HUMAN APOPTOTIC GENES

Benoit Chabot

Faculté de médecine et des sciences de la santé, Département de microbiologie et d'infectiologie, Université de Sherbrooke, Sherbrooke, Québec, Canada Inducing an apoptotic response is the the goal of most current chemotherapeutic interventions against cancer. However, intrinsic alterations in the apoptotic pathway are a hallmark of cancer cells that compromises the efficacy of chemotherapeutic agents. In addition, the activity of many apoptotic regulators is controlled by alternative splicing, sometimes to produce isoforms with opposing functions. Examples of this type are found in every category of apoptotic regulators including transmembrane receptors (e.g., Fas, FasL and LARD), adaptor molecules (e.g., Bcl-x, Bak, Apaf1, survivin, Mcl-1, TRAF2), caspases (e.g., caspases 1, 2, 6, 7, 8, 9) and executors (e.g., FLIP and ICAD). Cancer cells often display aberrant alternative splicing profiles that can help assign a signature with diagnostic, prognostic or functional value.

Very little is known about the impact of mainstream chemotherapeutic agents on the alternative splicing of apoptotic genes. Here, we have investigated the impact of twenty cancer agents on the alternative splicing of apoptotic genes. We find that many anti-cancer drugs can shift splicing from the anti-apoptotic Bcl-xL isoform to the pro-apoptotic Bcl-xS variant. However, the drugs that shift splicing vary in different transformed and cancer cell lines. Interestingly, we find that the shifting activity of several drugs in 293 cells require the first half of Bcl-x exon 2, suggesting that the putatively different modes of action of drugs converge on a common mechanism of splicing control. In a more global approach, we find that each anti-cancer agent tested can affect the alternative splicing of human apoptotic genes. In each cell line, drugs of the same class have a tendency to influence the splicing of a common set of targets. Globally however, the profile of splicing targets is determined primarily by the origin of the cell line rather than drug identity. Our study suggests that the impact of anti-cancer agents on alternative splicing may vary between different cancers and that this may contribute to drug efficacy.

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LOSS OF FUNCTION SCREENING USING A LENTIVIRAL RNAI LIBRARY TO IDENTIFY GENES THAT MODULATE CELLULAR RESPONSE TO

PACLITAXEL

Henry J George R&D Manager, Functional Genomics R&D, Sigma-Aldrich Corporation, St. Louis, MO 63103 The delivery of short hairpin RNA (shRNA) through use of lentiviral vectors is proving to be a powerful means to mediate gene specific RNA interference in mammalian cells. The RNAi Consortium (TRC) has created a library of 160,000 shRNA clones designed to target >15,000 human and >15,000 mouse genes. Through the utilization of a recombinant lentivirus delivery system, this library is demonstrating to be useful in targeting cell lines that are not amenable to small interfering RNA transfection, and in facilitating screens and experiments that require long-term knockdown that cannot be achieved by synthetic siRNA. After validation of this library and demonstration of its applicability in a broad array of cell types, we conducted a screen using a panel of shRNAs targeting tumor suppressor genes. We examined the effect of knocking out each of these tumor suppressors in a non-small cell lung cancer cell line prior to treatment with paclitaxel. This screen was effective in identification of genes involved in increasing sensitization or resistance to this chemotherapeutic. The availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. We believe that these results validate the usefulness of shRNA in loss of function screens designed to identify potential therapeutic targets and biomarkers.

SFRS1, THE GENE CODING FOR THE SPLICING FACTOR SF2/ASF, IS A PROTO-ONCOGENE

Rotem Karni, Elisa de Stanchina, Scott W. Lowe, David Mu, and Adrian R. Krainer Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA Alternative splicing plays an important role in cancer, partly by modulating the expression of many oncogenes and tumor suppressors, and also because inactivating mutations that affect alternative splicing of various tumor suppressor genes account for some of the inherited and sporadic susceptibility to cancer. We have tested the hypothesis that some of the principal factors that regulate alternative splicing events are causally involved in cancer. We found that the splicing factor SF2/ASF and its antagonist hnRNP A1 are upregulated in different sets of human tumors. In the case of SF2/ASF, this is sometimes due to increased copy number of its gene, SFRS1. SFRS1 resides on Chromosome 17q23, a region frequently amplified in breast cancer. Using retroviral transduction, we found that slight overexpression of SF2/ASF is sufficient to transform immortal rodent fibroblasts, which form sarcomas in nude mice. The increased expression of SF2/ASF modulates the sensitivity to apoptotic stimuli, and increases cell proliferation. In addition, SF2/ASF overexpression results in activation of downstream components of the PI3K/Akt/mTOR and Ras/MAPK signaling pathways, i.e., phosphorylation of S6K1 and eIF4E, respectively, bypassing upstream signaling. Inhibition of mTOR by rapamycin is sufficient to block the transformation of cells by SF2/ASF overexpression. As expected from its biochemical activities, SF2/ASF overexpression affects alternative splicing of transcripts from many endogenous genes, including several tumor suppressors and oncogenes. One of the key targets is S6K1 pre-mRNA, resulting in increased expression of an unusual isoform of this kinase with oncogenic activity. shRNA-mediated downregulation of either SF2/ASF or the novel S6K1 isoform is sufficient to reverse the transformed phenotype caused by SF2/ASF overexpression in vitro and in vivo, indicating that SF2/ASF plays a role in tumor maintenance. These findings demonstrate that an alternative splicing factor, SF2/ASF, can act as an oncoprotein by modulating alternative splicing of critical target genes, and is a potential target for cancer therapy.

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PROTEOMIC AND STRUCTURAL ANALYSIS OF THE SPLICEOSOME

Reinhard Lührmann

Max-Planck-Institut for Biophysical Chemistry, Göttingen, Germany The removal of non-coding intron sequences from a pre-mRNA to form mRNA (i. e.,pre-mRNA splicing) is an essential step in gene expression. In addition, the alternative splicing of pre-mRNA, which leads to the production of multiple unique mRNAs from a single gene, plays a major role in enhancing the proteomic complexity of most organisms. Splicing is catalysed by the spliceosome, a large ribonucleoprotein (RNP) complex, which is formed by the ordered interaction of the pre-mRNA with the U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) and more than 100 non-snRNP proteins. The spliceosome is a highly dynamic molecular machine that undergoes major structural changes during its assembly and catalytic action. Our research is primarily focussed on (i) determining the role of snRNP and spliceosomal proteins in the pre-mRNA splicing process and (ii) elucidating the 3D architecture of the snRNPs and the various spliceosomal complexes formed during the spliceosome’s assembly, catalysis and disassembly. To investigate biochemically and structurally the transition from the pre-catalytic spliceosomal B complex (containing all snRNPs) to the catalytic stepI spliceosome (complex C, containing substrate intermediates and the snRNPs U2, U5 and U6) we purified native B and C complexes under low stringency conditions, and determined their protein composition by mass spectrometry and immunoblotting techniques. These studies revealed dramatic changes in protein composition during the transition from B to C complex. Particularly interesting examples of protein dissociation and recruitment will be discussed. We have also started to analyse the role of posttranslational phosphorylation events of spliceosomal proteins in driving the spliceosomal cycle in addition to RNA helicases. In an attempt to identify small molecule inhibitors of pre-mRNA splicing in vitro, we tested a small library of known bio-active compounds regarding their effect on premRNA splicing. Several compounds could be identified which differentially inhibited spliceosome assembly at various stages prior to the first catalytic step. Mass spectrometry of the purified spliceosomes stalled in their assembly in the presence of the various inhibitors revealed significant differences in their protein composition when compared with the unstalled purified A or B complexes. These inhibitors will be valuable tools for investigating the role of certain proteins in the spliceosomal assembly cycle. Using site-directed hydroxyl radical probing of RNA we are also investigating the dynamics of RNA-RNA interactions during the action cycle of the spliceosome with the final goal to determine the higher order structure of the RNA network in the catalytic core of the spliceosome. Finally, in collaboration with Holger Stark and Markus Wahl (both MPIbpc) we are determining the 3D structure of purified snRNPs and spliceosomal complexes using cryo-electron microscopy and X-ray crystallography following a so-called hybrid approach. Selected examples will be discussed.

ALK GRANULES: AN ATTRACTIVE LINK BETWEEN IMPAIRED MRNA METABOLISM AND NPM-ALK MEDIATED TUMORIGENESIS

M. Fawal, F. Armstrong, H. Dupont, G. Delsol, B. Payrastre and D. Morello

Centre de Biologie du Développement, UMR 5547, Université Paul Sabatier, Toulouse Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a chimeric protein expressed in a subset of cases of anaplastic large cell lymphoma (ALCLs), for which constitutive expression represents a key oncogenic event. Despite numerous studies on signalling mediators, the molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK remain incompletely understood. The search for additional interacting partners of NPM-ALK led to the discoveryof

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AUF1/hnRNPD, a protein implicated in AU-rich element (ARE)-directed mRNA decay. AUF1 was immunoprecipitated with ALK both in ALCL-derived cells and in NIH3T3 cells stably expressing NPM-ALK or other X-ALK fusion proteins. AUF1 and NPM-ALK were found concentrated in the same cytoplasmic foci, that we called AG, for ALK granules, whose formation requires ALK phosphorylation. We will show that these granules contain mRNAs and will describe their dynamic link with two other well known cytoplasmic foci involved in mRNA degradation and storage, i.e. processing bodies (PBs) and stress granules (SGs). We propose that AGs function as transient mRNA transit/storage centres, enabling cells to proliferate despite hostile environmental changes and thus could be important actors of ALK oncogenicity.

TRANSLATIONAL CONTROL OF VEGF-A mRNA Bastide, A, Bornes, S, Karaa, SZ, Prado-Lourenco, L, Iacovoni, JS, Prats, AC, Touriol, C &

Prats, H INSERM U858, Université Paul Sabatier, IFR31, BP 84225, 31432 Toulouse Cedex 04, France. Vascular Endothelial Growth Factor (VEGF), is a growth and survival factor for endothelial cells, playing a key role in numerous physiological and pathological angiogenic processes throughout embryonic development and during adulthood. In the quiescent vasculature of adult organs, VEGF is produced at basal levels and protects endothelial cells from apoptosis. However, in a number of physiological situations, and particularly in cancers, VEGF level increases. Conversely, the absence of VEGF production is a characteristic feature of ischemia. Both the deletion of a single VEGF allele and VEGF overexpression result in embryonic lethality due to improper vascularization. These experiments on transgenic mice have clearly demonstrated that the expression level of VEGF is under tight regulatory control. We have previously shown that two independent internal ribosome entry sites, IRES A and IRES B, are present in the 5’ untranslated region of the human VEGF mRNA and control translation initiation at two alternative start codons, AUG 1039 and CUG 499, respectively. IRES A allows the maintenance of VEGF translation under hypoxic conditions in a cap-independent manner. IRES B, upstream of CUG 499, directs cap-independent translational initiation of a longer VEGF isoform (L-VEGF). The activities of these IRESes have been investigated in vivo using transgenic mice employing a bicistronic reporter vector strategy. Using severe hindlimb ischemia experiments, we found both VEGF IRESes A and B as well as the FGF-2 IRES were 5 times more active in the ischemic limb versus the control limb whereas the EMCV IRES was only 2-fold activated. Thus the angiogenic growth factors VEGF and FGF2 seem to have evolved IRES elements that not only enable their translation by 5’-cap independent mechanisms, but also allow enhancement of translation under ischemia, a stress condition whose physiological response requires the expression of these factors. We have also described the role of the mRNA splice variants on the use of the alternative CUG and AUG start codons. We have also identified an additional regulatory element, a very conserved microORF, located between the two start codons, and which efficient translation greatly participate to the repression of the AUG use. The relationship between 5’ and 3’ UTRs will be also discussed. In addition to its crucial role in tumor angiogenesis, VEGF, which is regulated at every conceivable stage of its production, thus represents a paradigm for gene regulation.

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Estrogen induction and overexpression of fibulin-1C mRNA in ovarian and breast cancer cells.

Pascal Pujol.

Institut National de la Sante et de la Recherche Medicale, CRLC Val d'Aurelle; Centre Hospitalier Universitaire de Montpellier, Hopital Arnaud de Villeneuve, Montpellier, France. Background : Fibulin-1 is an extracellular matrix protein induced by estradiol in estrogen receptor (ER) positive ovarian and breast cancer cell lines. Alternative splicing of fibulin-1 mRNA results in four different variants named A, B, C and D. We studied the relative expression of fibulin-1 mRNA variants and their estrogen regulation in human breast and ovarian cancer cells. Material, methods and results : In ovarian tissues and cancer cell lines, fibulin-1C and -1D are the predominant forms, whereas fibulin-1A and -1B are weakly expressed. We developed a competitive PCR assay based on coamplification of fibulin-1C and -1D to study the relative expression of these fibulin-1 variants in human ovarian samples. In ovarian cancer cell lines and ovarian cancer samples, there was a marked increase in the fibulin-1C:1D and fibulin-1C:HPRT mRNA ratios as compared to normal ovaries. In the BG1 estrogen receptor positive ovarian cancer cell line, fibulin-1C mRNA was induced by estradiol in a dose- and time-dependent manner. Transfection experiments using fibulin-1 promoter constructs indicate that 17beta-estradiol (E2) increases fibulin-1 gene transcription and that ERalpha is more potent than ERbeta to mediate E2 regulation of the transfected fibulin-1 promoter. Using SL2 cells devoid of specificity protein 1 (Sp1) and site-directed mutagenesis of GC boxes, we evidenced that the E2 regulation occurs through a proximal specificity protein 1 binding site. In addition, we show that fibulin-1C and -1D mRNAs, the two major fibulin-1 splicing variants, are differentially induced by E2. The induction of both mRNAs variants is direct and independent of a newly synthesized protein intermediate. Interestingly, actinomycin D chase experiments demonstrate that E2 treatment selectively shortens the fibulin-1D mRNA half-life. This indicates that estrogens affect differentially the stability of fibulin-1 variants and may explain the lower accumulation of fibulin-1D mRNA on E2 treatment. Conclusion : Our data show that estrogens, via ERalpha, are key regulators of fibulin-1 expression at both the transcriptional and posttranscriptional levels. The preferential induction of the fibulin-1C variant, which is overexpressed in ovarian and breast cancer, might play an important role in estrogen-promoted carcinogenesis.

SR PROTEIN SPLICING FACTORS AS TARGETS FOR THERAPY

Jamal Tazi Professor at University of Montpellier II (Institue of Molecular Genetics CNRS-UM II)

In humans the complexity of constitutive and alternative splice site recognition suggests multiple levels of regulation, each resulting from a combination of cis-elements and trans-acting factors adapting the enzyme responsible for intron excision “the spliceosome” to various situations. The significance of these observations, especially in regard to human pathologies, is hat there is a considerable number of disease-causing mutations in exons and introns that disrupt previously unrecognised auxiliary cis-elements as well as the well-known classical splice sites. Furthermore, viruses like the human immunodeficiency virus (HIV) uses a combination of several alternative 5’ and 3’ splice sites to generate more than 40 different mRNAs from its single transcribed genome pre-mRNA that are essential for its life cycle. Thus, targeting either conserved constituent of the spliceosome and/or regulatory sequences or factors that bind to them, holds great promise for future therapeutic action to correct aberrant splicing caused by these mutations (1). Our lab is currently

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developing an entirely new approach in which splicing factors are targeted by small chemical molecules. A large screen program has been conducted with the chemical library of the Curie Institute. Several molecules were found to counter the splicing stimulatory effect of individual members of the SR protein family and have been selected for further development in pathological splicing events (2). J. Tazi, S. Durand, Ph. Jeanteur. (2005). TIBS. 30, 469-478 J. Soret, N. Bakkour, S. Maire, S. Durand, L. Zekri, M. Gabut, W. Fic, G. Divita, C. Rivalle, D. Dauzonne, C. H. Nguyen, Ph. Jeanteur and J. Tazi.(2005) Proc Natl Acad Sci U S A, 102, 8764-8769.

IMPLICATION OF A NOVEL FORM OF HUMAN RNA POLYMERASE III IN THE FORMATION OF TUMORS

Valérie Haurie 1, Stéphanie Gaillard-Durrieu 1, Martina Prochazkova 2, Hélène Dumay-Odelot 1, Robert G. Roeder 3, Daniel Besser 4 and Martin Teichmann 1

1) INSERM U869; Institut Européen de Chimie et Biologie / Université Bordeaux 2; 2, rue Robert Escarpit; 33607 Pessac; FRANCE, 2 INSERM E347; 2) The Rockefeller University; 1230 York Avenue; New York, NY 10021; USA, 3) Max-Delbrück-Center; Robert-Rössle-Straße 10; D-13125 Berlin; GERMANY

In eukaryotes, transcription is carried out by three nuclear DNA-dependent RNA polymerases (I, II and III). Each of these RNA polymerases is specialized in transcribing specific sets of genes. RNA polymerase III (pol III) is involved in the transcription of small non-coding RNAs that intervene in splicing, translation and other processes. We identified a novel form of RNA polymerase III (pol IIIb) in human cells. This novel form differs in its subunit composition from the previously described form of human RNA polymerase III (pol IIIa). Both forms of human RNA polymerase III are active in transcription of all known pol III promoters in vitro. The novel form (pol IIIb) is present in all human tissues analyzed and its expression is neither affected during differentiation of human embryonic stem cells nor during controlled stepwise transformation of human fibroblasts. Transient, siRNA-mediated suppression of pol IIIb slows down growth of HeLa cells and clones, stably suppressing pol IIIb could not be obtained. These data indicate that pol IIIb is essential for viability of human cells. In contrast, pol IIIa levels are high in undifferentiated H1 human embryonic stem cells, but nearly undetectable after differentiation. Elevated pol IIIa levels are found in tumor cell lines and in fibroblasts that were at least partially transformed by papilloma virus E7 and SV40 small t proteins. Transient and stable knock-down of pol IIIa by siRNAs in HeLa cells is compatible with viability, but the suppression of pol IIIa strongly inhibits the ability of colony formation in soft agar assays. These results indicate that pol IIIa is not essential for the survival of human cells, but that it may play an important role in growth of undifferentiated cells.

APTAMERS: TOOLS AND PROBES.

Jean-Jacques Toulmé

INSERM U869 ARNA, “ARN: Régulations Naturelle et Artificielle”, Institut Européen de Chimie et Biologie, Université Victor Segalen, Bordeaux. Aptamers are oligonucleotides identified within randomly synthesized libraries through iterative

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selection/amplification steps. Aptamers can be raised against a wide range of targets (small molecules, peptides, enzymes, nucleic acids and even intact viruses or live cells). They generally display specific binding properties for the pre-determined target and are characterized by a high affinity. For proteins Kds in the low nanomolar range are frequent. Aptamers constitute interesting tools, rival antibodies for diagnostic purposes and can be considered for therapeutic applications (1).

Several examples will be described for different classes of targets. Aptamers have been selected against functional RNA structures of the Hepatitis C genome, the causative agent of a major human disease that occasionally leads to hepatocarcinoma (2). We also generated specific human RNase H inhibitors (3) and aptamers targeted to human Matrix MetalloProteinase 9 that are of interest for the development of probes for in vivo imaging (4). References

(1) The Aptamer Handbook, Functional oligonnucleotides and their applications (2005) S. Klussmann Ed., Wiley-VCH, 490 p.

(2) Toulmé, J. J., Di Primo, C., and Boucard, D. (2004) FEBS Lett 567, 55-62 ; Da Rocha Gomes, S., Dausse, E., and Toulmé, J. J. (2004) Biochem Biophys Res Commun 322, 820-826.

(3) Pileur, F., Andreola, M. L., Dausse, E., Michel, J., Moreau, S., Yamada, H., Gaidamakov, S. A., Crouch, R. J., Toulmé, J. J., and Cazenave, C. (2003) Nucleic Acids Res 31, 5776-5788.

(4) Toulmé, J.J., Da Rocha, S. and Dausse, E. (2007) Nucleic acids binding to ,MMP-9 and uses thereof. Patent to be filed.

ABERRANT EXPRESSION, AND POTENCY AS CANCER MARKERS/TARGETS,

OF SPLICING REGULATORS IN BREAST CANCER

Magali Lacroix-Triki and Stéphan Vagner

INSERM U563, Institut Claudius Regaud, 20-24 Rue du Pont St Pierre, 31054 Toulouse, France

SR « splicing regulator » and hnRNP « heterogeneous nuclear ribonucleoprotein » proteins are mRNA binding proteins that regulate several aspects of mRNA metabolism. Most of these proteins act at multiple steps, from splicing/polyadenylation processes to translational control.

Since ample evidences exist to demonstrate a connection between alternative splicing and cancer, we have examined the expression of several of these regulators in breast cancer.

Immunohistochemistry experiments were performed using breast carcinoma tissue microarrays representing 240 patients with 9 years mean follow-up. We have shown that high hnRNP A1 expression correlates with reduced overall survival and reduced relapse-free survival in patients with breast cancer. Several features of SR protein expression in breast tumours will also be discussed.

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NOM PRENOM SOCIETE POSTE VILLE TELEPHONE MAIL

ANCZUKOW Olga Laboratoire Génétique Moléculaire, Signalisation et Cancer UMR5201

PhD student Lyon 04 78 77 72 73 olga.anczukow@recherche.univ-lyon1.fr

AUGEREAU Patrick Inserm researcher Montpellier 04 67 61 24 07 p.augereau@valdorel.fnclcc.fr

AZADIAN Pascal MicroBioChips Business development Paris 02 32 34 98 48 microbiochips@hotmail.fr

BADIA Eric INSERM FAC MCU Montpellier 04 67 61 24 08 e.badia@valdorel.fnclcc.fr

BAILLE Olivier SIGMA-ALDRICH Biotech Specialist Lyon 04 74 82 29 08 Olivier.Baille@europe.sial.com

BAKKOUR Nadia IGMM PhD student Montpellier 04 67 61 36 32 nadia.bakkour@igmm.cnrs.fr

BALLEREAU Marc N/A Ordino (Andorre) 00 33 667 85 53 21 mballereau@gmail.com

BASYUK Eugenia CNRS postdoc Montpellier 04 67 61 36 46 eugenia.basyuk@igmm.cnrs.fr

BECLIN Christophe IBDML-CNRS life science Marseille 04 91 26 93 41 beclin@ibdml.univ-mrs.fr

BEROUD Rémy SIGMA-ALDRICH

BERTRAND Edouard CNRS Group leader 04 67 6136 50 bertrand@igmm.cnrs.fr

BISCH Antoine IGMM student Montpellier 06 83 98 63 97 antoine.bisch@laposte.net

BOIREAU Stéphanie IGMM - CNRS UMR5535 Post-Doc Montpellier 06 19 68 74 92 stephanie.boireau@igmm.cnrs.fr

BORDONNE Remy CNRS chercheur Montpellier 04 67 61 36 47 remy.bordonne@igmm.cnrs.fr

CACERES Javier F. MRC Human Genetics Unit Group leader 0044/131/4678426 javier.caceres@hgu.mrc.ac.uk

CAIZERGUES-FERRER Michèle LBME CNRS Toulouse 05 61 33 59 88 ferrer@ibcg.biotoul.fr

CAVAILLES Vincent INSERM U824 DR2 CNRS Montpellier 04 67 61 24 05 v.cavailles@valdorel.fnclcc.fr

CHABOT Benoit Universite de Sherbrooke Group leader/Professor 001/819/5645295 b.chabot@courrier.usherb.ca

CHABRIER Nathalie Sigma-Aldrich Sales Development Specialist Lyon 04.74.82.28.21 Nathalie.Chabrier@sial.com

CHALBOS Dany Inserm U826 Montpellier 04 67 61 24 32 d.chalbos@valdorel.fnclcc.fr

CHEBLI Karim cnrs ingenieur Montpellier 04 67 61 36 18 karim.chebli@igmm.cnrs.fr

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CHEN KUO CHANG Murielle inm student Montpellier 06 09 24 88 42 chen@montp.inserm.fr

CHOFARDET Patrick

CLAIR Philippe Institut de Génomique Fonctionnelle Montpellier 04 67 14 29 81 philippe.clair@igf.cnrs.fr

COMMES Thérèse Université Montpellier2 MCU Montpellier 04 67 14 42 36 commes@univ-montp2.fr

CONDAMINE Pascal RNAworks Montpellier 04 67 59 36 08 p.condamine@rnaworks.net

CONNAN Emmanuelle RNAworks Chef de projet R&D Montpellier 04 67 59 36 08 e.connan@rnaworks.net

CRAPEZ Evelyne CRLC Val d'Aurelle PhD Montpellier 04 67 61 30 48 ecrapez@valdorel.fnclcc.fr

CREMER Evelyne Cancéropôle GSO Manager Toulouse 05 62 74 45 71 evelyne.cremer@canceropole-gso.org

DA ROCHA GOMEZ Sonia INSERM U869 post doc Bordeaux 05 57 57 10 14 sonia.darocha@bordeaux.inserm.fr

DARFEUILLE Fabien INSERM U386 CR Bordeaux 05 57 57 45 09 fabien.darfeuille@bordeaux.inserm.fr

De TOLEDO Marion IGMM - CNRS UMR5535 Montpellier 04 67 61 36 85 marion.detoledo@crbm.cnrs.fr

DEL RIO Maguy CRLC Val D'Aurelle Scientist Montpellier 04 67 61 37 45 mdelrio@valdorel.fnclcc.fr

DESJOBERT Cécile INSERM U563 Toulouse 05 62 74 45 3 Cecile.Desjobert@toulouse.inserm.fr

DESMETZ Caroline CHU Arnaud de Villeneuve PhD, AHU Montpellier 04 67 61 24 12 caroline.desmetz@univ-montp1.fr

DEVAUX Agathe Universite de Bordeaux II Post-doc Bordeaux 05 57 57 48 98 agathe.devaux@bordeaux.inserm.fr

DUONG Vanessa Inserm U824 Montpellier 04 67 61 24 0 v.duong@valdorel.fnclcc.fr

DURAND Sébastien IGMM Montpellier

FARGES Charlotte Cancéropôle GSO Assistante communication-coordination

Toulouse 05 62 74 45 51 charlotte.farges@canceropole-gso.org

FERNANDO Céline IGMM M2 student Montpellier 06 30 57 35 97 celine.fernando@igmm.cnrs.fr

FIC Weronika IGMM phD student Montpellier 04 67 61 36 32 weronika.fic@igmm.cnrs.fr

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GALLONI Mireille University Montpellier 2 Biology Heath-Biology Engineering

Montpellier 04 67 14 93 69 mgalloni@univ-montp2.fr

GEORGES Henry SIGMA-ALDRICH

GOLZIO Muriel CNRS CR Toulouse 05 61 17 58 13 muriel.golzio@ipbs.fr

GONGORA Céline CNRS CR Montpellier 04 67 61 24 13 celine.gongora@valdorel.fnclcc.fr

GUETTARI Nadia SIGMA-ALDRICH

GUILHEM Frederic INSERM U 869 PhD Student Bordeaux 05 40 00 30 44 f.guilhem@iecb.u-bordeaux.fr

GUILLOUX Elisabeth

HAURIE Valerie INSERM 889 Bordeaux 05 57 57 17 71 valerie.haurie@bordeaux.inserm.fr

JACQUEMIN-SABLON Hélène Inserm Chercheur Bordeaux 05 57 57 48 21 helene.jacquemin-sablon@gref.u-

bordeaux2.fr

JALAGUIER Stéphan INSERM Montpellier 04 67 61 24 06 s.jalaguier@valdorel.fnclcc.fr

JUGE Francçois IGMM Montpellier

KORNBLIHTT Alberto R. Universidad de Buenos Aires

Group leader/Professor 0054/11/4/5763386 ark@fbmc.fcen.uba.ar

KRAINER Adrian Cold Spring Harbor Laboratory

Group leader/Professor 001/516/3676417 krainer@cshl.edu

LALOO BenoŒt INSERM U889 GREF Bordeaux 05 57 57 48 99 zwyx.bl@gmail.com

LE GUEDARD Sandie IURC-Laboratoire de Génétique Moléculaire Montpellier 04 67 41 53 60 sandie.le-guedard@mintp.inserm.fr

LEFEVRE Stéphane INSERM U869 PhD student Bordeaux 05 40 00 30 62 stephan.lefevre@iecb.u-bordeaux.fr

LEJEUNE Fabrice IGMM Researcher Montpellier 04 67 61 36 32 fabrice.lejeune@igmm.cnrs.fr

LEROY Olivier INSERM U827 PhD Montpellier 04 67 41 53 60 olivier.leroy@montp.inserm.fr

LOUBERSAC Julie Montpellier 2 etudiante Montpellier 06 75 50 49 52 julie_loubersac@yahoo.fr

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LUEHRMANN Reinhard Max Planck Institute of Biophysical Chemistry

Group leader/Professor 0049/551/2011405 reinhard.luehrmann@mpi-bpc.mpg.de

MAILLOT Gérard INSERM U563 ICR Toulouse 06 76 56 77 32 gerardchristian.maillot@laposte.net

MARCEL Virginie Internal Agency for Research on Cancer PhD sudent Lyon 04 72 73 83 90 marcel@iarc.fr

MILLEVOI Stefania inserm Toulouse 05 61 42 42 42 millevoi.stefania@claudiusregaud.fr

MORELLO Dominique Toulouse 05 61 55 64 73

MORILLON Pauline INSERM PhD student Bordeaux 05 40 00 30 44 p.morillon@iecb.u-bordeaux.fr

NEEL Henri CNRS Montpellier 04 67 61 36 46 henry.neel@igmm.cnrs.fr

NELSON Elisabeth Equipe avenir Inserm Centre Hayem IUH Paris

01 53 72 22 39 e.nelson@voila.fr

NIRDÉ Philippe CRLC - Inserm U826 Montpellier 04 67 61 24 17 p.nirde@valdorel.fnclcc.fr

OMAR el FAROUK Zouani Institut de Génétique Moléculaire de Montpellier Montpellier 04 67 61 36 64 zouani.omar@hotmail.fr

PAGANIN-GIOANNI Aurélie CNRS PhD Student Toulouse 05 61 17 59 98 aurelie.paganin@ipbs.fr

PARCELIER Aude Equipe avenir Inserm Centre Hayem IUH Paris

01 53 72 22 39 aude_parcelier@yahoo.fr

PRATS Hervé INSERM Toulouse 561322144 pratjh@toulouse.inserm.fr

PUJOL Pascal

REIGADAS Sandrine inserm U869 PhD Bordeaux 05 57 57 10 14 sandrine.reigadas@bordeaux.inserm.fr

ROSENBAUM Jean INSERM U889, Universite Bordeaux 2 Bordeaux 05 57 57 15 94 jean.rosenbaum@gref.u-bordeaux2.fr

RUFFLE Florence GET (équipe IGH) Montpellier 04 67 14 42 3 fruffle@univ-montp2.fr

SABATHE Fabrice

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SAROT Emeline RNAworks Chef de Projet R et D Montpellier 04 67 59 36 08 e.sarot@rnaworks.net

SAULOT Vincent MicroBioChips CEO Paris 06 09 93 79 28 microbiochips@hotmail.fr

TAZI Jamal CNRS Group leader Montpellier 04 67 61 36 85 tazi@igmm.cnrs.fr

TEICHMANN Martin IECB / INSERM U869 Bordeaux 05 40 00 30 53 Martin.Teichmann@bordeaux.inserm.fr

TOULME Jean-Jacques Institu Européen de Chimie et Biologie - U869 Pessac 05 40 00 30 34 jean-jacques.toulme@bordeaux.inserm.fr

TOURIOL Christian INSERM U858 - Equipe 15 Toulouse 05 61 31 21 44 touriol@toulouse.inserm.fr

TUFFERY-GIRAUD Sylvie IURC-Laboratoire de Génétique Moléculaire PhD Montpellier 04 67 41 53 61 sylvie.tuffery@montp.inserm.fr

VAGNER Stephane Inserm Group leader, DR2 inserm Toulouse 05 67 69 63 11 vagner@toulouse.inserm.fr

VERHEGGEN Céline CNRS Chargé de recherche Montpellier 04 67 61 36 62 verheggen@igmm.cnrs.fr

VEYRIER Anne INSERM phD student Toulouse 05 61 42 42 42 poste 44-31 veyriera@toulouse.inserm.fr

VILLALBA GONZALEZI Martin CNRS-IGMM CR1/assistant

professor Montpellier 04 67 61 36 67 martin.villalba@igmm.cnrs.fr

WATRIN Marguerite IECB Etudiante en thŠse Bordeaux 05 40 0030 77 m.watrin@iecb.u-bordeaux.fr

WIEZLAK Maria IGMM Student Montpellier 04 67 6136 32 maria.wiezlak@igmm.cnrs.fr

ZEKRI Latifa IGMM-CNRS Ph. D. Student Montpellier 04 67 61 36 32 latifa.zekri@igmm.cnrs.fr

CHAABIHI Hassan AGATE Bioservices Ales 699239786 hchaabihi@agatebioservies.com

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2) "Cell Biology, Signaling and Alternative Splicing"

Bariloche, Province of Río Negro, Argentina.

November 29-30, 2007

Organised by J. Cáceres and A. Lamond

A stimulating and informative interdisciplinary focus meeting was held in the Patagonian town of Bariloche, Argentina. The primary aim of the meeting was to review new technological developments of value to the NOE groups and to the projects underway within the NOE, to inform ourselves of exciting new developments in the field from experts outside the NOE and to evaluate progress within EURASNET and discuss opportunities for fruitful new collaborations and for new strategic directions. This IFM greatly benefited from taking place immediately following a closely related meeting “Gene Expression and RNA Processing” organized by EURASNET partners Alberto Kornblihtt and Anabella Srebrow and by Tom Misteli, and to which a number of EURASNET partners contributed. Because these two meetings were effectively integrated and run one after the other, with all participants attending both meetings, this clearly enhanced the degree of interaction and discussion that took place and added extra value to the EURASNET participants, who contributed to both meetings. A survey of key discussions that took place in these meetings is given below. The meeting was very well attended by EURASNET groups, including Reinhard Lührmann, Javier Caceres, Stefan Stamm, Angus Lamond, Jamal Tazi, Chris Smith, Karla Neugebauer, Guiseppe Biamonti, Alberto Kornblihtt, Anabella Srebrow and Tito Baralle. In addition, it was very helpful that the advisory board members Joan Steitz, Michael Rosbash, Robert Singer, Jim Dahlberg, Mariano Garcia Blanco, and Adrian Krainer also attended, made presentations and actively participated in discussions and review activities with the NoE group leaders. Many leading groups in the RNA processing and gene expression field from the USA and Europe also attended and presented their latest work, including Tom Cech, Gideon Dreyfuss, Tom Misteli, Robert Tjian, James Manley, Doug Black, Xiang-Dong Fu, Kristen Lynch, Oliver Rando, Nick Proudfoot, Melissa Moore, Jim Dahlberg, Don Rio, Tom Blumenthal, Brent Graveley, David Bentley, Benoit Chabot and Steve Buratowski. Exciting new developments in the area of cell biology that can be applied to the study of RNA splicing factors and the mechanisms of alternative RNA splicing in vivo were presented. For example, Don Rio’s lab (Berkeley, CA) has used a variety of methods to analyze alternative splicing factors in Drosophila Schneider cells, including splicing-sensitive microarrays, bioinformatic searches for splicing factor binding sites using SELEX motifs, and RIP-Chip with immunopurified nuclear RNP complexes to define binding regions on the associated pre-mRNAs using whole genome tiling microarrays. They also used RNA interference in Drosophila in conjunction with splice junction microarrays to shed light on how many alternative splicing events a given factor controls. The results are consistent with the model that sequence-specific RNA-protein recognition can occur in a combinatorial fashion to control both unique and common transcript pools that undergo alternative splicing. Many current efforts are geared toward the identification of the pre-mRNA targets of known alternative splicing factors. A lack of global experimental approaches has so far held the field back. Chris Smith (Cambridge, UK) addressed this by comparing quantitative proteomics and two different microarray platforms for the identification of alternative splicing events altered by knockdown of PTB and nPTB in HeLa cells. The double knockdown was essential, because nPTB is switched off by a PTB induced exon-skipping event that leads to NMD. Surprisingly, there was not a large degree of overlap between the events identified by the proteomic and array approaches, showing that both can provide insights into how regulatory factors like PTB impact gene expression at multiple levels. How signaling impacts alternative splicing is often mentioned but not understood in depth.

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While Doug Black (Los Angeles, CA) talked about how neuronal activity influences splice site selection, Anabella Srebrow (Buenos Aires) addressed the mechanism of feedback from extracellular matrix and growth factors to the splicing machinery. Kristen Lynch (Dallas, TX) focused on how networks of regulated splicing are controlled in response to T cell stimulation. Her lab developed an in vitro assay to recapitulate signal-induced alternative splicing of CD45 pre-mRNA and demonstrated that PSF is specifically recruited upon stimulation, leading to hyper-repression of the exon. A global investigation by microarray has provided additional examples of signal-induced splicing regulation in T cells, in which signal-regulated exons were found to share sequence elements bound by an overlapping set of factors (Ip et al., 2007). Benoit Chabot (Sherbrooke, Canada) implicated protein kinase C in the splicing repression of the 5’ splice site of the pro-apoptotic Bcl-x isoform. His group has identified a region in the Bcl-x pre-mRNA that confers responsiveness to PKC inhibitors in 293 cells. The action of several anticancer drugs, which require the same cis-acting element, suggests that this region represents a converging platform for a variety of signals. Stefan Stamm (Lexington, KY) reported that at least ten splicing factors have a protein phosphatase 1 (PP1) binding site in their RRM. This finding links well-established signaling pathways regulating the activity of PP1 to the spliceosome. Spliceosomal snRNAs hang onto a diverse constellation of shared and snRNP-specific proteins important for the assembly and stability of snRNPs as well as the assembly and function of the spliceosome, which ultimately attains the size and complexity of the ribosome at the pinnacle of its assembly cycle. The characterization of these dynamic RNPs has relied on technological developments, notably in mass spectrometry. In recent years the protein composition of snRNPs and spliceosomal intermediates has been determined and structural studies have begun. Angus Lamond (Dundee, UK) presented a novel integrative approach, which combines quantitative mass spectrometry for high throughput protein identification with quantitative measurements of protein localisation, dynamics and protein interaction partners. Stable isotope labeling was used to measure the distribution of 3,000 cell proteins between the cytoplasmic, nucleoplasmic and nucleolar compartments illustrated the power of this "spatial proteomics" technique. What has eluded splicing biochemists has been the isolation of a minimal spliceosomal complex, capable of carrying out catalysis. Reinhard Lührmann (Göttingen, Germany) described elegant biochemistry that has led to the achievement of this goal. For the first time, spliceosomal complexes affinity purified on truncated pre-mRNA substrates were shown to catalyze exon ligation in the absence of added factors. A salt-stable core complex was thereby identified and characterized, showing that splicing intermediates together with the U2, U5, and U6 snRNAs along with only a small subset of spliceosomal proteins constitutes the catalytic center. Surprisingly, all of the spliceosomal snRNPs are substantially destabilized during the assembly and function of the spliceosome. Thus, in contrast to the telomerase RNP, the protein components of snRNPs and the spliceosome position the snRNAs and pre-mRNA for catalysis. And unlike the ribosome, this dynamic behavior of the spliceosome requires that snRNPs continually be reassembled in order to function in subsequent rounds of splicing. Two talks highlighted novel aspects of how spliceosomal snRNPs are made. Gideon Dreyfuss (Philadelphia, Pennsylvania) focused on the role of the SMN complex, which guides the formation of a heteroheptameric ring of Sm proteins on U1, U2, U4, and U5 snRNAs. Having established an assay to assess the capacity of cells to assemble the Sm ring, it was shown that reduction of SMN by RNAi methods decreases the rate of snRNP assembly. that allows for in assembly (Wan et al., 2005). SMN genes are mutated in patients with spinal motor atrophy (SMA) and it has been mysterious how tissues are differentially sensitive to reduction in SMN complex levels. In an SMA mouse model, the Dreyfuss lab now finds tissue specific decreases in snRNA expression that could lead to tissue-specific changes in splicing. Adrian Krainer’s work (Cold Spring Harbor, NY), aiming to repair the SMA defect, uses an antisense-oligonucleotide (ASO)-tiling method to systematically screen for intronic regulatory elements in SMN transcripts. Targeting of identified regions with ASOs in human-SMN2-transgenic mice strongly increased hSMN2 exon 7 inclusion in transgenic animals, demonstrating the therapeutic potential of ASOs. Coming from the transcriptional angle, Karla Neugebauer (Dresden, Germany) presented data that levels of the U6 snRNA, an RNA polymerase III transcript, are determined by RNA polymerase II (Pol II) activity. Pol II was found to accumulate upstream of human U6 genes where its activity was linked to histone modifications, such as

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acetylation. Taken together, cellular levels of all protein and RNA components of the spliceosome are ultimately controlled by RNA polymerase II, while the rate of snRNP assembly likely contributes by determining their stability. The formal presentations and subsequent discussions led to many plans for future collaborations within the NOE, including new ideas for using high throughput technologies such as SILAC based mass spectrometry and deep sequencing techniques to characterise protein–protein and protein-RNA interactions that are essential for splicing and for splicing regulation. Discussions were also held to consider what scope was offered for commercial exploitation of the new technologies and the new reagents that have been developed within EURASNET, including the role of the spin out reagents and services company Dundee Cell Products. It was agreed that a follow up meeting would be held in January 2008 in Berlin, to be organised by Reinhard Lührmann, to capitalise on these discussions and to solidify and expand plans emerging from these discussions for new projects and collaborations within the NOE. List of Participants: Didier Auboeuf* (Paris), Francisco Baralle* (Trieste), Andrea Barta* (Vienna), Eric Batsché (Paris), David Bentley (Denver), Giuseppe Biamonti* (Pavia), Douglas Black (Los Angeles), Tom Blumenthal (Boulder), Graciela Boccaccio (Buenos Aires), José Luis Bocco (Córdoba), Steve Buratowski (Cambridge, USA), Javier Cáceres* (Edinburgh), Tom Cech (Boulder), Benoit Chabot (Sherbrooke), James Dahlberg (Madison), Robert Darnell (New York), Gideon Dreyfuss (Philadelphia), Xiang-Dong Fu (La Jolla), Andrea Gamarnik (Buenos Aires), Mariano García-Blanco (Durham), Mauro Giacca (Trieste), Brenton Graveley (Farmington), Joaquín Espinosa (Boulder), Klemens Hertel (Irvine), Steve Innocente (Edinburgh), Alberto Kornblihtt* (Buenos Aires), Adrian Krainer (Cold Spring Harbor), Angus Lamond* (Dundee), Reinhard Lührmann* (Göttingen), Kristen Lynch (Dallas), James Manley (New York), Javier Martínez (Vienna), Bill Marzluff (Chapel Hill), Tom Misteli (Bethesda), Melissa Moore (Boston), Karla Neugebauer* (Dresden), Franco Pagani (Trieste), Javier Palatnik (Rosario), Carol Prives (New York), Nick Proudfoot (Oxford), Oliver Rando (Worcester), Don Rio (Berkeley), Mike Rosbash (Boston), Marcelo Rubinstein (Buenos Aires), Rob Singer (New York), Chris Smith* (Cambridge, UK), Anabella Srebrow* (Buenos Aires), Stefan Stamm* (Erlangen), Joan Steitz (New Haven), Carles Suñe (Granada), Jamal Tazi* (Montpellier), Robert Tjian (Berkeley). *EURASNET Members

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Two Linked Meetings

"Gene Expression and RNA Processing" Nov 26 – 29, 2007

International Centre for Genetic Engineering and Biotechnology (ICGEB) - Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (UBA) – IFIBYNE-CONICET - Agencia Nacional de Promoción Científica y Tecnológica

(ANPCyT)

"Cell Biology, Signaling and Alternative Splicing" Nov 29 – 30, 2007

Interdisciplinary Focus Meeting of the European Alternative Splicing Network (EURASNET)

Hotel Amancay – Bariloche – Province of Río Negro – Argentina

Organizers

Invited Speakers

Topics

Participants

Deadline

Fellowships

Contact

Javier Cáceres (Edinburgh), Alberto Kornblihtt (Buenos Aires), Angus Lamond (Dundee), Tom Misteli (Bethesda) and Anabella Srebrow (Buenos Aires). Francisco Baralle* (Trieste), David Bentley (Denver), Giuseppe Biamonti* (Pavia), Adrian Bird (Edinburgh), Douglas Black (Los Angeles), Ben Blencowe (Toronto), Tom Blumenthal (Boulder), Graciela Boccaccio (Buenos Aires), José Luis Bocco (Córdoba), Steve Buratowski (Cambridge, USA), Javier Cáceres* (Edinburgh), Maria Carmo-Fonseca* (Lisbon), Tom Cech (Boulder), Benoit Chabot (Sherbrooke), James Dahlberg (Madison), Robert Darnell (New York), Gideon Dreyfuss (Philadelphia), Xiang-Dong Fu (La Jolla), Andrea Gamarnik (Buenos Aires), Mariano García-Blanco (Durham), Mauro Giacca (Trieste), Brenton Graveley (Farmington), Klemens Hertel (Irvine), Alberto Kornblihtt* (Buenos Aires), Adrian Krainer (Cold Spring Harbor), Angus Lamond* (Dundee), Reinhard Lührmann* (Göttingen), Kristen Lynch (Dallas), James Manley (New York), Tom Misteli (Bethesda), Melissa Moore (Boston), Karla Neugebauer* (Dresden), Franco Pagani (Trieste), Carol Prives (New York), Nick Proudfoot (Oxford), Oliver Rando (Worcester), Mike Rosbash (Boston), Marcelo Rubinstein (Buenos Aires), Rob Singer (New York), Chris Smith* (Cambridge, UK), Anabella Srebrow (Buenos Aires), Stefan Stamm* (Erlangen), Joan Steitz (New Haven), Jamal Tazi* (Montpellier), Robert Tjian (Berkeley), Juan Valcárcel* (Barcelona), Martín Vázquez (Buenos Aires) * EURASNET Members

Chromatin and nuclear organization, transcriptional regulation, RNA processing, transcription/processing coupling, mechanisms of mRNA splicing and regulation of alternative splicing

Registration is limited to 60 participants selected from applications which should include a complete application form, a max. 3-page CV and a 300-word abstract in English intended for a poster presentation. Application forms can be requested by e-mail or obtained from the website XXXXXXXXXXXX. Registration fee plus full board accommodation (4 nights, twin share) at the Hotel Amancay is US$ 400. Please send applications to silceriani@gmail.com before 15 June 2007.

Forty fellowships covering the registration fee plus full board accommodation (4 nights, twin share) at the Hotel Amancay and partial reimbursement of the transportation Buenos Aires - Bariloche - Buenos Aires are available. Preference will be given to graduate students and post- docs.

Meeting manager: Miss. Silvina Ceriani. Email: silceriani@gmail.com.

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BARILOCHE TIMETABLE

MONDAY 26 NOV.

TUESDAY 27 NOV.

WEDNESDAY 28 NOV.

THURSDAY 29 NOV.

FRIDAY 30 NOV.

9:00-9:30 9:30:10:00

10:00-10:30 10:30-10:45 10:45-11:00

Mauro Giacca Marcelo Rubinstein

José Luis Bocco Joaquín Espinosa*

Carol Prives*

8:30-9:00 9:00-9:30

9:30-10:00 10:00-10:30 10:30-10:45

Klemens Hertel

Karla Neugebauer Alberto Kornblihtt Michael Rosbash Steve Innocente*

9:00-9:30 9:30-10:00 10:00:10:30 10:30-10:45

Tito Baralle Chris Smith

Benoit Chabot Andrea Barta*

9:00-9:30 9:30-10:00

10:00-10:30

Reinhard Lührmann Stefan Stamm

Jamal Tazi

11:00-11:30 COFFE BREAK 10:45-11:15 COFFE BREAK 10:45-11:15 COFFE BREAK 10:30-11:00 COFFE BREAK

11:30-12:00 12:00-12:30 12:30-13:00

Gideon Dreyfuss

Graciela Boccaccio Joan Steitz

11:15-11:30 11:30-11:45 11:45-12:15 12:15-12:45 12:45-13:15

Eric Batsché*

Didier Auboeuf* Steve Buratowski

Nick Proudfoot David Bentley

11:15-11:45 11:45-12:00 12:00-12:15 12:15-12:45

Javier Cáceres Javier Palatnik* Javier Martínez*

Tom Steitz

11:00-11:30 11:30-12:00 12:00-12:30 12:30-13:00

Adrian Krainer Xiang-Dong Fu Kristen Lynch

Anabella Srebrow

10:00-16:00

REGISTRATION

13:00-15:00 LUNCH 13:00-15:00 LUNCH 13:00 LUNCH ICGEB MEETING

Gene expression and RNA processing

EURASNET MEETING

Cell Biology, signaling and alternative splicing

16:00-17:00

Opening lecture

Tom Cech

15:00-15:30 15:30-16:00 16:00-16:30 16:30-16:45

Andrea Gamarnik Tom Blumenthal Brent Graveley

Don Rio*

15:00-15:30 15:30-16:00 16:00-16:30 16:30-17:00

Giuseppe Biamonti Mariano G. Blanco

Doug Black Franco Pagani

17:00-17:15 Short break 16:45-17:15 COFFE BREAK 17:00-17:30 COFFE BREAK 17:15-17.45 17:45-18:15 18:15-18:45

Robert Tjian Tom Misteli

Oliver Rando

17:15-17:45 17:45-18:15 18:15-18:45 18:45-19:00

Melissa Moore Jim Dahlberg Jim Manley

William Marzluff*

13:30-18:30

BOAT TRIP

17:30-18:00 18:00-18:30 18:30-19:00

Rob Singer

Angus Lamond Maria Carmo Fonseca

19:00-20:30 CONCERT 19:30-21:00 DINNER 19:30-21:00 DINNER 19:00-21:00 POSTER SESSION 20:30 WELCOME

RECEPTION 21:00-onwards POSTER SESSION 21:00-

onwards POSTER SESSION 21:00 “ASADO”

(BARBECUE DINNER)

*Short talks

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3) EURASNET Interdisciplinary Focus Meeting Biophysical Methods and Functional Genomics Tools in Alternative Splicing , Berlin Jan 20 – 23, 2008 Harnack Haus der Max-Planck-Gesellschaft Program Sunday Jan 20

18.30 – 20.00 Dinner

20.00 – 22.00 Evening session:

Work packages WP7 Molecular Characterization of Splicing Substrates – C. Branlant, WP9 Complexity of Spliceosomal Proteomes – R. Lührmann, WP10 Post-translational Modification and Dynamic Regulation – J. Cáceres

Monday Jan 21 Session on biophysical methods 9.00 – 9.30 Marina Rodnina, University of Witten Through the kinetics window: looking at the ribosome functions by fluorescence and FRET 9.30 – 10.00 Stefan Jacobs, MPIBPC Göttingen High resolution microscopy: Looking into mitochondria 10.00 – 10.30 Thomas Orth, TU Dresden

Elucidating the RNA interference pathway in human cells by FCS and FCCS in vivo

10.30 – 11.00 Manfred Roessle, EMBL Hamburg Biological Small Angle Scattering - Low Resolution Protein Structure Determination in Solution

11.00 – 11.30 Coffee break 11.30 - 13.00 Continuation of discussions on WPs 7, 9, 10

WP13 Co-transcriptional Mechanisms of Alternative Splicing – K. Neugebauer

13.00 – 14.30 Lunch 14.30 – 18.00 WP8 Genome-wide Analyses of Splicing Regulation – C. Smith WP6 In silico approaches to alternative splicing – G. Ast WP4 The Alternative Splicing Database – S. Stamm (Coffee break 16.30 – 17.00) 18.00 – 19.00 WP14 Chemical Biology and Therapeutics – J. Tazi/A. Lamond 19.00 – 20.30 Dinner

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20.30 – 22.00 WP16 Conferences and meetings – R. Lührmann for J. Beggs WP18 Career Development – J. Kjems WP19 Public Understanding of RNA Biology – A. Barta WP20 SMEs and Technology Transfer – A. Lamond Tuesday Jan 22 9.00 – 10.30 WP1 The EURASNET Web site – J. Brown

WP2 Sharing Resources, Technology and Reliable Protocols – I. Eperon

WP17 Staff Exchange and Training – A. Krämer 10.30 – 11.00 Coffee break 11.00 – 13.00 WP12 Mis-splicing and Disease – F. Baralle 13.00 – 14.30 Lunch 14.30 – 16.30 WP12 Mis-splicing and Disease (continued) WP11 Function of Splicing Factor Isoforms – J. Valcárcel WP15 Development of Enabling Technologies – B. Séraphin 16.30 – 17.00 Coffee break 17.00 – 17.30 Holger Stark, MaxPlanck Institute Göttingen Studying dynamics of macromolecular complexes by cryo-EM

17.30 – 19.00 WP21 Reachout to the Broader RNA Community – J. Brown

19.00 – 20.30 Dinner 20.30 – 22.00 WP5 Ensuring Durability – M. Carmo-Fonseca WP22 Management – R. Lührmann Final discussions and conclusions Wednesday Jan 23 Departure after breakfast Report of discussions and decisions The meeting in Berlin served a dual purpose. On one hand network members had the opportunity to familiarize themselves with state of the art biophysical methods that can readily or with minor modifications be applied to research in alternative splicing. The second purpose of this PI-only meeting was an intense brainstorming on the future directions of the network. Given that the 2007 report is due, this was an excellent opportunity to reflect on the experience of the previous two years and to decide on the redesign and reorientation of work package tasks, to discuss weaknesses and to make decisions on new deliverables in advance of this report. Although this was a meeting arranged at short notice, attendance was excellent, with 35 of 40 members present. In the biophysical session Marina Rodnina provided deep insights into our gradually advancing knowledge of the kinetics of the ribosomal machinery using fluorescence technologies. The research on spliceosomal complexes will profit from the approaches

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and experiences collected with this other important protein-RNA complex. Stefan Jacobs gave a presentation of state of the art technology in high resolution microscopy, which recently advanced into resolution ranges unheard of only a few years ago. These studies can possibly be adapted to the needs of studies localizing spliceosomal complexes in the living cell. Thomas Orth provided an overview of recent studies in the molecular biology of the RNA interference pathway, a technology which is already widely used in alternative splicing research. Manfred Roessle provided an overview on the technique of small angle scattering in biological research. This technique can provide data sets of particle structures measured in solution. Such data sets can provide a valuable complement for data generated with other structure elucidation techniques. Holger Stark, one of the leading experts in the development of cryo-electronmicroscopy and in spliceosomal structures, gave an overview how the rapidly evolving computing power will revolutionize the picture processing in this type of EM and how this will also affect splicing research. During the PI-session particular emphasis was given to a discussion of rapidly developing technologies in in silico approaches and the genome-wide analysis of alternative splicing. The updates provided by network members Ast and Smith had an important impact on the future design of work packages 6 and 8 and the deliverables therein. An additional focus was the impact alternative splicing has on the understanding of disease processes and possible therapeutic approaches. Members Tazi, Baralle and Schümperli provided updates on progress made in these approaches. Network member Lamond instructed the audience on how to possibly commercialize results and knowledge created during EURASNET research, this based on his extensive experience of such commercialization activities at the University of Dundee. New YIP member Diana Baralle gave profound insights into the mechanisms and structures of the medical and genetic research community and how it would be possible to establish close contacts and cooperation with this community. This discussion as well had an immediate impact on the planning of future events and deliverables. Furthermore, the new, improved design of the EURASNET presence was discussed in conjunction with the new initiatives in Public Understanding of RNA Biology activities and reach out activities to the broader RNA community (Brown, Barta). Member Carmo-Fonseca gave an extensive overview on funding opportunities within the 7th Framework. The Coordinator R. Lührmann used the opportunity to instruct the network members on the task of efficiently compiling the 2007 Annual Report.

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REPORTS ON WORKSHOPS HELD IN 2007

1) EURASNET Workshop on Alternative Splicing in Plants

Friday May11th 2007

Carry-le-Rouet, France

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EURASNET Workshop on Alternative Splicing in Plants The workshop was organised by John Brown, Andrea Barta and Artur Jarmolowski. The aims of the workshop were to:

• Raise awareness of the importance of alternative splicing among plant scientists; • Demonstrate the need for increased research in this area; • Present the work of the EURASNET members working on plant alternative splicing; • Describe the particular issues facing plant scientists in understanding alternative

splicing and its effects and consequences. The workshop was arranged to coincide with an international meeting of plant scientists involved in post-transcriptional regulation of gene expression. The meeting was the 6th in a series of meetings entitled “Post-transcriptional Regulation of Plant Gene Expression” which is held every two years. As such, the workshop was particularly relevant and provided an ideal opportunity to target plant scientists from Europe and around the world working on RNA control (Annex I). The workshop consisted of a series of seminars from EURASNET members and invited speakers followed by round table discussions. The programme of seminars (Annex II) was designed to provide an introduction to alternative splicing followed by detailed research and issues facing plant scientists in this area. In particular, Jamal Tazi (EURASNET) gave a presentation on alternative splicing in animals and disease. This put the whole programme into context by describing examples of splicing-related diseases and introducing gene therapy and the discovery of chemicals which specifically disrupt splicing. Robert Fluhr and Craig Simpson (EURASNET) presented methods of studying alternative splicing of multiple genes which was followed by a presentation on bioinformatics of alternative splicing from Dirk Holste. Dirk Holste presented work on bioinformatic analysis of animal alternative splicing and approaches relevant to plants – this again reinforced the importance of alternative splicing in eukaryotes and the current state of play for plants. Talks from Mariya Kalyna (EURASNET) and Artur Jarmolowski (EURASNET) highlighted the work of the plant EURASNET groups and their joint project in analysing the effects of trans-acting factors on alternative splicing. The programme was rounded off by talks looking at the interaction between splicing and alternative splicing and processing of intronic non-coding RNAs, and trans-splicing in chloroplasts (Echeverria and Goldschmidt-Clermont). Following the seminars, there were three round table discussions for interested parties. The discussions were on bioinformatics of plant alternative splicing, methods to quantify changes in alternative splicing (tiling arrays, RT-PCR) and a session for the EURASNET labs only to plan experimentation. Bioinformatics of plant alternative splicing. Databases exist for alternative splicing in plants. However, these are incomplete and the work of the plant EURASNET groups has identified many novel alternative splicing events in a relatively small number of analysed genes. On this basis, it is clear that current estimates of the prevalence of alternative splicing in plants are definitely underestimates. This is largely due to relative low numbers of ESTs. Thus although bioinformatic approaches are available and are being further developed for animal systems (Dirk Holste), the major bottleneck in alternative splicing in splicing in

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plants is the discovery of AS events. Discussion then covered high-throughput sequencing techniques (454; Solexa) and how these could be applied to discovering AS events. Methods to quantify changes in alternative splicing. Robert Fluhr has used both the whole genome tiling arrays for Arabidopsis and the new Affymetrix tiling arrays. The whole genome arrays were good at defining intron retention but had difficulty in detecting exon skipping and alternative 5’ and 3’ splice site selection. The Affymetrix tiling arrays had similar results and required extensive bioinformatics to access the hybridization data for individual oligos along the length of a gene. RF had to prepare his own programmes to examine the data – these are not very transferable. The utility of the tiling arrays to be able to quantify changes in alternative splicing will clearly require a great deal of development, refinement and validation. The RT-PCR method developed by the EURASNET groups is reproducible and able to monitor changes in alternative splicing but is limited to a few hundred events. Both methods are limited by the lack of knowledge of alternative splicing events coming back to the need for an extensive discovery phase. Ultimately a high-throughput system able to quantify alternative splicing changes is required and will probably involve elements of both systems. EURASNET labs – experimentation. Members of the three plant labs discussed details of planned experiments using the RT-PCR AS panel, data analysis issues and RNA preparation. Reaction to the Workshop We received only positive reactions to the EURASNET Workshop on Alternative Splicing. Many of the comments expressed new interest and excitement in the area and that alternative splicing as a regulator of gene expression is very unappreciated. Many attendees had noticed that their genes of interest are alternatively spliced and discussed their observations with members of the EURASNET labs. Two groups have also contacted us to work together on particular alternatively spliced genes. Conclusions The Workshop fulfilled all of its objectives in raising awareness of the importance of alternative splicing and of some of the issues in alternative splicing among plant scientists. The mixture of presentations on plant and animal systems was extremely valuable and the contribution of the EURASNET labs was a key factor in covering the breadth of interest in alternative splicing.

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ANNEX I - Participants Alboresi, A. Marseille, France Bailet-Serres, J. California, USA Barta, A. Vienna, Austria Brown, J. Dundee, Scotland, UK Burgyan, J. Godollo, Hungary Caffarri, S. Marseille, France Caranta, C. Montfavet, France Chan, E. Auckland, New Zealand Choquet, Y. Paris, France Crespi, M. Gif-sur-Yvette, France Crete, P. Marseille, France Dalmas, F. Marseille, France Devert, A. Marseille, France Dielen, A.S. Bordeaux, France Doonan, J. Norwich, UK Dunoyer, P. Strasbourg, France Echeverria, M. Perpignan, France Etheve, N. Perpignan, France Fabres, N. Marseille, France Fray, R. Nottingham, UK Gagliardi, D. Strasbourg, France Goldschmidt-Clermont, M. Geneva, Switzerland Haraguchi, Y. Hokkaido, Japan Hienerwadel, R. Marseille, France Hohn, T. Basel, Switzerland Holste, D. Vienna, Austria Huang, J. St. Louis, USA Jarmolowski, A. Poznan, Poland Kalyna, M. Vienna, Austria Kawamura, K. Nara, Japan Kockerols, M. Marseille, France Kruszka, K. Warsaw, Poland Kufel, J. Warsaw, poland Lacombe, S. Montpelier, France Lagrange, T. Perpignan, France Lanet, E. Marseille, France Laporte, P. Gif-sur-Yvette, France Laufs, P. Versailles, France Lecampion, C. Marseille, France Lewandowska, D. Dundee, Scotland, UK Makelainen, K. Helsinki, Finland Makinen, K. Helsinki, Finland Mallory, A. Versailles, France Marin, E. St Paul-lez-Durance, France Maule, A. Norwich, UK Meyer, C. Versailles, France Miller, W. Iowa, USA Moreau, M. Versailles, France

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Pacak, A. Poznan, Poland Pierrat, O. Norwich, UK Pontvianne, F. Perpignan, France Redondo, E. Clermont-Ferrand, France Robaglia, C. Marseilles, France Ryabova, L. Strasbourg, France Shchepetilnikov, M. Strasbourg, France Silhavy, D. Godollo, Hungary Simpson, C. Dundee, Scotland, UK Small, I. Perth, Australia Sobkowiak, L. Poznan, Poland Song, H.-S. North Carolina, USA Szarzynska, B. Poznan, Poland Taliansky, M. Dundee, Scotland, UK Tazi, J. Montpelier, France Terzi, L. Dundee, Scotland, UK Thiebauld, O. Strasbourg, France Vaucheret, H. Versailles, France Veit, B. Palmerston North, New Zealand Wypijewski, K. Dundee, Scotland, UK Zakrzewska, M. Warsaw, Poland Ziegler-Graff, V. Strasbourg, France

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ANNEX II

EURASNET Workshop on Alternative Splicing in Plants

Friday 11th May, 2007

Programme Chairperson: Artur Jarmolowski (Poznan) Alternative splicing in plants and animals 9.00 – 9.15 Andrea Barta (Vienna) Introduction to splicing and alternative splicing in plants 9.15 – 9.45 Jamal Tazi (Montpellier) Alternative splicing in animals and disease Analysis of plant alternative splicing 9.45 – 10.15 Robert Fluhr (Rehovot) Dynamics of plant alternative splicing as viewed by comparative dbEST and Tiling array analysis. 10.15 – 10.30 Craig Simpson (Dundee) An RT-PCR panel to monitor multiple AS events Coffee Chairperson: Andrea Barta (Vienna) Bioinformatic approaches to alternative splicing 11.00 – 11.25 Dirk Holste (Vienna) How computational sequence analysis can help to understand RNA splicing specificity Trans-acting factors 11.25 – 11.40 Maria Kalyna (Vienna) Highly conserved alternative splicing events in plant specific SR protein genes 11.40 – 11.55 Artur Jarmolowski (Poznan) The role of nuclear cap-binding protein complex in alternative splicing of plant pre-mRNA Other splicing systems 11.55 – 12.10 Manuel Echeverria (Perpignan) Intronic non-coding RNAs and splicing 12.10 – 12.30 Michel Goldschmidt-Clermont (Geneva) Trans-splicing in chloroplasts Lunch

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2.30 – 4.30 Round table discussions (Chaired by John Brown, Andrea Barta, Artur Jarmolowski) 1) Bioinformatics of plant alternative splicing 2) Methods to quantify changes in alternative splicing (tiling arrays, RT-PCR) 3) Experimental planning (EURASNET labs)

2. EURASNET Workshop: “Alternative Splicing and Bioinformatics”

Vienna, Austria, July 19-20, 2007

Organised by Eduardo Eyras and Andrea Barta

OVERVIEW Alternative RNA splicing (AS) does not fit one drawer alone, but is about the interplay of genomes and genomics approaches, regulatory networks, and evolution. It has emerged as a ubiquitous and dynamic mechanism of genetic regulation, and this workshop aimed to establish a permanent forum to meet and discuss about this exciting research topic collaboratively. This workshop was somewhat special, because it was held immediately prior to both the annual International Conference on Intelligent Systems in Biology (ISBM is the major ISCB conference) and the European Conference on Computational Biology (ECCB), July 21-27. The centre of Vienna provided a fitting location to disseminate research and interact with the international research community. The meeting was organized in four major sessions: I) Regulatory mechanisms of splicing, II) Evolution of alternative splicing, III) Alternative splicing and function, and IV) Genomics and bioinformatics of splicing. The program featured twenty-one talks and included one poster session with about 20 posters, which took place at the end of the first day. There were six plenary talks, including three EURASNET speakers: Juan Valcarcel (Centre de Regulacio Genomica, Barcelona), Larry Chasin (Columbia U, New York), Ben Blencowe (U Toronto, Ontario), Christopher Smith (Cambridge U, England), Al Zahler (U Santa Cruz, California), and Jamal Tazi (U Montpellier II, Montpellier). In addition, early career researchers and students were provided the opportunity to present their work, either as an oral or as a poster presentation. The selection of the short talks and posters was conducted by organizing submitted abstracts into main sections and establishing a reviewing process. Based on the interest of talks and posters, interaction between scientists and the very positive received feedback, we can report that the workshop was a highly successful event. SUMMARY OF TALKS We describe next some of the presentations in the meeting. In his plenary talk, Larry Chasin discussed the occupation of RNA with splicing cis-regulatory exon motifs. The talk discussed several aspects of the identification and function of such motifs in primates and investigated their evolution, which was indicative of purifying selection against the disruption (creation) of enhancer (silencer) motifs. He further described the design, utilization and initial analysis of synthetic exons, to reengineer the mechanism of the “splicing code” in a simplified construct that demonstrated complex AS patterns from the combination of motifs in various permutations studies. Chris Smith presented and discussed selected examples of “AS-NMD switches” that act in the case where regulated splicing leads to the production of mRNAs that are degraded via the NMD pathways, and further illustrated a global approach for analysis of AS that incorporated the use of quantitative proteomics to study the consequences of splicing factor knockdown. Different model systems were used by Alan Zahler

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and Maria Kalyna, who investigated patterns and mechanisms of AS in nematodes (C. elegans) and plants (A. thaliana), respectively. Alan Zahler’s talk addressed the utilization of phylogenetic conservation of sequences flanking AS exons to identify motif likely to function in splicing regulation, which led to new motifs with subsequent experimental confirmation, in addition to the identification of their nuclear RNA binding proteins. The talk further discussed splicing-sensitive DNA microarrays to survey several hundred exon-skipping events during nematode development, showing interesting examples of developmental stage-specific regulation, as well as to explore changes in splicing in strains carrying mutations in AS regulatory proteins. Jamal Tazi’s talk was related to the human “RNA pathologies” as a critical feature of disease-causing mutations in exons and introns that disrupt splice sites or splicing auxiliary motifs. He described a new small-molecules approach to target splicing factors, the outcome of which identified several promising molecules that affect individual SR proteins. These methodologies are now under further development to innovate therapeutics correcting aberrant splicing in pathological events. Michael Hiller discussed in his talk the importance of the structural context of binding sites for splicing regulatory proteins and investigated splicing enhancing and silencing motifs, by analysis of the extent of base pairing within a motif. He provided computational and experimental evidence for the preference of such motifs for single strandedness, by comparing the preference for verified motifs with artificially simulated data and testing predictions in an experimental minigene system. Ralf Bortfeldt and Karol Szafranski discussed non-canonical patterns and their implications, by using computational sequence analysis and experimental validation assays to demonstrate the frequent utilization of tandem donors with overlapping splice site information (RB) or employment of TG/ dinucleotides as splice sites in U2-dependent introns (KS). Both Mihaela Zavolan and Uwe Ohler discussed in their talks the lines of evidence for the association between transcription initiation and internal splicing (MZ) and the possible implications between transcript diversity at 5'- and 3'-ends (UO), the latter including the accessibility of microRNA binding sites. Databases for AS often stand at the beginning of our investigations and the continuous development and upgrades of such data repositories are an important topic on their own right. Three talks, given by Elenore Whitfield (for the ASTD platform), Graziano Pesole (ASPIC software), and Shoba Ranganathan (database for G. gallus), reported about the latest developments in data integration efforts. More specifically, Graziano Pesole described the abundance of isoforms in the human ENCODE genes and their potential to expand the proteome by about one order of magnitude.

ANNEX I – List of participants (81) Last Name First Name Organization Barta Andrea MFPL, U Vienna Blencowe Benjamin University Of Toronto Bortfeldt Ralf H. Friedrich Schiller University Calley John Lilly Research Labs Caragea Doina Kansas State University Castle John Rosetta Inpharmatics / Merck & Co Chasin Lawrence Columbia University, NY Clark Tyson Affymetrix, Inc Courtot Melanie CERTH - Institute Of Agrobiotechnology Daemen Anneleen K.U.Leuven D'Antonio Lawrence Ramapo College Of New Jersey Dawe Adam University of Western Cape De Sousa Corvelo André Pompeu Fabra University De Souza Sandro Ludwig Institute For Cancer Research

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Dixon Richard Leicester University Dong Xianjun Bergen University Ermakova Ekaterina Institute For Information Transmission Problems Eyras Eduardo Pompeu Fabra University Ferreira Elisa Napolitano Hospital AC Camargo Gautheret Daniel Université Paris Sud Gelfand Mikhail Institute For Information Transmission Problems, RAS Grosse Ivo IPK Gatersleben Hegyi Hedi Inst Of Enzymology Hellmann Ines University Of Copenhagen Hiller Michael University Freiburg Holste Dirk Institute of Molecular Pathology Hotz-Wagenblatt Agnes German Cancer Research Center (DKFZ) Kalyna Maria MFPL, Vienna Biocenter Kane Vanessa College Of Science Keilwagen Jens IPK Gatersleben Kim Nam Shin Molecular Biology Institute, University of California, Los A Koscielny Gautier European Bioinformatics Institute Kull Meelis University Of Tartu Langer Wolfram Bayer Schering Pharma AG Mattick John U Queensland Majewski Jacek McGill University Mangiulli Marina University Of Bari-Italy Mollet Ines Instituto de Medicina Molecular Morre D. James Purdue University Morre Dorothy Purdue University Muino Jose M Institute of Plant Genetics-PAN Nahkuri Satu Imb Nakken Sigve Centre For Molecular Biology And Neuroscience & Institute Of Ohler Uwe Duke University Ong Cheng Soon Friedrich Miescher Lab. And MPI For Biol. Cybernetics Pawlowski Marcin International Institute Of Molecular And Cell Biology In War Pesole Graziano University Of Bari Ranganathan Shoba Macquarie University Rasche Axel MPI Molecular Genetics Rätsch Gunnar Friedrich Miescher Laboratory Richardson Dale University Of Cologne Riethoven Jean-Jack University Of Nebraska-Lincoln Ritchie William Inserm Satagopam Venkata P. Embl Schramm Gerrit Roche Diagnostics Schweitzer Anthony Affymetrix Seemann Stefan Faculty Of Life Sciences, University Of Copenhagen Severing Edouard Ibert Wur Shia Wei-Chung Feng Chia University Shin Youngah Ewha Womans Univ. Shovlin Claire Imperial College London Sinha Rileen Leibniz Institute For Age Research - Fritz Lipmann Institute Smith Christopher Cambridge University Sonnenburg Soeren Fraunhofer Institute FIRST Souvorov Alexandre National Center For Biotechnology Information

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Szafranski Karol Fritz Lipmann Institute - Leibniz Institute For Age Research Tazi Jamal University Of Montpellier

Thangavel Alphonse Thanaraj CRS4

Tomiuk Stefan Miltenyi Biotec GmbhH Tress Michael Liam Cnio Valcárcel Juan Center For Genomic Regulation Van Ham Roeland Plant Research International Vukusic Ivana University Of Cologne/ Institute For Genetics Whitfield Eleanor European Bioinformatics Institute Woodwark K. Cara Wellcome Trust Sanger Institute Wright Frank Biomathematics & Statistics Scotland Zahler Alan University Of California At Santz Cruz, CA Zavolan Mihaela Biozentrum Basel

Organizing committee Invited speaker Additional session chair

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3. EURASNET Workshop: High Throughput Applications in Biology

Edinburgh, Scotland, September 4-5, 2007.

Organised by Jean Beggs

REPORT The purpose of this workshop was to provide an update on recent developments in high throughput technologies and applications that might be useful to members of the NoE. Although this was an independent workshop, it benefited from taking place immediately after the Inaugural Symposium of the Edinburgh Centre for Systems Biology. The Symposium gave valuable insights into systematic approaches to studying metabolic processes at the genomic and whole cell level, as well as developments in the emerging field of synthetic biology. The EURASNET workshop included presentations on a number of high throughput approaches relevant to RNA and RNA-protein interaction studies. High throughput sequencing and alternative splicing: At the EURASNET workshop, there were representatives present from several companies with displays of equipment, including robots for large-scale genetic screens (Singer) and for massive parallel sequencing (Roche 454/FLX). Jean Beggs and John Brown had discussions with the representatives from Roche about the possibilities for analysing alternative splicing by massive parallel sequencing of cDNAs. As a consequence, Roche has agreed to perform two trial experiments, using mRNA from plant cells, provided by John Brown (Dundee), and from human cells, provided by Chris Smith (Cambridge). This will be extremely helpful in assessing this new technology for splicing studies. In particular, the results from the analysis of the human RNA will be directly comparable with the results from the Affymetrix microarray trials. It is hoped that some of these results may be presented at the meeting in Krakow. Overview of the workshop presentations: Christina Smolke, California Institute of Technology, USA. “Biomolecular engineering/ Riboswitches” Professor Smolke gave a stunning presentation on how to use RNA molecules that function as riboswitches to control gene expression. She is using these as tools to devise new metabolic pathways. An example of a riboswitch controlling alternative splicing was recently published in Nature. David Baulcombe, University of Cambridge, UK. “Small RNA silencing Networks in Plants” Professor Baulcombe described the discovery of complex networks of small RNAs that control plant development. He presented evidence for a link between RNA silencing and the control of flowering that involves modification of chromatin structure and gene silencing at specific loci. Interestingly, factors have recently been found that seem to be involved in both mRNA splicing and RNAi regulation of heterochromatin. Jussi Taipale, University of Helsinki, Finland. “Systematic RNAi screens for cell cycle and growth regulators” Professor Taipale presented vast amounts of data his group has produced to investigate Drosophila development, in particular, the aberrant activation of the Hedgehog and Wnt signaling pathways that lead to tumorigenesis. His group has set up a high throughput centre, which will allow somatic cell genetic analysis of 10 000- 40 000 samples using RNAi or cDNA expression. Mark Bradley University of Edinburgh, UK. “High throughput chemical biology”

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Professor Bradley gave an outstanding talk about how combinatorial chemical and peptide libraries can be used in molecular zip-coded arrays, and the development of novel polymers as surface coatings on chips to permit the highly specific selection of particular cell types. There are endless applications for these tools, especially for the analysis of peptide interactions or identification of RNA binding peptides. Jamal Tazi, CNRS, Montpellier, France. “Small molecule screens for splicing inhibitors” Professor Tazi explained how pre-mRNA splicing can cause disease, due to altered dosage of splicing factors or to sequence changes in target genes. He described high throughput screens to identify small molecule inhibitors that may lead to new disease therapies. Paul Ko Ferrigno, Leeds Institute of Molecular Medicine, UK. “Towards miniaturised, highly multiplexed electronic protein microarrays” Dr Ko Ferrigno explained how very large peptide libraries containing more than 108 random 10-20mer peptides have been created in the form of peptide aptamers that can have highly specific affinity for binding to target molecules. He also explained how these can be arrayed and electronically addressed for highly sophisticated lab-on-a-chip experiments. Partner J. Beggs has subsequently agreed a collaboration with Dr Ferrigno to search for peptide aptamers that affect U5 snRNP development and splicing.

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SYMPOSIUM: FROM SYSTEMATIC TO SYNTHETIC BIOLOGY UNIVERSITY OF EDINBURGH

MICHAEL SWANN BUILDING, KING’S BUILDINGS

3rd to 5th September 2007

Organisers: Mike Tyers and Jean Beggs

Monday 3rd September 17.15 Inaugural Lecture: Mike Tyers,

CH Waddington Chair of Systems Biology. “Charting the Genetic Landscape”

Inaugural Symposium of the Centre for Systems Biology at Edinburgh

Tuesday 4th September 09.00 – 09.30 Registration 09.30 – 10.10 Andrew Millar, University of Edinburgh, UK.

“Unwinding the biological clock with systems biology”

10.10 – 10.50 Drew Endy, MIT, Cambridge, USA.

“Synthetic biology” 10.50 – 11.20 Break 11.20 – 12.00 Mike Tyers, University of Edinburgh, UK.

"Size control: a systems level problem" 12.00 – 12.40 Joerg Stelling, ETH, Zurich, Switzerland.

“Analysis and synthesis of biological networks” 12.40 – 14.10 Lunch Sponsored by Roche Posters and Sponsors’ displays

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14.10 – 14.50 Angelika Amon, MIT, Cambridge, USA. “Systematic analysis of aneuploidy”

14.50 – 15.30 Hans Lehrach, MPI of Molecular Genetics, Berlin, Germany.

“Vertebrate genomics” 15.30 – 16.10 Break EURASNET Workshop: High Throughput Applications in Biology

16.10 – 16.50 Christina Smolke, California Institute of Technology, USA.

“Biomolecular engineering/ Riboswitches” 16.50 – 17.30 David Baulcombe, University of Cambridge, UK.

“Small RNA silencing Networks in Plants” 19.00 EURASNET Dinner Wednesday 5th September 09.30 – 10.10 Jussi Taipale, University of Helsinki, Finland.

“Systematic RNAi screens for cell cycle and growth regulators” 10.10 – 10.50 Mark Bradley University of Edinburgh, UK.

“High throughput chemical biology” 10.50 – 11.20 Break 11.20 – 12.00 Jamal Tazi, CNRS, Montpellier, France.

“Small molecule screens for splicing inhibitors” 12.00 – 12.40 Paul Ko Ferrigno, Leeds Institute of Molecular Medicine, UK.

“Towards miniaturised, highly multiplexed electronic protein microarrays” SPONSORS:

Singer Instruments

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ANNEX II - Participants

EURASNET WORKSHOP: High Throughput Applications in Biology

4th & 5th September 2007

Tatsiana Auchynnikava University of Edinburgh Welllcome Trust for Cell Biology Edinburgh UK Pablo Arrisi MRC Human Genetics Unit Chromosomes and Gene Expression Edinburgh UK David Barrass University of Edinburgh WCCB Edinburgh UK Jean Beggs University of Edinburgh Edinburgh UK John Brown Scottish Crop Research Institute Genetics Dundee UK Javier Caceres MRC Human Genetics Unit Chromosomes and Gene Expression Edinburgh UK Olivier Cordin University of Edinburgh ICB Edinburgh UK Vanessa Cristão University of Edinburgh ICB Edinburgh UK Jochen Deckert Max Planck Institute of Biophysical

Chemistry Department of Cellular Biochemistry Göttingen Germany

Parastoo Ehsani University of Edinburgh ICB Edinburgh UK Richard Grainger University of Edinburgh ICB, Swann building Edinburgh UK David Greenberg Hebrew University of Jerusalem Biological Chemistry Jerusalem Israel Michael Grote Max Planck Institute for Biophysical

Chemistry Cellular Biochemistry Goettingen Germany

Sonia Guil MRC Human Genetics Unit Chromosomes and gene expression Edinburgh UK Daniela Hahn University of Edinburgh Cell Biology Edinburgh UK Steve Innocente University of Edinburgh ICB Edinburgh UK Jenny Long MRC Human Genetics Unit Chromosomes and Gene Expression Edinburgh UK Dasa Longman MRC Human Genetics Unit Human Genetics Unit Edinburgh UK Martin Reijns University of Edinburgh Wellcome Trust Centre for Cell Biology Edinburgh UK Timur Samatov Max Planck Institute for Biophysical

Chemistry Department of Cellular Biochemistry Göttingen Germany

Craig Simpson Scottish Crop Research Institute Genetics Dundee UK Jamal Tazi CNRS Montpellier France

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Work Package 17 Staff Exchange and Training Lead Participant: Angela Krämer

month planned achieved deliverables: 13 By month 18, to have provided the equivalent of 40 PMT

of training, collectively by network laboratories (person month training)

18 partially

14 First Workshop on RNA biology techniques by month 12 12 postponed 116 Continue and further develop the use of travel bursaries for

student and postdoc exchanges (also for necessary intra-workpackage meetings)(40 PMT/18 month) (month 30).

30 partially

Objectives of the work package • To increase the technical competence of PhD students and postdocs in the EURASNET by giving them opportunities to learn state-of-the-art techniques in other member laboratories.

• To encourage staff exchange among EURASNET laboratories for the purpose of training specific techniques.

• To promote scientific collaborations, in particular those of a multidisciplinary nature, by supporting the exchange of personnel between EURASNET member laboratories. Short description of activities 13 PMT provided by network laboratories. The number of staff exchanges increased from seven in 2006 to 12 in 2007 resulting in 5.2 PMT. Twelwe PhD students and post-docs from seven member groups in Germany, Sweden, Italy, France, Portugal, Poland and Argentina (1b, 4, 10a, 12c, 16, 18, 20) visited five groups in Germany, Spain and the UK (1a, 2, 9, 14, 21) for purposes of training and/or collaborations (for details see Table). All participants submitted short reports after the exchange and especially the younger participants were very enthusiastic about their training experience abroad. 116 Develop the use of travel bursaries for student and postdoc exchanges. Given the low number of PMT for staff exchanges in 2006, EURASNET members, SAB and the external reviewer suggested at the 2007 annual meeting that travel bursaries could be requested for small intra-workpackage meetings, whenever the complexity of workpackage projects and discussions among participants required such meetings. Two intra-workpackage meetings were held in 2007. Travel bursaries were requested by nine participants from five network laboratories for the ExonArray Workshop in Cambridge, UK. Another eight bursaries from three groups were requested for participation of the Plant Intra-Workpackage Meeting in Dundee, UK. One group recruited through the YIP in 2007 was present with two collaborators at Cambridge. These activities add up to 1.6 PMT. Together with the staff exchange and training in 2006 a total of ca. 14 PMT has been achieved by month 24, which is well below the original goal for deliverable 13 of 40 PMT/18 months. Problems and explanations for delays, postponements etc. 13 and 116 Although the number of exchanges increased during the second year of existence of the network as predicted in the report of 2006, the goal of 40 PMT has not been reached after 24 months. This could in part be explained by the fact that many exchanges are relatively short (12 days on average in 2007). Contrary to 2006, when staff exchange was mainly used for training purposes, exchanges in 2007 also involved newly established collaborations. We expect that further collaborations will be initiated and thus more groups will take advantage of the staff exchange programme. However, it can be anticipated that the goal of 90 PMT/60 months will not be achieved. The possibility to request travel bursaries for participation at intra-workpackage meetings only became available after month 18. Thus far, participants at two intro-workpackage meetings (in September and November 2007) have benefited from travel bursaries. The possibility to use travel bursaries for such activities has been advertised in emails to all network participants. It is expected that more groups will take advantage of these bursaries as workpackages evolve. 14 Postponed due to too many meeting commitments in 2006/2007.

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Work Package 18 Career Development

Lead Participant: Jørgen Kjems

month planned achieved deliverables: 16 Establishing joint PhD committees for PhD students in the

EURASNET by month 15 15 15

117 2 Web-based seminars in 2007 about “Effective scientific communication" (month 24).

24 24

118 Career development related round table discussions with representatives of both academia and industry (month 30).

30 29

119 Career Development Workshop in conjunction with 1st International EURASNET meeting, Krakow – Poland (month 29)

29 29

143 Joint PhD committees for PhD students in the EURASNET (continuation of del. 16, month 30).

30 24

Objectives of the work package • To prepare young scientists for careers in science, teaching and industry

• To facilitate the transition from a post-doctoral fellow to an independent group leader

• To give young scientists the opportunity to raise the profile of their research and their research groups, and to establish lasting contacts/collaborations among the young generation of European scientists Short description of activities 16 Joint PhD committees. There have been several examples of joint PhD committees and supervision/mentorship of PhD students between EURASNET members: Ian Eperon (partner 17) is official supervisor for a student working in Tito Baralle’s laboratory in Trieste (partner 10a). Jamal Tazi (partner 12a) and Karla Neugebauer (partner 1b) have been external participants on two 2 PhD thesis defenses in Angela Krämer's lab (partner 13) Albrecht Bindereif (partner 11) participated in a Ph.D. committee in Reinhard Lührmann’s group (partner 1a) 117 "Effective scientific communication" seminars. A general seminar about career development was given by Jørgen Kjems (partner 19) at the EURASNET Ile Bendor Meeting 2007. The seminar slides were posted on the web side. 118 Career development related round table discussions with representatives of both academia and industry. A round table career development lunch has been organized for the career development workshop in Krakow, May 2008. EURANET partners will function as mentors at this event. 119 Career Development Workshop in conjunction with the 1st International EURASNET meeting, Krakow 2008 A program for the career development workshop in Krakow has been organized and a speaker list has been confirmed. 143 Joint PhD committees for PhD students in the EURASNET We expect an increase of the numbers of EURASNET members in PhD committees in the future. Problems and explanations for delays, postponements etc. None

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Work Package 19 Public Understanding of RNA Biology Lead Participant: Andrea Barta

month planned achieved deliverables: 17 A webpage for science and society issues (coordinated with

the EURASNET homepage) by month 15 15 continued

19 A training workshop on public understanding of science for EURASNET scientists by month 18

18 29

144 Collection of teaching material about RNA biology (for high school students, university students of various levels and for the general public) (continuation of del.18, month 30)

30 on target

Objectives of the work package • To inform the public about the important role of RNA processing in mechanisms of human disease and for the developmental program of organisms, and to explain in general terms the major aims and results of the NoE consortium.

• To discuss with a general audience issues of ethical and social concern raised by progress in the RNA splicing field and to explain the impact of this research for future medical intervention and disease management.

• To educate and support scientists in the NoE to enhance their skills in communication with the public, media and politicians. Short description of activities 17 Webpage for science and society issues. On this webpage alternative splicing in general and special mechanisms and topics with the focus on human disease and health are presented in a form comprehensible to the layman. (topics are splicing in general; alternative splicing and mechanisms; impact of alternative splicing on neurological diseases). Outstanding findings and publications of the NoE members are presented and explained. The EURASNET webpage is currently reorganized. It will be rearranged in a way that people visiting the webpage can specifically select for the section of their interest. A link will lead laymen to another path than scientists, medical doctors or teachers. Rearrangement of the EURASNET webpage is ongoing. 19 A Training workshop on public understanding of science. A media workshop with the aim to provide media training for network members is currently organized. In the framework of the First International EURASNET Conference on Alternative Splicing (23rd -25th May 2008 in Krakow) this media training will take place. Today’s scientists must possess the ability to communicate with the media, decision-makers and the general public. EURASNET members are encouraged to learn how to get their scientific message across to the public and/or the media. Therefore special media training is provided addressing tasks like formulating a press release, interview training and discussion training with the focus on the social and ethical matters of science. Invited speakers for the workshop are Elisabeth Waigmann (dialog<>gentechnik, Vienna), Andrew Moore (Science and Society Programme Manager, EMBO) and Phil Taylor, Head of Communications and Information Services, SCRI. A media writing contest is already set up to train the participants to interact with the media and communicate their research data to lay people. EURASNET members participating in the media workshop are strongly encouraged to compose a press release describing their recent research data. The press releases should be based on the author’s submitted scientific abstracts or on recent publications of their scientific work. An expert jury will evaluate criteria such as newsworthiness, clarity of language, engaging presentation of topic and format of the press release. The workshop is designed to show scientists how best to talk to both journalists and the general public. The main focus of the workshop will be on bringing the scientific message across to the media in a press release. This deliverable will be completed by month 29. 144 Collection of teaching material about RNA biology. A variety of slides and information leaflets are available on the EURASNET webpage in the “splicing info” domain. Each teaching block offers tables of content and short introductions for easier orientation. The information content reaches from introduction in the biochemistry of RNA in general to special topics of complex RNA biology and metabolism – including alternative splicing and also applications of RNA molecules in modern medical treatment. Addition of information to the EURASNET webpage (splicing info domain) is ongoing.

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Problems and explanations for delays, postponements etc. 17 Continued efforts but delays due to website restructuring. 19 The media training workshop was planned for the first international EURASNET conference on alternative splicing. As this event will take place in May 2008 (in Krakow, Poland), deliverable 19 will be completed by month 29 only. Public scientific officer

At the annual meeting in Ile de Bendor (April 2007), Claudia Panuschka (Vienna) was appointed public

scientific officer. She is in the process of generating a brochure describing the members and aims of the

EURASNET as well as topics connected to alternative splicing. Another task of the public scientific officer is to

directly communicate to the webpage master in Dundee for consistent report on PUS activities like press

releases, accessible for EURASNET members and the public.

PUS activities The EURASNET webpage

Results of recent publications of EURASNET members which are suitable to be presented in layman’s

words are accessible on the EURASNET webpage in the form of a short press text. This text should lead the

interested audience to the full text version of the publication. Therefore, direct access to the MedLine link is

provided. The webpage is currently rearranged in a way that people visiting the webpage can select for the

section of their interest. Laymen are for example lead to another path than scientists, medical doctors or

teachers.

A brochure for the general public describing the EURASNET, a further development of the leaflet

(deliverable 20)

The EURASNET leaflet provides a description on basic and general topics of alternative splicing with

a special focus on the impact of alternative splicing on human health. It also contains information on the NoE,

like organisation structure and the main objectives. This first leaflet is designed to support the members of the

network in their communication with the public, politicians or journalists. It provides a concise overview of the

EURASNET and is a mean to quickly introduce the target groups to the topic. The leaflet was produced in a

first edition of 2.500 copies and sent to EURASNET members. It is distributed as a communication tool among

the public, politicians and journalists. Furthermore the leaflet has been provided at several scientific conferences

to propagate the knowledge of the EURASNET. Preparation of the updated version of the leaflet was delayed

awaiting confirmation of the five new YIPs to be appointed in the second round of selection in 2007. The

confirmation was not received until December 2007 (month 24). The updated version of the EURASNET leaflet

will be available by month 30.

Extended brochure for specific target groups like medical doctors, teachers and pupils

The more extended EURASNET brochure is supposed to inform the public about the impact of

alternative splicing on life in general, and to improve the awareness and understanding of the activities, aims

and results of EURASNET in particular. Target groups should be informed about the important role of RNA

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processing in mechanisms of human disease as well as in the developmental programme of higher organisms.

Furthermore, a detailed insight into research topics and fields of all the network participants should be given.

Presented articles will be richly illustrated with pictures, schemes and drawings for an easy understanding of the

topic. Target groups to be reached were selected to act as “amplifiers”. These are medical doctors, teachers

(especially biology teachers, intending to further instruct their pupils about the topic of alternative splicing) and

higher educated pupils by themselves. Additionally, a broader audience should be reached, depending on PUS

activities that are organized by participating members at their places. Therefore the brochure should be

translated into several languages. The brochure is also intended to act as a more elaborate introduction to the

network (including all 40 research groups). It will be produced in a first edition of 2000 pieces each in German

and English. The extent of the brochure will be about 25 pages. The brochure will be completed by month 34.

Press conferences

Guiseppe Biamonti was involved in a local press conference in Sanremo at October 20, 2007 with the

Italian Minister of Health.

Press releases in local media or magazines

Albrecht Bindereif wrote a short summary on alternative splicing for GenomXpress (in German). This

Brochure is intended to help to get projects funded by the German Genome Project (NGFN-II) and is composed

in a modality understandable for the general public (2004: p.12-14; 2007: p. 64-65).

Jørgen Kjems took part in a TV-news broadcast on January 19, 2007. The audience was approximately

1.000.000 Danish laymen. On September 8, 2007 a radio programme reaching about 50.000 people in Denmark

was broadcasted, the topic of the broadcast was HIV-1 including alternative splicing.

The EURASNET was presented in “The Parliament”, a journal published twice a month, dealing with the

current European affaires. It is distributed to all the MEP's and senior members of the Commission, ministries of

all EU member countries, and major European institutions throughout Europe. In the issue of January 28, 2008 a

policy focus on World cancer day (February 4) was published. The EURASNET was presented as one full page

advertisement (see Annex 1). In the February 11, 2008 issue a policy focus on Health Equality was presented.

For EURASNET the same information was shown.

Organisation of Open house events and visits (pupils)

On March 14, 2007 an event called “Open University” took place in at the University of Aarhus with

Jørgen Kjems. 300 people were visiting this event; gene expression including alternative splicing was the topic.

On October 4, 2007 500 high school pupils visited the University of Aahrus. They were introduced to several

RNA biology topics. In the framework of an “Open University” day 300 laymen were introduced to Gene

expression in general and alternative splicing in particular on November 6, 2007.

Jean Beggs: The Wellcome Trust Centre for Cell Biology at Edinburgh repeated two of its more regular

outreach activities. In May 2007 a science activity tent at the Parents Like Us Festival on Leith Links was

organised. (Activities, information desks and experiments included “creepy crawlies”, red cabbage water pH

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experiments, “float or sink”, making fossils (from Plaster of Paris), “whose mummy am I?” and a “build a cell –

beetle drive”).

In August 2007 the Wellcome Trust Centre for Cell Biology at Edinburgh ran the third Cell Biology “Kickstart

Workshop” for high school students. The participants were from schools in Ediburgh, Dumfries and Galloway.

Short talks were given on ribosomes, microscopy, splicing and cellular dynamics. A microscopy-based practical

was carried out, studying development of spermatocytes. Additionally, an annual Teachers’ Open Day was held

at the Wellcome Trust Centre for Cell Biology at Edinburgh.

John Brown gave several talks to pupils visiting the Scottish Crop Research Institute. The presentations

included information about gene expression and alternative splicing.

Mihaela Zavolan took part in the Biozentrum Open House event in Zürich presenting her data on alternative

splicing.

Artur Jarmolowski gave a talk during the X Poznan (Poland) Festival of Science and Art (October 10, 2007).

The topic was: RNA rules! at the Adam Mickiewicz University, Poznan.

Seminars for a broader audience

Reinhard Lührmann presented the EURASNET on the occasion of the EICOS meeting in Götting

2007. This is an event for dissemination of science to science journalists.

Hermona Soreq gave a number of public (layman) talks about her work on alternative splicing and the brain.

Among them talks on July 25, 2007 at the Hebrew University (Jerusalem, Israel) and on January 4, 2008 at the

Land of Israel Museum (Tel Aviv, Israel).

Davide Gabellini gave a short presentation about his work at an “Open day” – event for the general public

(October 20, 2007). This event was called “Open doors for Neurosciences” and took place at the Stem Cell

Research Institute, Milano. The topic of his talk was “FSDH and the fact that aberrant alternative splicing could

be at the basis of the disease”.

Albrecht Bindereif introduced the concept of EURASNET and European research funding to his new first-year

students in a mentoring seminar.

Chris Smith gave a talk to an audience of group leaders, post docs, students, technicians and department

administrators working in different scientific fields with the title “Taking a global view of alternative splicing”.

Cambridge January 12, 2007.

Francisco Baralle presented the activities of the NoE to the members of the IIMCB, Warsaw (Poland) on the

occasion of the International Advisory Board meeting (June 1-2 2007; Warsaw, Poland).

Karla Neugebauer was invited to a gradient student retreat at Schloss Ringberg (June 13-15, 2007). She gave a

talk about her work and spent two days with the pre-docs.

Andrea Barta introduced alternative splicing and disease and the EURASNET in her graduate programme in

RNA biology in Vienna. She gave several talks to the graduate students about the topic.

Others

Daniel Schümperli had a visit of a delegation with families of patients affected with Spinal Muscular

Atrophy from France. The delegation included two affected children and a delegate from AFM, the Association

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Française Contre les Myopathies (AFM) which is funding his research on SMA. In the same context the work

done by his lab was briefly described in the bimonthly AFM brochure “Vaincre les Myopathies” (numéro 131;

novembre/decembre 2007).

He had two meetings in Paris with AFM representatives, other labs funded by AFM working on SMA and SMA

family members on May 3 (Daniel Schümperli and Julien Marquis) and November 17 (Julien Marquis).

Daniel Schümperli is performing music with a trio named “Hugo hat Töne”, converting DNA sequences into

music. They played three concerts/performances: One in August 27 in Bern; two other concerts were played in

the context of European Science Festival WONDERS organised by EUSCEA (European Science Event

Association) on September 8, Vilnius, Lithuania and December 8, Helsinki, Science Museum Heureka. “Hugo

hat Töne” produced a CD with pieces based on musical transpositions of nucleotide and protein sequences and

other kind of biological information.

On all these occasions the EURASNET leaflet were displayed.

The FEBS congress of 2007 was co-organised by Andrea Barta in Vienna. At this meeting, the EURASNET

was propagated by displaying the leaflet at the congress venue.

Annex 1: EURASNET advertisement in “The Parliament”

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115

Work Package 20 SMEs and Technology Transfer Lead Participant: Angus Lamond

month planned achieved deliverables: 23 Setting up a special screening initiative to

identify and characterize RNA-splicing modulators as lead compounds for novel anti-cancer, anti-fungal and anti-viral therapies (month 18).

18 24

120 Update the basic fact file for all NoE members, providing advice and detailed information on the potential legal and practical issues confronting academic researchers who wish to commercialize their work (month 30).

30 24

145 Transfer of IP, reagents (e.g. antibodies, plasmids and cell lines) and technology from NOE member laboratories into the commercial sector (month 12).

30 24

Objectives of the work package • To provide NOE members with advice, information and training that will alert them to commercial opportunities in their research field and enhance their ability to interact successfully with the private sector.

• To facilitate the commercial exploitation of reagents, technology and intellectual property in the field of RNA splicing and cognate areas; to forge commercial links between NOE members and existing European companies.

• To enhance the commercial viability of a spin-out drug discovery company using RNA splicing factors as drug targets. Short description of activities 23 Identify and characterize RNA-splicing modulators as lead compounds. Groups 1A (Lührmann) and 22 (Lamond) have worked on assay development to facilitate chemical compound screening for small molecule inhibitors of RNA splicing factors. The standard methods used for splicing assays involve polyacrylamide gel electrophoresis and autoradiography, techniques which are too time consuming and expensive for high throughput screening of large chemical libraries and small molecule inhibitor collections. An assay has been developed in which RNA substrates are linked to the surface of microtitre wells and a splicing mix added using robotic automated transfer and where spliced RNA products are detected using a molecular beacon probe and a fluorescence readout. This solid phase method has been validated and is currently undergoing final optimization and refinement. Group 12A (Tazi) have identified a chemical inhibitor of SR protein splicing factors and are characterizing its effect in vivo on tissue culture cells and developing the inhibitor to increase its potency. 120 Update the basic fact file for all NoE members. It was decided that the initial assessment for deliverable 120 needed to be revised to improve the quality of the final product delivered. A basic Commercialization Fact File was created during months 3-9, primarily through activities from Group 22 (Lamond), with input from a business consultant, Dr Howard Marriage (Director and Founder of a business development consultancy, “Biotech Innovation and Future Health” (www.bifhit.com)). The factfile was distributed to all Eurasnet groups and feedback and comments invited to revise and improve the document. During months 15-18 a second document was prepared, again predominantly from the Lamond group, with input from a business consultant, to add a detailed overview of the issues and practical details involved in the Strategic Planning required for launching a Biotech Spinout or Startup Venture. This document expanded the previous content of the Commercialisation Factfile and was distributed to all the NOE groups. Copies are available in PDF form for distribution via the Eurasnet website. Thus we feel that this deliverable has been met and a high quality and detailed document generated. Nonetheless, our plan is to allow comments

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and feedback to be provided throughout the full period of the NOE to facilitate further extension and updating of the information in the Commercialisation Factfile. 145 Transfer of IP, reagents and technology. Several EURASNET groups have been involved in technology transfer activities during months 1-12. Group 22 (Lamond) has now launched a new spin-out company, Dundee Cell Products (www.dundeecellproducts.com), which provides reagents and services. Current products include mammalian cells and cell fractions, splicing extracts, RNAase Inhibitor, antibodies, plasmids and cell lines of relevance to RNA processing and the work of the NOE. Dundee Cell Products offers these products to NOE groups at discounted price. Group 11 (Bindereif) for example has tested and purchased from DCP nuclear extracts for RNA splicing assays. Proteomics services and reagents for SILAC MS analysis, including isotope labelled cell extracts, are also available to NOE groups at discounted rates. Dundee Cell Products also provide a route to market for commercialization of reagents generated by NOE member laboratories. So far Group 16 (Carmo-Fonseca) has transferred to Dundee Cell Products hybridoma cells producing monoclonal antibodies to spliceosome proteins (U2AF) and to the Cajal body marker protein p80 coilin for them to market. Discussions and negotiations are currently underway with other NOE groups to arrange for other reagents to be commercialised by Dundee Cell Products. Group 13 (Schümperli) has made steps to obtain a patent for a a novel U7-based tool to enhance exon 7 inclusion in SMN2, a potential therapeutic target gene for Spinal Muscular Atrophy (SMA). The invention cannot be patented by itself since data on the tool have already been published. However, the idea is to try to obtain a patent in collaboration with the group of Dr. Martine Barkats at Genethon, Evry, France who are developing vectors capable of delivering the tool to motor neurons. It is not yet certain whether this strategy will lead to an accepted patent. Additionally, Group 13 (Schümperli) is collaborating with the groups of Prof. Christian Leumann, Department of Chemistry and Biochemistry, University of Bern and that of Dr. Luis Garcia, Institut de Myologie Paris, France on tricyclo-DNA applications to exon skipping in Duchenne Muscular Dystrophy (DMD). The tricyclo-DNA chemistry itself is no longer patentable, again for reasons of prior disclosure, but the collaborating groups hope to obtain a patent for the special application of their technology to DMC.

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Objectives of the work package

Short description of activities

Work Package 21 Reachout to the Broader RNA Community Lead Participant: John Brown

month planned achieved deliverables: 121 Send EURASNET brochure and information to

non-EURASNET RNA researchers in Europe – encourage exchange of information on meetings, advertising new positions etc. (month 18)

18 28

122 Prepare updated posters with YIP information for distribution to partners institutions and for use at national and international scientific meetings. (month 18)

18 27

123 Invite non-EURASNET RNA researchers to workshops/meetings to support interactions and collaborations. (month 24)

24 24

124 Maintain updated contact list of RNA researchers and groups in Europe. (month 30)

30 30

• To identify and make contact with other EU-funded groups and other relevant organizations in Europe, to produce a database of e-mail contacts of their members. • To establish contact with these members and make them aware of our existence and our aims. • To promote exchange of expertise within this extended network. • To collect and disseminate information about conferences and workshops on relevant topics. • To identify sources of grant funding for applications to extend these activities • To identify commercial organisations to support and promote RNA research

121 EURASNET brochure. The preparation of the new EURASNET brochure (by WP19 – Public understanding of Science) was delayed awaiting confirmation of the five new YIPs to be appointed in the second round of YIPs in 2007. This confirmation was not received until December 2007 (month 24). The preparation of the list of European RNA researchers (Deliverable 124) has been used to send information on EURASNET activities. The original plan underpinning this deliverable was to update the highly successful EURASNET brochure and to send it to the European RNA researchers with an accompanying letter introducing the EURASNET network and inviting interaction. The distribution of the new brochure to the non-EURASNET RNA researchers was to act as an introduction to the complete network of 40 groups and would be accompanied with a letter from EURASNET management providing information and directing scientists to the websites and meetings/workshop programmes. This deliverable will now be completed by month 28. 122 Updated posters with YIP information. The preparation of the updated EURASNET poster was delayed awaiting confirmation of the five new YIPs to be appointed in the second round of YIPs in 2007. This confirmation was not received until December 2007 (month 24). This deliverable will now be completed by month 27 and will be distributed to members and placed on the website for EURASNET members to display in home organisations and to take to scientific meetings. 123 Invite non-EURASNET RNA researchers to workshops/meetings to support interactions and collaborations. Information on upcoming EURASNET workshops and meetings in 2008 has been sent to the current list of non-EURASNET RNA researchers. 124 Contact list of RNA researchers. The list of European RNA researchers has been added to by all the EURASNET members and forms the basis of communicating EURASNET activities such as meetings and workshops. It currently contains contact information for 131 scientists. The Public Scientific Officer has prepared a full e-mail list for the efficient dissemination of information about EURASNET activities such as meetings and workshops. This deliverable will continue throughout the project.

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Problems and explanations for delays, postponements etc.

The whole of the EURASNET network are continually involved in promoting the importance of alternative splicing to the wider RNA community. Apart from the activities of this work package in preparing and maintaining a list of non-EURASNET RNA scientists in Europe, other dissemination WPs, and in particular, WP16 – Conferences/meetings, are engaged in transferring knowledge and practical skills in RNA biology and alternative splicing. The outreach activities to the wider RNA community occur at different levels but involve all of the EURASNET member labs. Importantly, many postdocs and PhD students in the EURASNET labs have made presentations at meetings and workshops and are actively encouraged to engage in these activities. The first year report suggested that it was necessary for efforts to be taken to quantify the intensity of the activities of the Network and to establish a structure for ensuring repeated requests for information to be made at regular intervals. In this regard, the change in the management of the EURASNET website (see WP1) and the establishment of a team to co-ordinate the activities of WP1 (website), WP19 (Public Understanding of Science) and this WP (WP21 – Broader outreach to the wider RNA community) will address these suggestions. We have already designed a form where the many different activities are separated into different classifications allowing members to quickly input this information which is then collated. The form has been used in gathering information for the 2007 report but will require some refinement. Information is fed directly to the Management team. The first year report also pointed out the necessity of the Network to function, not only at the European level, but also at the international level. This is also reflected in the information requested. One of the main routes of disseminating information on alternative splicing and EURASNET to the wider RNA community is in the publication of refereed papers and the publication of abstracts or conference proceedings (reflecting presentation of research in the form of talks and posters at national and international meetings) all of which acknowledge EU-FP6 funding and carry the EURASNET logo.

Publications acknowledging EURASNET - 2007

Activity Number Refereed publications from one EURASNET lab 60 Refereed publications from interactions between more than one EURASNET lab

9

Conference proceedings involving one EURASNET lab 59 Conference proceedings from interactions between more than one EURASNET lab

15

The number of publications clearly reflects the scientific activity of the Network. The publications involving more than one EURASNET lab which have been achieved already in the second year of the Network demonstrates the increasingly active interactions among the groups and the added value which the network is bringing to this community.

121 delay awaiting confirmation of the five new YIPs 122 delay awaiting

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The wide range of other activities in communication alternative splicing research and EURASNET include presentations at EURASNET workshops, national RNA groups or meetings, the organisation by EURASNET members of national and international meetings and presentations at these meetings, invited seminars, interactions with industry, consultancy and discussions with scientists/clinicians involved in therapy for diseases due to altered splicing or alternative splicing and talks/discussions to RNA-orientated students to promote interest and future careers in the field of alternative splicing. These activities are quantified below and illustrated with some examples

Broader outreach to the wider RNA community - 2007

Activity Number Presentations at workshops 40 Presentations at national RNA groups or meetings 14 Organisation of national/international meetings 15 Presentations at national/international meetings 31 Invited seminars 35 Presentations/consultancy to industry/SMEs 57 Consultancy to therapeutic groups 5 Education of RNA focused students 32 Workshops: EURASNET has organized a number of workshops for molecular biology students and postdoctoral scientists, clinicians and clinical molecular biologists, and scientists working on other areas of RNA biology or wider biological science or medicine. The workshops (see WP16 report).provide knowledge and information on alternative splicing as well as practical skills (e.g. splicing analyses, cell biology techniques etc) based on the outstanding expertise of EURASNET members. For example, the workshop on alternative splicing in plants, held in France in May 2007 aimed at raising awareness of the importance of alternative splicing to plant scientists involved in gene regulation. The workshop included a presentation by Jamal Tazi on human alternative splicing and disease to place the research into a broad context for the plant audience. National RNA groups or meetings: many countries have national RNA groups who organize meetings for RNA scientists. For example, RNA UK, Swiss RNA Workshop, SifrARN (France), the Scottish RNA Club etc organize meetings annually or biannually. EURASNET members and their labs are key contributors to these meetings and present their current research in alternative splicing to other RNA scientists. For example, Angela Krämer and Dani Schümperli organise the Swiss RNA meeting. National/international meetings: the annual meeting of the RNA Society and the Cold Spring Harbor meeting on RNA splicing brings together RNA scientists from around the world. EURASNET members are often involved in the organization and running of these meetings (e.g. Reinhard Lührmann, Andrea Barta) and many other members attend these meetings to present their work on alternative splicing. In addition, EURASNET members attend national and international meetings with broader focus on cell biology, gene expression, biochemistry etc. and present their research on alternative splicing at these meetings. Invited seminars: EURASNET members are invited to give seminars in Universities and research organizations across Europe and internationally. Seminars describe the latest research in alternative splicing and reach audiences who work on RNA or gene expression as well as different areas of biology. Talks to industry: although less frequent, some EURASNET members have given presentations to SMEs and industry to describe alternative splicing and develop collaborations, and are involved in the setting up of SMEs. For example, Didier Auboeuf gave the Sanofi-Aventis Seminar on the “Development of bioinformatics tools for the study of the transcriptome at exon level” at the SANOFI Genetic Center, Evry. Rienhard Lührmann presented a comprehensive picture of the enormous potential of RNA and in particular alternative splicing research to an audience of about 300 representatives from chemical companies, the medical community and RNA researchers at the 2007 EURASNET IFM on Cancer and RNA. Angus Lamond has been instrumental in the setting up of an SME – Dundee Cell Products – which commercialise some reagents and products for alternative splicing research. Bertrand Seraphin organizes a course to train teachers, medical personnel, technicians and researchers on RNA as part of "continuous development" of professionals from private companies, public employees etc. and the topics include alternative splicing.

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Consultancy to therapeutic/diagnostic groups: a key function of some EURASNET members is to consult for groups involved in developing diagnostic methodologies for examining whether and to what extent alternative splicing is involved in disease phenotypes; similarly some EURASNET members are involved as experts in alternative splicing advising groups addressing therapeutic approaches to correcting splicing or alternative splicing defects in disease. For example, Ian Eperon is the expert consultant to a group examining antisense technologies to combat muscular dystrophy and Francesco and Diana Baralle consult on Neurofibromatosis for the BIRD Onlus Foundation, Longare, Italy. Education of RNA focused students: Most EURASNET members are involved in teaching broad aspects of molecular and cell biology, biochemistry to students. In some cases, the focus of PhD retreats or lecture sessions is alternative splicing with the aim of promoting the importance of alternative splicing research and attracting students into this area.

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5.2 Consortium Management (Work package 22) Management (Göttingen) was actively involved in the planning and organization of the 2007 Annual Meeting at the Ile de Bendor, France. Management organized the open call for the second round of YIP integration, the selection process within the YIP committee, and forwarded the relevant material for the selection process to the Scientific Advisory Board. Minutes of the Annual Conference and full documentation of the YIP selection was forwarded to the EC. Management prepared the documents for YIP integration and forwarded the signed documents to the EC for an amendment. The new YIPs were integrated by January 1, 2008. Management (Göttingen) prepared the Annual Report for 2007 (month 13-24) and submitted the report in March 2007. This small delay was due to the slow return of financial documents from several members and the need to include the results of the Berlin IFM on Biophysical Methods which Management (Göttingen) organized (January 2008, but part of the integration commitment for 2007). This meeting was also used to discuss future directions of EURASNET and required time consuming writing and data collection. The steering committee convened frequently to discuss major administrative decisions and scientific problems of the network. Major discussions and decisions in 2007 centered around the YIP selection, the planning of the Annual Meetings at Ile de Bendor and in Krakow (2008), the microarray initiative and the organization of small molecule screening. The Göttingen based management team served as a hub for the distribution of help on all scientific, financial and legal questions, in particular during the reporting period, and on matters of meeting and workshop activities. Management organized the evaluation of legal aspects of the microarray initiative. The team maintained the contacts to the EC, the SAB, the network members and their labs, their legal and financial departments. Göttingen managed all financial traffic within the network including travel reimbursement. The Barcelona based management team was in charge of dissemination activities and was heavily involved in organizing several of the network's workshops and meetings. They again played a crucial role in organizing the Krakow International EURASNET meeting 2008. Juan Valcárcel, together with Chris Smith, continues to have a leading role in all Network decisions related to microarray technologies. The Erlangen based Management team was in charge of integrating activities, in particular related to the Web site. These duties have been transferred to EURASNET member John Brown (participant 14) effective July 01, 2007. To fully describe the scope of the integrating management activities we include here the full description of their proposal.

EURASNET Website Proposal Introduction SCRI, Dundee has been asked by the management of the European Alternative Splicing Network of Excellence (EURASNET) to take over responsibility for the EURASNET website. The advantages of this function being hosted at SCRI are 1) SCRI has a strong Communications and IT infrastructure to establish and support the website, 2) John Brown leads the Workpackage (WP21) on outreach to the RNA community, 3) John Brown has excellent interactions with Andrea Barta who leads the Public Understanding of Science Workpackage (WP19), and 4) the group of Angus Lamond is also located in Dundee. The intention is therefore to establish the website on a strong basis of interaction among EURASNET groups. Editorial proposal The EURASNET website will: State that EURASNET is a focal point for alternative splicing research in Europe as part of a world-wide community

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Publicise and raise awareness of the importance of alternative splicing and how it impacts human health and disease Provide information about the Network and how it is organised the research of the Network the activities of the Network the successes of the Network Provide information on alternative splicing and its link to human disease to scientists, clinicians, the general public, university teachers school teachers Act as a repository of information on shared resources Requirements The requirements of EURASNET for an overhauled website with regular and improved information content to meet the objectives of the Network as described in Annex 1 can be met by the following objectives:

• develop a new design for the website, • incorporate a full Content Management System (CMS), • CMS to have capability for interactive use by all or selected members of EURASNET with a built-in

approval system, • automatic e-mail alerts/newsletter to members when new information is mounted (will increase use by

network Members), • overhaul the organisation of the public website and Member’s only area, • searchable website. • single-click access points for targeted user groups (scientists, clinicians, general public), • organisation of information gathering from EURASNET members (in association with the Public

Understanding of Science function – Claudia Panuschka - Vienna), • mount and update information on a regular basis, • provide technical expertise to maintain and update the technical capabilities of the website, • searchable databases of resources in Member’s only area • EURASNET blog – noticeboard in Member’s only area where comments, questions, notes can be

posted and members can respond • Events calendar (public and private)

Management of the Website The management structure to ensure that the EURASNET website is successfully managed in terms of content, design, development and maintenance is:

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John Brown – to oversee all EURASNET website activities and development at SCRI. Integrating Manager (Lorraine Wakefield) – Lorraine Wakefield is SCRI’s Online and Information Services Officer and oversees and manages the day-to-day running of the SCRI website. She would take the same project management responsibility for the EURASNET website (20% of her time). ‘EURASNET webmaster’ (Dominika Lewandowska) will be employed on a part-time basis (40%) for three years and two months. She is currently a part-time postdoctoral scientist (60%) working on the EURASNET project. As such she is a molecular biologist with experience in splicing and alternative splicing with an interest in computing. Her role as webmaster will be managed by John Brown and Lorraine Wakefield (SCRI’s Online and Information Services Officer). The primary responsibilities of this role is to ensure that the website content is up-to-date, correct and accessible; to liaise with Claudia Panuschka, Public Science Office (Vienna) to prepare content targeted at key user groups and to regularly contact EURASNET members both with information and with requests for information (news, publications, meetings, job adverts etc.). The webmaster will also be the first line in highlighting any defects/enhancements to the website and working with SCRI IT to deliver the software to rectify them. Claudia Panuschka – EURASNET Public Understanding of Science Officer (based in Vienna). She will liaise closely with the Webmaster to gather and provide information and prepare scientific content. Technical Assistant (part-time – 50%) Responsible for technical computing side of the website and liaison with SCRI IT on hardware/software, routine maintenance and trouble-shooting. Short-term Contracted Website developer – The design and development of the initial release of the EURASNET website is best achieved by a professional website developer. This is the most cost-effective way to generate initial designs for the website and to develop the system. The website development phase will be under the management of Robert McCreary (SCRI IT services) who will ensure adherence to the specification and computing best practices (20% of time from October to December 2007) and oversee the IT interface for the remainder of the contract (10%) of time. Continual updates/meetings – regular meetings to discuss progress, problems, new developments etc will be held with John Brown and the web team.

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Release Strategy The launch of the new EURASNET website will be in March 2008:

Oct07 Nov07 Dec07 Jan08 Feb08 Mar08 Information architecture 1 Define Standards and processes 2 Document requirements and sign off

Design Design mock-ups Template decision Content Editorial proposition Prepare new content Migrate content Technology Define Code templates and requirements

Test and launch Staffing Recruit Train as required

1 Information Architecture – analysis of the information currently on the existing EURASNET website; strengths and weaknesses in the existing website; best way to present information to target audiences; recommendations on how to progress a refined website development. (see Annex I for example) 2 Design Brief and Outline Specification / Requirements Analysis – along with the design of the website, definition of necessary requirements of the website will be made. This will have to be agreed and signed off by the EURASNET management and designer before development. Initial design mock-ups – exploration of potential designs will be circulated to EURASNET management for agreement.

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Annex I – Example of information architecture - potential site map Having been pointed in the direction of the Epigenome Network of Excellence website and with the experiences of recently setting up the new SCRI website, we would suggest a EURASNET Home page with: Home page EURASNET Network Upcoming events Alternative Splicing What is alternative splicing? …for biologists …for clinicians …for the general public Alternative splicing and disease Available technologies Exon arrays RT-PCR Gene therapy approaches Links to EURASNET publications (e.g. pamphlet) List of sub-directories List of Subdirectories Home About Us EURASNET network Alternative splicing Overview Member profiles Joint Programme of Activities Research Programme Protocols Protocol database Tools and Resources Resource databases (plasmids etc) Link to Alternative splicing databases Education Simplified teaching material for teachers Presentations News and Events Conferences Events and Workshops Calendar News Member Publications EURASNET Intranet Log in (Member website) Calendar Shared resources Plasmids Antibodies Oligonucleotides Teaching material for students Blog for discussion of active topics Opportunities Jobs and PhD studentships Training Contact Us

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Additional coordination and intra-work package meetings Attached to the Berlin IFM on Biophysical Methods the network had a one day session dedicated to discussions on the future directions of EURASNET research. The outcome of this discussion is included as Section 15.1 of this report. As coordinator we felt this to be an appropriate tool to encourage and promote an even higher level of integration within the network. The close supervision of the steering committee of all decisions made relating to high-throughput fund financed activities and the extensive discussions among members of the research options this fund offers make sure that money is only spent on highly integrated research projects. Interactions between two or more network members are described in the diagram on page 240. WP13 members met after the 2007 Ile de Bendor Annual Meeting for an Intra-work package meeting. The network members working with plants (Barta, Brown and Jarmolowski) are working as a highly integrated unit (see page 223-224 of the report). The plant groups organized a special inter-work package meeting in Dundee Nov 8-11, 2007 (8 travel bursaries for students and postdocs used). Work package 8 organized an intra-work package meeting (Cambridge UK Sep 14, 2007) on exon array technology, which also served as an important decision-making event of the microarray initiative (9 travel bursaries for students and postdocs used). Participant Jean Beggs, in January 2008, organized a workshop of the british EURASNET members to discuss ways in which to promote alternative splicing research in the UK RNA community and the UK generally. Formal changes in the network As reported in the WP12 report, participants 13 (Schümperli) and 17 (Eperon) are now associated with WP14. As described in the WP1 report the lead participant of WP1 is now participant 14 (Brown), while the lead participant in WP2 is now participant 3 (Stamm).

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6 TABLE OF DELIVERABLES MONTH 13-30

Joint programme of activities (18 months period, month 13 - 30) Del. no.

Deliverable name WP no.

Date due

(proj. month)

Actual/Forecast delivery date

Lead participant

17 A webpage for science and society issues (coordinated with the EURASNET homepage) by month 15

19 15 continued

Barta

16 Establishing joint PhD committees for PhD students in the EURASNET by month 15

18 15 15 Kjems

74 Validation by Q-RT-PCR of 40-50 AS changes predicted by ExonHit SpliceArrays and Affymetrix ExonArrays

8 16 16 Smith

11 2007 Annual Meeting by month 16. 16 16 16 Beggs

9 Generation of a webpage dedicated to disseminating information for NoE members about future funding

5 18 14 Carmo-Fonseca

59 Experimental setup and optimization for D2 bombardment feasibility studies

15 18 18 Séraphin

19 A training workshop on public understanding of science for EURASNET scientists by month 18

19 18 29 Barta

121 Send EURASNET brochure and information to non-EURASNET RNA researchers in Europe – encourage exchange of information on meetings, advertising new positions etc.

21 18 28 Brown

23 Setting up a special screening inititive to identify and characterize RNA-splicing modulators as lead compounds for novel anti-cancer, anti-fungal and anti-viral therapies (continued)

20 18 24 Lamond

122 Prepare updated posters with YIP information for distribution to partners institutions and for use at national and international scientific meetings.

21 18 27 Brown

60 Construction of plasmids for use in establishing cell lines that stably express a tagged pre-mRNA (continued)

15 18 24 Séraphin

125 Report on decisions made during the Annual Meeting 2007

22 18 18 Lührmann

33 Agreement on pre-mRNA substrates, nuclear extracts and cell lines; computer analysis of secondary structures and distributions of known preferred binding sites for SR and hnRNP proteins.

7 18 postponed Eperon

13 By month 18, to have provided the equivalent of 40 PMT of training, collectively by network laboratories (person month training)

17 18 partially

Krämer

8 First incorporations of curated information from databases generated by individual laboratories into the ASD structure and report of discussions between ASD staff and computational groups on implementation of data accessfor large-scale computational studies and assembly of combined databases.

4 19 20 Apweiler

113 Four Interdisciplinary Focus Meetings in 2007

16 24 16-25 Beggs

128

115 Three workshops on areas of strategic relevance

16 24 17-21 Beggs

117 2 Web-based seminars in 2007 about “Effective scientific communication

18 24 24 Kjems

123 Invite non-EURASNET RNA researchers to workshops/meetings to support interactions and collaborations.

21 24 24 Brown

126 Write amendment for Annex I to integrate winners of the second YIP recruiting round

22 24 24 Lührmann

127 Prepare Annual Report for 2007 period 22 25 28

5 Start selection of the second group of five YIP awards by month14 (Annual Meeting, France). Integrate successful candidates by month 25.

3 25 25 Lührmann

58 Extending the network's repertoire of available technologies through integrating suitable candidates during the 2nd YIP recruiting round

15 25 24 Séraphin

114 First International EURASNET Meeting, Krakow – Poland

16 29 29 Beggs

119 Career Development Workshop in conjunction with 1st International EURASNET meeting, Krakow – Poland

18 29 29 Kjems

61 Optimization of database content, in particular through linking of already available content

1 30 24 Stamm

62 Improve content intended for the general public

1 30 24 Stamm

63 Software improvements, bug-fixes and more user-friendly interface

1 30 24 Stamm

130 Web pages containing protocols and useful information regarding RNAi, shared databases and analysis programs (continued)

2 30 24 Eperon

131 Updated list available to all participants describing the network's access to services, shared facilities and technical expertise via collaborative arrangements (continued)

2 30 24 Eperon

132 Improved tools for combined searches and user-friendly access to the ASD database; Integration of additional species to ASTD (continued).

4 30 30 Apweiler

64 Using the exon index generated by the ASTD pipeline for human, mouse and rat, we aim to annotate exons with experimental properties to capture the microarray data that is available from individual laboratories within the Network.

4 30 Replaced by different approach.

See WP 4 discussion

Apweiler

65 The Stamm lab will concentrate on verifying the bioinformatically predicted targets for snoRNAs. This will be done by two approaches: a) direct verification of ‘promising-looking’ hits and b) construction of a custom-made DNA array that detect all the predicted changes in splice site usage

6 30 25 Ast

66 The Zavolan lab in collaboration with the Stamm lab will validate some of our initial predictions snoRNA prediction targets, based on which we would like to further improve the model to predict targets for other orphan snoRNAs

6 30 25 Ast

129

67 The Bork lab will identify conserved and therefore functionally constrained alternative transcripts in divergent Metazoa. We will also use information on conservation to identify associated regulatory sequences

6 30 25 Ast

68 The Ast lab will compile a unified dataset of splicing regulatory elements and examine the minimum/maximum potential regulatory sequences. Also, we will examine the level of alternative splicing among metazoan

6 30 25 Ast

69 Further analysis of local RNA secondary structures and effects on splicing

7 30 24 Eperon

70 Complete identification of components in complexes and assessment of roles in the functions of influential sequences

7 30 24 Eperon

71 Expansion of work on detailed analysis of RNA-protein complexes formed with recombinant protein and repeat sequences or regulatory sequences, including structural analysis.

7 30 24 Eperon

72 Identification of more in vivo targets of the functional activity of splicing regulatory proteins (to be subsumed in WP8).

7 30 delayed Eperon

73 Development of single molecule methods to follow the kinetics of splicing and factor association.

7 30 24 Eperon

133 A web-based resource of validated techniques for global analyses of splicing (continued)

8 30 24 Smith

134 Feasibility testing of key experimental approaches (continued)

8 30 24 Smith

75 Comparison of different data analysis procedures for extracting AS information from ExonArrays

8 30 21 Smith

76 Exon junction microarray analysis of effects of CBC knockdown upon alternative splicing

8 30 24 Smith

77 Validation of SF1 CLIP targets to test whether SF1 is an alternative splicing factor. CLIP of U2AF65 to compare binding sites with SF1 and application of CLIP to hnRNP L

8 30 24 Smith

78 The expanded AS RT-PCR panel will be used to address a) the effects of individual factors in mRNA biogenesis, b) the effects of stress on AS events and levels of trans-acting factors, c) the link between AS and nonsense-mediated decay (NMD), and d) combinatorial control of alternative splicing of subsets of transcripts.

8 30 24 Smith

79 The ability of new Arabidopsis genome tiling arrays from Affymetrix to detect and report on AS will be examined

8 30 24 Smith

80 Genome-wide identification and analysis of promoter-dependent exons in human

8 30 24 Smith

81 Complete the analyses of the protein composition of RNP complexes formed on the wildtype/mutant exon 11 of the LMNA pre-mRNA, the wildtype/mutant exon 5 of the MCAD pre-mRNA, the SRp55 ESE, the DMPK CUG repeats, and the HIV-1 A7 site and its regulatory region (including an upstream RRE)

9 30 24 Lührmann

82 Perform MS2 affinity selections with complexes formed on the second important

9 30 24 Lührmann

130

and highly regulated acceptor site of HIV-1 RNA, site A3

83 Mass spectrometry analyses of spliceosomal complexes from adenovirus infected cells, RNP complexes formed in the presence and absence of the rev protein on RRE-containing model constructs

9 30 In progress Lührmann

84 Determine whether different types of cells can be used for the preparation of splicing active nuclear extracts

9 30 24 Lührmann

85 Investigate the role of the RES complex, and in particular, the human factors homologous to the yeast RES subunits and of the EJC and associated factors in splicing regulation

9 30 24 Lührmann

86 Mass-spectrometric analysis of Prp8p protein-protein crosslinks, nuclear Brr2-containing complexes, and other yeast protein complexes involved in splicing with the aim of identifying new, possibly post-translationally modified, factors involved in this process

9 30 24 Lührmann

87 Measure the elongation rate of RNA polymerase II in vivo by imaging techniques using FRAP (fluorescence recovery after photobleaching)

10 30 24 Cáceres

88 The functional significance of adenoviral L4-33K phosphorylation and stability will be investigated by mutational analysis. Establish the effect of PKA on alternative splicing and a potential subcellular colocalization between PKA and splicing regulatory proteins

10 30 24 Cáceres

89 Develop FRET microscopy techniques to compare protein-protein interactions in the spliceosome and in nuclear speckles. Analyze by FRET-FLIM the possible interactions of U1 with SF2/ASF, at the HIV-1transcription site

10 30 24 Cáceres

90 Use the tap-tagged SRp30 protein to select and MS-analyze ribonucleoprotein complexes in nSBs

10 30 delayed Cáceres

91 Investigate the function of hPrp4 kinase in splicing employing a combined cell biological and biochemical approach

10 30 24 Cáceres

92 Investigate the effect of the RNAi-mediated silencing of SRPK1 and SRPK2 on the subnuclear distribution of snRNPs and other splicing factors using fluorescence microscopy methods

10 30 24 Cáceres

93 Analyse the post-translational modifications of spliceosomal proteins at defined functional stages of the spliceosome, with particular emphasis on phosphorylation

10 30 on target Cáceres

94 Continue experiments to elucidate relationships between post-translational modifications of p68 and specific functions in different steps of the gene expression process (e.g. transcription, splicing, or export)

10 30 24 Cáceres

95 Further optimization of microarray data analysis

11 30 20 Valcárcel

96 Analysis of the effects of knocking down specific isoforms in cellular function

11 30 24 Valcárcel

97 Application of microarrays to the study of effects of knocking down splicing factor isoforms

11 30 24 Valcárcel

131

98 Complete the assessment of AS in plant splicing factors and prepare genetic material for analysis of AS isoforms in plants and analysis using the AS RT-PCR panel

11 30 24 Valcárcel

136 Generation of diagnostic and bioinformatics tools useful to identify mutations in humans that affect splicing (continued)

12 30 24 Baralle

137 Databases (genes vulnerable to inactivating mutations due to the presence of distant branch points, splicing factors and related proteins) and update of motif database at the EBI with mutations seen in diseases studied by the network (continued)

12 30 24 Baralle

138 RNAi of trans-acting factors to modulate aberrant splicing (continued)

12 30 24 Baralle

139 Initial description of co-transcriptional spliceosome assembly in vivo in yeast and design of systems/mutants in yeast for analysis of co-transcriptional spliceosome assembly mechanisms (continued)

13 30 24 Neugebauer

140 Feasibility study of splicing factor ChIP in mammalian cells (continued)

13 30 12 Neugebauer

142 Investigation of relationship between transcription elongation rates and alternative splicing, through coordinated development of reporter systems and compilation of transcriptional regulators that effect alternative splicing changes (continued)

13 30 24 Neugebauer

141 Examination of the kinetics of RNA synthesis and co-transcriptional association of GFP-tagged versions of splicing regulators (e.g. SR proteins) to the SAT III locus, including investigation of the effect of SAT III RNA synthesis on alternative splicing of heterologous gene transcripts (continued)

13 30 24 Neugebauer

99 Examination of the kinetics of cyclin D1 and PS2 RNA synthesis and RNA splicing in response to a transcriptional stimulus. (continued)

13 30 24 Neugebauer

100 Use pol II elongation inhibitors and Pol II mutants to study how elongation impacts alternative splicing.

13 30 24 Neugebauer

101 Determine the specific step at which transcription is affected by Prp45p mutants.

13 30 24 Neugebauer

102 Investigate the requirement of the RNA Pol II CTD for mRNA release from the site of transcription and alternative splicing of human IKBKAP exon 20.

13 30 24 Neugebauer

103 Determine the recruitment patterns of distinct GFP-tagged SR proteins to a variety of selected genes (intronless, constitutively spliced intron-containing, and alternatively spliced) (ChIP) and to the SATIII locus (fluorescence microscopy).

13 30 24 Neugebauer

104 Comparison of c-fos and cyclin D1 genes with respect to: splicing factor recruitment (ChIP), spliced levels on chromatin (ChRIP), and spliced levels attached to RNA pol II.

13 30 24 Neugebauer

105 Feasibility study of application of yeast 3-hybrid system to identify RNA binding proteins interacting with SATIII transcripts.

13 30 24 Neugebauer

106 Investigation of feedback from splicing to 13 30 24 Neugebauer

132

transcription. 107 Analysis of chemical derivatives of the five

newly identified inhibitors of pre-mRNA splicing

14 30 24 Tazi

108 Purify SR protein associated complexes and probe SR protein/drug interaction with photo-affinity molecules

14 30 24 Tazi

109 Adaption of the in vitro pre-mRNA splicing assay with a fluorescence readout to additional pre-mRNA substrates

14 30 24 Tazi

110 Reduce the amount of RNP starting material for successful routine cross-linking experiments to less than 10 pmol

15 30 24 Séraphin

111 Selective identification of cross-linked sites in various protein-RNA complexes by online nanoLC-Electrospray Ionization (ESI) MS/MS and off-line 2-dimensional nanoLC with subsequent MALDI-ToF/ToF analysis

15 30 24 Séraphin

112 Increase of the protein-RNA cross-linking yield by a factor 2 - 3 using a frequency quadrupled Nd-Yag Laser

15 30 24 Séraphin

116 Continue and further develop the use of travel bursaries for student and postdoc exchanges (also for necessary intra-workshop meetings)

17 30 partially Krämer

118 Career development related round table discussions with representatives of both academia and industry

18 30 29 Kjems

143 Joint PhD committees for PhD students in the EURASNET (continued)

18 30 24 Kjems

144 Collection of teaching material about RNA biology (for high scool students, university students of various levels and for the general public) (continued)

19 30 on target Barta

120 Update the basic fact file for all NoE members, providing advice and detailed information on the potential legal and practical issues confronting academic researchers who wish to commercialize their work

20 30 24 Lamond

145 Transfer of IP, reagents (e.g. antibodies, plasmids and cell lines) and technology from NOE member laboratories into the commercial sector (continued)

20 30 24 Lamond

124 Maintain updated contact list of RNA researchers and groups in Europe.

21 30 30 Brown

128 Steering Committee Meetings at month 15,18, 21, 24, 27, 30

22 30 15-30 Lührmann

129 Quarterly network e-mail at months 15, 18, 21, 24, 27, 30

22 30 15-30 Lührmann

135 An agreed coordinated strategy for subsequent analyses of global splicing factor activity (continued)

8 30 16 Smith

N6 Computational structural model of splicing regulators hnRNP-L and hnRNP-L-like in complex with RNA

6 30 30 Ast

N7 Computational modeling and experimental testing of novel RNA binding domains

6 30 30 Ast

133

N8 Use minigene splicing assay to study the influence of mutations in neurofibromatosis type 1 exon/intron 29 splicing

12 30 30 Baralle

N9 Analysis of the role of PTB in pseudoexon splicing

12 30 30 Baralle

N10 Determine the structures of hnRNP F, hnRNP L and Etr3 in their free state and in complex with RNA using NMR spectroscopy

7 30 24 Branlant

134

7 UPDATED PLAN FOR USING AND DISSEMINATING THE KNOWLEDGE Section 1 – Exploitable knowledge and its use At this point of time EURASNET cannot present exploitable results, defined as knowledge having a potential for industrial or commercial application in research activities or for developing, creating or marketing a product or process or for creating or providing a service. After now two years into the network it still appears to be too early to discuss such issues in detail. Furthermore the legal implications of doing so must not be underestimated. Whenever exploitable knowledge is discussed, partners need to consider the advice of their local legal departments. This situation will change over time: once a final version of the Commercialization fact file has been agreed, this file can actively support partners in their commercialization efforts. The EURASNET management will encourage members to take an active role in contributing to exploitable knowledge in cooperation with their legal departments. Although it is still necessary to proceed cautiously, substantial progress has been made over the last reporting period. This progress is primarily linked to the activities of participant 22 (Angus Lamond) and in particular to his negotiations with the Dundee Screening Facility described in WP 20. Section 2 – Disseminating of knowledge Network participants during the last reporting period have been involved in a plethora of disseminating activities either organized by EURASNET or by other socities. Both the scientific community and the more general public have been addressed. Here we only list those activities which are outside of dissemination deliverables and don’t represent individual national activities. Presentations at international meetings The Coordinator Reinhard Lührmann presented the EURASNET NoE to an international audience of RNA researchers at the International Meeting of the RNA Society, to an international audience of RNA researchers at the 2007 FEBS Meeting in Vienna, and at the European Science Foundation (ESF) Workshop 2007, Berlin. On the occasion of the 2007 EURASNET IFM on Cancer and RNA he presented a comprehensive picture of the enormous potential of RNA and in particular alternative splicing research to an audience of about 300 representatives from chemical companies, the medical community and RNA researchers. EURASNET was also introduced to a group of international science journalist on the occasion of EICOS Meeting Göttingen 2007. Section 3 – Publishable results The same considerations outlined in Section 1 apply to this section. The consortium is not ready to publicize and has not yet taken the appropriate measures to protect the IPR. List of Publications in 2007 Boon, K.L., Grainger, R.J., Ehsani, P., Barrass, J.D., Auchynnikava, T., Inglehearne, C. and Beggs, J.D. (2007) prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nature Struc. Mol. Biol. 14:1077–83. Boireau,S., Maiuri,P., Basyuk,E., de la Mata,M., Knezevich,A., Pradet-Balade,B., Bäcker,V., Kornblihtt,A., Marcello,A. and Bertrand,E. (2007) The transcriptional cycle of HIV-1 in real-time and live cells. J. Cell Biol., 179, 291-304. Fic,W., Juge,F., Soret,J. and Tazi,J. (2007) Eye development under the control of SRp55/B52-mediated alternative splicing of eyeless.PLoS ONE, 2(2):e253.

135

Gabut, M., Dejardin, J., Tazi, J. and Soret, J. (2007) The SR family proteins B52 and dASF/SF2 modulate development of the Drosophila visual system by regulating specific RNA targets. Mol.Cell Biol.,27, 3087-3097. Buratti,E., Dhir, A., Lewandowska, M.A., and Baralle, F.E. (2007) RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events. Nucleic Acids Res 35:4369-83 Buratti, E., Stuani, C., De Prato, G., and Baralle F.E. (2007) SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer. Nucleic Acids Res 35:4359-68 Buratti, E., Chivers, M., Královičová, J., Romano, M., Baralle, M., Krainer, A.R., and Vořechovský, I. (2007) Aberrant 5′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 35:4250-63 Custódio N, Vivo M, Antoniou M, Carmo-Fonseca M. (2007) Splicing- and cleavage-independent requirement of RNA polymerase II CTD for mRNA release from the transcription site. J Cell Biol. 179: 199–207. Asparuhova MB, Marti G, Liu S, Serhan F, Trono D, Schümperli D. (2007) Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping. J Gene Med. 9:323-34. Bakkour N, Lin YL, Maire S, Ayadi L, Mahuteau-Betzer F, Nguyen CH, Mettling C, Portales P, Grierson D, Chabot B, Jeanteur P, Branlant C, Corbeau P, Tazi J. (2007) Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance. PLoS Pathog. 3:1530-9. Lenz C. Kühn-Hölsken E. & Urlaub H. (2007) Detection of protein–RNA cross-links by nanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring (MRM) experiments, J. Am. Soc. Mass Spec. 18, 869–881 Kühn-Hölsken E, Dybkov O, Sander B, Lührmann R, Urlaub H., (2007). Improved identification of enriched peptide–RNA cross-links from ribonucleoprotein particles (RNPs) by mass spectrometry. Nucleic Acids Res. 35:e95 Listerman, I., Bledau, A.S., Grishina, I., and Neugebauer, K.M. (2007) Extragenic Accumulation of RNA Polymerase II Enhances Transcription by RNA Polymerase III. PLoS Genet. 3: e212. Amit, M., Sela, N., Keren, H., Melamed, Z., Muler, I., Shomron, N., Izraeli, S. and Ast, G. Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene. (2007) BMC Mol Biol. 8: 109. Lev-Maor, G., Goren, A., Sela, N., Kim, E. Keren, H. Doron-Faigenboim, A., Leibman-Barak, S., Pupko, T., and Ast, G. (2007) The “Alternative” Choice of Constitutive Exons throughout Evolution. PLoS Genet. 3: e203. Alberstein, M., Amit, M., Vaknin, K., O'Donnell, A., Farhy, C., Lerenthal, Y., Shomron, N., Shaham, O., Sharrocks, A.D., Ashery-Padan, R. and Ast, G. (2007) Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing. RNA 13:1988–1999. Sela, N., Mersch, B., Gal-Mark, N., Galit Lev-Maor, G., Hotz-Wagenblatt, A., and Gil Ast, G. (2007) Comparative analysis of transposed element insertion within human and mouse genomes reveals Alu's unique role in shaping the human transcriptome. Genome Biol. 8: R127. Lev-Maor, G., Rotem Sorek, R., Erez Y Levanon, E.Y., Paz, N, Eisenberg, E. and Ast, G. (2007) RNA-editing-mediated exon evolution. Genome Biol. 8: R29. Marazziti, D., Mandillo, S., Di Pietro, C., Golini, E., Matteoni, R., and Tocchini-Valentini, G.P. (2007) GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs. Proc Natl Acad Sci U S A. 104: 9846–9851

136

Puig, O., Bragado-Nilsson, E., Koski, T., and Séraphin, B. (2007) The U1 snRNP-associated factor Luc7p affects 5′ splice site selection in yeast and human. Nucleic Acids Res. 35: 5874–5885 Dorothée, M., Maiuri, P., Boireau, S., Bertrand, E., Knezevich, A., Marcello, A., and Basyuk, E. (2007) A real-time view of the TAR:Tat:P-TEFb complex at HIV-1 transcription sites. Retrovirology. 2007; 4: 36 Simpson, C.G., Fuller, J., Maronova, M., Kalyna, M., Davidson, D., McNicol, J., Barta, A. and Brown, JWS (2007) Monitoring changes in alternative precursor messenger RNA splicing in multiple gene transcripts. Plant J 53: 1035-1048 Guil, S., and Javier F Cáceres, J.F. (2007) The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nat Struct Mol Biol.14:572-3 Lykke-Andersen, S., Piñol-Roma, S., and Kjems, J. (2007) Alternative splicing of the ADAR1 transcript in a region that functions either as a 5′-UTR or an ORF. RNA13: 1732–1744 Spellman, R., Llorian, M., and Smith, C.W.J. (2007) Crossregulation and Functional Redundancy between the Splicing Regulator PTB and Its Paralogs nPTB and ROD1. Mol Cell. 27(3): 420–434 Perry, C., Pick, M., Podoly, E., Gilboa-Geffen, A., Zimmerman, G., Sklan, E.H., Ben-Shaul,Y., Diamant, S., and Soreq, H. (2007) Acetylcholinesterase/C terminal binding protein interactions modify Ikaros functions, causing T lymphopenia. Leukemia. 21:1472-80 Kehat, R., Zemel, E., Cuenca, N., Evron, T., Toiber, D., Loewenstein, A., Soreq, H. and Perlman, I. (2007) A Novel Isoform of Acetylcholinesterase Exacerbates Photoreceptors Death after Photic Stress. Invest Ophthalmol Vis Sci. 48:1290-7 Skrisovska, L., Bourgeois, C.F., Stefl, R., Grellscheid, S.-N., Kister, L., Wenter, P., Elliott, D.J., Stevenin, J., and Allain, F.H-T. (2007) The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction. EMBO Rep. 8: 372–379 Listed are only those publications which appeared in print version in 2007 and acknowledge EURASNET support. The names of EURASNET members appear in red.

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8 TABULAR OVERVIEW OF ALL RESOURCES EMPLOYED ON THE PROJECT

The last column shows the estimate of person-months of permanent staff working on the project.

No. Partner Eligible costs Non-EU funded personnel costs (€)

Estimated costs for other resources deployed by the project (€)

Estimated total costs

PM of permanent staff (non-EU funded)

1A MPG 211.523,60 184.000 150.000 545.523,60 49 1B MPG 38,590.56 90,000.00 60,000.00 150,000.00 45 1C MPG 44.002,24 100.000 60.000 204.002,24 24 2 CRG 59.893,81 140.000 220.000 419.893,81 18 3 UERLN 71.804,35 102.308,00 75.700,00 249.812,35 72 4 UU 61.412,08 28.800 53.000 143.212 3 5A EMBL 77.205,11 8.850,00 2.000,00 88.055,11 1.5 5B EMBL 98.505,19 32.539,57 1.600,00 132.644,76 3 6 TAU 47.715.84 140.000 100.000 287.715,84 13 7 ICGEB 87.849,46 110.774,00 18.160,00 216.783,46 40 8 MUW 50.577,89 83.650,00 30. 000,00 164.227.89 4 9 UEDIN 52.778,60 149.625,00 192.123,36 394.526,96 29 10A CNR FC 10B CNR FC 11 LUG 39.690,88 78.000 50.000 167.690,88 2 12A CNRS FC 12B CNRS FC 12C CNRS FC 12D CNRS FC 13 UNIBE 8.480 180.500 67.600 256.580 12 14 SCRI 66.088,08 80.000 12.000 158.088,08 24 15 MRC FC 16 IMM 71.452,08 75.000 80.000 226.452,08 48 17 UNILEIC 63.237,20 174.114,33 160.449,48 397.801,01 37.5 18 AMU 36.035,58 9.604,04 10.000 55.639,62 9 19 UAAR 41.368,48 70.000 50.000 161.368,48 14 20 FCEN-UBA 30.448,05 80.000 187.200 297.648,05 8 21 UNIGE 40.845,48 106.000 44.500 191.345,48 6 22 UNIVDUN 62,275.02 39,960.50 20.000 108,537.08 10 23 UCAM-DBIOC 39.729,19 222.285 81.878 343.892,19 42 24 HUJI 56.267,25 30.000 45.000 131.267,25 15 25 CERBM-GIE 50.464 49.000 0 99.464 20.5 26 INSERM FCF 27 FCSR 46.573,54 83.000,00 60.000 189.573,54 0 28 ISB-SIB 23.011,45 43.169 0 66.180,45 11

138

9 TABULAR OVERVIEW OF MAJOR COST ITEMS Cost Budget Follow-up Table

Contract No. 518238 Acronym EURASNET budget actual costs (€)

period 1 period 2 period 3 period 4 period 5

Participant Type of expenditure (as defined by

participant) e a1 b1 c1 d1 e1

1A Total Person-month 121 116.5 Personnel costs 95.560,98 97.615,94 consumables 20.296,08 1.648,31 training 0 0 equipment 0 0 travel 60.714,75 3.785,47 overhead 35.314,36 35.253,93 other (the rest) 0 74.594,95 total costs * 211.886,17 212.898,60 1B Total Person-month 63 60 Personnel costs 17.088 18,450.00 consumables 15.743,40 13,708.80 training 0 0 equipment 360 0 travel 0 0 overhead 6.638.28 6,431.76 other (the rest) 0 0 total costs * 39.829,68 38,590.56 1C Total Person-month 16 25 Personnel costs 9.575,72 23.124,15 consumables 7.651,31 13.544,38 training 0 0 equipment 0 0 travel 0 0 overhead 3.445,41 7.333,71 other (the rest) 0 total costs * 20.672,44 44.002,24 2 Total Person-month 52 60 Personnel costs 56.321,79 49.911,51 consumables 37.808,93 0 training 0 0 equipment 0 0 travel 28.733,76 0 overhead 11.264,36 9.982,30 other (the rest) 0 total costs * 134.128,84 59.893,81 3 Total Person-month 50.5 23.0 Personnel costs 71.238,10 30.304,08 consumables 30.788,40 15.643,52 training 9.465,05 0,.00 equipment 12.779,75 10.125,68 travel 3.147,61 3.763,68 overhead 18.094,49 11.967,39 other (the rest) 0 0 total costs * 145.513,40 71.804,35 4 Total Person-month 31 34 Personnel costs 27.792,02 43.867,23

139

consumables 6.284,02 7.009,45 training 0 0 equipment 0 0 travel 0 300,06 overhead 6.815,11 10.235,34 other (the rest) total costs * 40.891,15 61.412,08 5A Total Person-month 8.5 21 Personnel costs 16.371,75 64.036,05 consumables 0 0 training 0 0 equipment 0 0 travel 0 301,54 overhead 3.274,35 12.867,52 other (the rest) 0 total costs * 19.646,10 77.205,11 5B Total Person-month 12 12 Personnel costs 74.778,32 78.951,12 consumables 0 64,61 training 0 0 equipment 0 0 travel 2.785,21 3.071,93 overhead 15.512,71 16.417,53 other (the rest) 0 total costs * 93.076,24 98.505,19 6 Total Person-month 45.5 13.5 Personnel costs 1.504,41 9.626,91 consumables 7.571,96 24.166,67 training 0 equipment 2.909,47 711,62 travel 484,29 1.878,18 overhead 2.587,48 7.952,64 other (the rest) 467,27 3.379,82 total costs * 15.524,89 47.715,84 7 Total Person-month 39.8 39.8 Personnel costs 20.800,00 35.473,64 consumables 0 0 training 0 16.748,50 equipment 0 0 travel 246.24 20.985,74 overhead 4.209,25 14.641,57 other (the rest) 0 0 total costs * 25.255,49 87.849,46 8 Total Person-month 12.8 17,8 Personnel costs 19.192,02 37.038,39 consumables 19.140,00 876,05 training 0 1.205,00 equipment 369,58 369,58 travel 0 2.659,22 overhead 8.027,27 8.429,65 other (the rest) 1.437,27 total costs * 48.166,64 50,577,89 9 Total Person-month 35.5 35.0 Personnel costs 2,093.41 9583.64 consumables 12,851.27 32262.47 training 0 0 equipment 0 0 travel 797.92 2136.06 overhead 3,148.52 8796.43

140

other (the rest) 0 0 total costs * 18.891,12 52778.60 10A Total Person-month 32.5 32.5 Personnel costs 26.024,09 26.492,74 consumables 22.873,19 30.056,40 training 0 equipment 0 722,80 travel 518,00 1.209,66 overhead 23.207,66 other (the rest) 438,44 total costs * 49.853,72 81.689,26 10B Total Person-month 23 23 Personnel costs 50.643,11 60.305,15 consumables 0 0 training 0 0 equipment 0 0 travel 0 0 overhead 44.363,36 52.827,31 other (the rest) 0 0 total costs * 95.006,47 113.132,46 11 Total Person-month 14.5 37 Personnel costs 0 30.941,24 consumables 32.563,81 2.134,49 training 0 0 equipment 0 0 travel 0 0 overhead 6.512,76 6.615,15 other (the rest) 0 0 total costs * 39.076,57 39.690,88 12A Total Person-month 57 17 Personnel costs 66.186,40 51.695,15 consumables 13.398,72 8.507,08 training 0 0 equipment 0 0 travel 339,00 5.855,09 overhead 15.984,82 13.211,46 other (the rest) total costs * 95.908,22 79.268,78 12B Total Person-month 21 29,14 Personnel costs 95 991,14 125.391,66 consumables 0 5.631,99 training 0 0 equipment 0 0 travel 330,00 2,80 overhead 19264,28 26.205,29 other (the rest) 0 total costs * 115.585,42 157.231,74 12C Total Person-month 21.34 19,49 Personnel costs 93.524,62 92.996,28 consumables 0 5.786,30 training 0 0 equipment 0 0 travel 0 5.220,09 overhead 18.704,92 20.800,54 other (the rest) 0 total costs * 112.229,54 124.803,21 12D Total Person-month 6 25,67 Personnel costs 22.831,01 100.066,22

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consumables 0 190,22 training 0 0 equipment 0 0 travel 0 233,05 overhead 4.566,20 20.097,89 other (the rest) 0 total costs * 27.397,21 120.587,38 13 Total Person-month 74 74 Personnel costs 0 0 consumables 40.924.20 8480 training 0 0 equipment 0 0 travel 1.480.00 overhead 6.505.20 other (the rest) 2.302.20 total costs * 51.211,60 8480 14 Total Person-month 18.2 27,9 Personnel costs 17.092,33 19.061,94 consumables 12.171,03 30.172,19 training 0 equipment 0 4.647,49 travel 1.302.24 2.146,40 overhead 6.113,12 10.060,06 other (the rest) 0 total costs * 36.678,73 66.088,08 15 Total Person-month 45 45 Personnel costs 0.00 8704.67 consumables 42,134.39 13810.25 training 0.00 equipment 0.00 travel 5,489.37 5884.23 overhead 0.00 5895.81 other (the rest) 1,240.98 1079.89 total costs * 48.864,74 35,374.85 16 Total Person-month 34.5 28 Personnel costs 4.195,00 36.752,00 consumables 34.390,54 12.480,61 training 0 0 equipment 541,02 0 travel 1.963,58 10.310,79 overhead 8.383,39 11.908,68 other (the rest) 826,83 0 total costs * 50.300,36 71.452,08 17 Total Person-month 37.5 37.5 Personnel costs 8,481.63 43389.22 consumables 5.896,07 5539.92 training equipment 38.319,97 travel overhead 10.539,53 9785.84 other (the rest) total costs * 63.237,20 58714.98 18 Total Person-month 14 14 Personnel costs 3.210,51 0 consumables 24.079,98 21.872,52 training 703,75 0 equipment 0 0 travel 7.976,61 8.157,13

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overhead 7.203,88 6.005,93 other (the rest) 48,57 0 total costs * 43.223,30 36.035,58 19 Total Person-month 42.5 42.5 Personnel costs 20.637,28 16.533,36 consumables 7.269,08 14.297,00 training 0 0 equipment 0 0 travel 1.394,30 3.643,38 overhead 5.860,13 6.894,74 other (the rest) 0 0 total costs * 35.161,00 41.368,48 20 Total Person-month 9.5 9.5 Personnel costs 0 0 consumables 44.734,90 27.742,95 training 0 0 equipment 0 0 travel 0 2.705,10 overhead 6.111,90 0 other (the rest) 0 0 total costs * 50.846,80 30.448,05 21 Total Person-month 31.3 Personnel costs 16.132,46 7398.30 consumables 13.099,14 27997.36 training 0 equipment 0 travel 1.638,19 -1357.76 overhead 6.077,86 6807.58 other (the rest) -480.48 total costs * 36.467,17 40845.48 22 Total Person-month 27 52 Personnel costs 41.212,43 49,616.23 consumables 0 0 training 0 0 equipment 0 0 travel 2.793,00 2,279.62 overhead 8.801,08 10,379.17 other (the rest) 0 0 total costs * 52.806,51 62,275.02 23 Total Person-month 54.5 78 Personnel costs 10.683,96 21.350,64 consumables 21.282,89 10.515,88 training 0 0 equipment 0 0 travel 750,33 1.241,14 overhead 6.543,43 6.621,53 other (the rest) 0 0 total costs * 39.260,61 39.729,19 24 Total Person-month 29.5 13.9 Personnel costs 8.150,75 25.056,00 consumables 14.093,34 1.617,49 training 0 0 equipment 0 0 travel 4.305,44 4.295,25 overhead 5.309,91 9.284,61 other (the rest) 0 0 total costs * 31.859,44 56.267,25 25 Total Person-month 20.5 20.5

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Personnel costs 26.211,00 29.737 consumables 15.240,50 12.599,72 training 0 0 equipment 0 0 travel 0 429,28 overhead 7.461 7.698 other (the rest) 0 0 total costs * 48.912,50 50.464,00 26 Total Person-month 14 25 Personnel costs 719,54 2811,07 consumables 5058,83 26159,33 training 0 0 equipment 0 0 travel 0 1843,12 overhead 1155,67 6162,70 other (the rest) 0 0 total costs * 6.934,04 36976,22 27 Total Person-month 6.5 15 Personnel costs 0 0 consumables 0 27.025,90 13.079,80 training 0 0 equipment 0 0 travel 0 885,55 overhead 0 5.582,29 other (the rest) 0 0 total costs * 0 46.573,54 28 Total Person-month 11 12 Personnel costs 19.176,20 28275.18 consumables 0 training 0 equipment 0 travel 0 overhead 3.835,25 5655.36 other (the rest) 0 total costs * 23.011,45 33930.54

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10 JUSTIFICATION OF MAJOR COST ITEMS AND RESOURCES Participant 1A – Reinhard Lührmann, Coordinator a) Work performed during the period • WP2

The Lührmann lab provided protocols, expertise and biological research material to several netwqork members.

• WP3

R. Lührmann supervised and successfully completed the final second YIP recruitment for EURASNET.

• WP9

In a collaboration between Partner 12a and Partner 1a (R. Lührmann) it was shown by both in vitro and ex vivo experiments that the single point mutation (GGC>GGT) in exon 11 was sufficient to induce utilisation of a cryptic 5' splice site in exon 11Partner 1a (R. Lührmann) investigated complexes that assemble in vitro on intron-exon-intron constructs. Partner 1a (Lührmann) in collaboration with partner 1c (Urlaub) have now expanded these studies to other organisms (yeast, Drosophila) in order to gain deeper insight into the evolutionary conservation and/or diversity of spliceosomal proteomes. Partner 1b (B. Seraphin) in collaboration with the group of group of H. van Tilbeurg (Orsay) and partner 1a (R. Lührmann in collaboration with M. Wahl) performed biochemical analyosis of the architecture of the yeast RES complex and solved the crystal structure of the Pml1p subunit.

• WP10 The Lührmann investigated the function of the human Prp4 kinase and investigated the role of phosphorylations of the tri-snRNP protein, hPrp28.

• WP14

The Lührmann group characterized the mechanism of action of various HDAC and HAT inhibitors as novel inhibitors of pre-mRNA splicing. They also collaborated with Partner 12a, 12c, Partner 25 in the research on Hutchinson-Gilford progeria syndrome (HGPS). The Lührmann group also worked on read-out assays for high throughput screening.

• WP15 Performed D2 bombardment feasibility studies. • WP17

The Lührmann lab provided extensive training for other EURASNET labs within the travel bursary program.

• WP20

Groups 1A (Lührmann) and 22 (Lamond) have worked on assay development to facilitate chemical compound screening for small molecule inhibitors of RNA splicing factors.

• WP21 R. Lührmann promoted the EURASNET concept during several national and international events.

• WP22

The Göttingen based management team supervised all research, integration and dissemination activities of the network, organized the 2006 report, steering committee meetings, handled all travel reimbursements and financial transactions. It provided support for all legal and financial problems of the network and served as the hub for network information.

145

b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 97.615,94 consumables 1.648,31 training 0 travel bursaries program

10.784,08

Annual Meeting 2007 29.239,85 YIP ads 3.230 travel reimbursement 30.307,60 equipment 0 travel management 3.785,47 audit 1.375

adjustments -341,58 = total direct costs 177.644,67 + overhead 35.253,93 = total eligible costs 212.898,60 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Jochen Deckert [WP 9] 3 PM 5.876,83 Euro Marc Schneider [WP 9] 6 PM 23.507,34 Euro Consumables Laboratory supplies 1.356,79 Euro Travel 545,40 Euro Marc Schneider traveling to Eurasnet Annual Meeting 2007. • Integration travel reimbursement for IFM Lisbon 2007 and

for Annual meeting 2007 29.762, 20 Euro

YIP Program advertisements 3230 Euro

146

• Dissemination training bursaries paid through the coordinator are part

of the dissemination program 10.784,08 Euro Annual Meeting 2007 Ile de Bendor (meeting venue) 29.239,85 Euro • Management Personnel Reinhard Rauhut 12 PM 68.231,77 Euro Scientific Network Manager Consumables bureau material 291,52 Euro Travel 3.785,47 Euro Transfers from escrow account (Coordinator) to members as reported in Distribution_report file: 8.196,72 € transferred to member Maria Carmo-Fonseca (participant 16) for Workshop 2006 Lisboa 10.000,00 € transferred to member Alberto Kornblihtt (participant 20A) for 2007 IFM Bariloche 25.000,00 € transferred to Angus Lamond (Dundee small molecule screening facility) (participant 22) 20.000,00 € transferred to member Jamal Tazi (participant 12A) for IFM 2007 Cancer & RNA 20.000,00 € transferred to member John Brown (participant 14) for Workshop 2007 on plant splicing 13.000,00 € transferred to member Andrea Barta (participant 8) for Workshop 2007 Bioinformatics Vienna 60.000,00 € transferred to member John Brown (participant 14) HTE fund RT-PCR panel plants 9.591,78 € transferred to member Stefan Stamm (participant 3) for Workshop Bioinformatics Erlangen 2006 Summe 165.788,50 Euro

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Participant 1B – Karla Neugebauer a) Work performed during the period WP8: In a collaboration between the Neugebauer and Valcarcel labs, the cap binding complex (CBC) was been depleted from HeLa cells by RNAi. Because CBC plays a role in exon definition, by recruiting the U1 snRNP to the cap-proximal 5' splice site, we wanted to test for the possibility that CBC is important in alternative splicing. A predoctoral student (Marta Pabis) from the Neugebauer lab in Dresden traveled to Barcelona with the RNA samples resulting from three biological replicates in which CBC proteins were depleted by at least 80%. There she produced labeled probes, hybridized them to the custom-designed splice junction arrays developed by the Valcarcel lab, hybridized and scanned them, and finally extracted the data. Together with Claudia Ben-Dov of the Valcarcel lab, she determined that only two candidate alternative splicing events could be identified. Marta was unable to validate these AS events by RT-PCR, so we conclude that at least under the conditions of this experiment, we were unable to detect a role for the CBC in AS. WP13: Our lab established splicing factor ChIP in yeast and mammalian cells; this includes the entirely novel strategy of ChIP of alternative splicing factors, which are expressed as tagged versions from stably transfected BACs. This has turned out to be an improvement over the use of antibodies and has therefore led to a number of new collaborations within the workpackage. Collaborative project with Seraphin on the role of Pol II in U1 snRNP recruitment to nascent RNA is complete, with a manuscript in progress. We also collaborated with Biamonti to screen through a number of splicing factors for co-transcriptional association with the SAT III locus. This work is also now a manuscript in preparation. b) Major cost items with justification

Participant 1B Type of expenditure Budget total costs personnel 18,450.00 consumables 13,708.80 training 0 equipment 0

travel 0 = total direct costs 32,158.80 + overhead 6,431.76 = total eligible costs 38,590.56 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items Research Personnel Marta Pabis [WP 13] 12 PM (= 1,920 hours) 15,000 Euro Inna Grishina [WP 13] 3 PM (= 480 hours) 3,450 Euro Consumables Antibodies 3,238.81 EUR Chip, Q-PCR reagents 6,212.73 EUR Yeast strains 308.00 EUR Chemicals + misc 39,49.26 EUR

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Participant 1C – Henning Urlaub a) Work performed during the period WP2: Establishing protocols for sample preparation of splicesomal complexes using pre-cast gels prior to mass spectrometric analysis. Establishing protocols for the rapid enrichment of phosphopetides using self-made TiO2 columns. Establishing protocols for the chemical stable isotope labeling of peptides derived from in-gel-digest for relative quantitation by mass spectrometry. WP15: Set up of novel mass spectrometric method to detect cross-linked peptide RNA oligonucleotide conjugates derived after hydrolysis of UV irradiated complex with ribonucleases and endoproteinases. Feasibility studies for the TiO2 enrichment and subsequent mass spectrometric analysis of cross-linked peptide-RNA conjugates derived from UV irradiated and subsequently hydrolyzed RNP particles. Generation of computational interface (in collaboration with partner 28, Mihaela Zavolan) that enables the identification of cross-linked protein from a database based on the accurate mass of the cross-linked peptide-RNA oligonucleotide conjugate. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 23.124,15 consumables 13.544,38 training equipment

travel = total direct costs 36.668,53 + overhead 7.333,71 = total eligible costs 44.002,24 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Florian Richter [WP 15] 12 PM 23.124,15 Euro Consumables Chemicals 8.821,33 Euro Laboratory supplies 4.723,05 Euro

Florian Richter is a PhD student working on the development of cross-link identifications between proteins and RNA via mass-spectrometry (WP15). Consumables include general lab chemicals and minor replacement parts for mass spectrometry machines.

149

Participant 2 – Juan Valcarcel a) Work performed during the period • WP2 Shared resources, technology and reliable protocols related to splicing-sensitive microarrays. • WP3 Member of the YIP pre-selection and selection panels • WP5

Part of application for funding of splicing-sensitive microarray work in a systems biology framework. Initiated Spanish splicing network (Plired) involving academics and industry

• WP9

Characterized the role of spliceosomal components and their complexes (U2AF, SPF45, RBM5, TIA-1 / TIAR, PTB, Raver-1) in alternative splicing.

• WP10

Characterized signaling route leading to regulation of splicing factor TIA-1 and its target gene Fas.

• WP11

Led work in this workpackage and contributed splicing microarray experiments in Drosophila and mammalian cells. Characterized the function of TIA-1 isoforms.

• WP14

Collaborative work to explore transcriptome changes under conditions of reduced elongation rate of RNA pol II.

• WP15

Participated in the design and set up of splicing microarray validation experiment. Contributed to the development of these technologies.

• WP17 Host to three collaborative exchanges with other EURASNET groups. • WP18 Provided advice to PhD students and postdocs from EURASNET labs on career moves. • WP20

Contract with Marcelino Botín Foundation for IP protection and commercialization of the research activities of the group.

• WP21 Lectured on RNA biology and alternative splicing in high-schools.

150

• WP22 Deputy coordinator and member of the Stering Committee of EURASNET. b) Major cost items with justification

Participant 2 Juan Valcarcel

Type of expenditure Budget total costs

personnel 49.911,51 consumables 0 training 0 equipment 0

travel 0 = total direct costs 49.911,10 + overhead 9.982,30 = total eligible costs 59.893,81 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Claudia Ben-Dov [WP 11] 1.320 hours 31.584,11 Euro Consumables N/A Travel N/A • Management Personnel Bruna Vives 670 hours 18.327,40 Euro Travel N/A Justification: Dr. Claudia Ben-Dov has carried out microarray design optimization and data analysis for various groups in the consortium (WP11). Bruna Vives carried out secretarial work for dissemination activities coordinated through the Barcelona EURASNET outspost.

151

Participant 3 – Stefan Stamm a) Work performed during the period • WP1 maintenance and further development of the web page, integration of additional content, debugging WP2 Setting up a protocol web interface, preparation of the first practical course in splicing and disease • WP4 generation of a clean set of splicing regulatory motifs that were described in the literature, these data were used to show that exon regulatory elements are preferentially in single stranded regions. • WP6 the data from WP4 were used to show that exon regulatory elements are preferentially in single stranded regions, which was published in Hiller, M., Zhang, Z., Backofen, R., and Stamm, S. (2007). pre-mRNA secondary structure and splice site selection. PLOS Genetics 3, 2147-2155. • WP8 usage of affymetrix and exonhit chips to determine alternative splicing. Published in Novoyatleva, T., Heinrich, B., Tang, Y., Benderska, N., Butchbach, M. E., Lorson, C. L., Lorson, M. A., Ben-Dov, C., Fehlbaum, P., Bracco, L., Bollen, and Stamm. (2008). Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing. Hum Mol Genet, 52-70. • WP10 we could demonstrate that PP1 directly binds to the RRM of splicing factors, published in: Novoyatleva, T., Heinrich, B., Tang, Y., Benderska, N., Butchbach, M. E., Lorson, C. L., Lorson, M. A., Ben-Dov, C., Fehlbaum, P., Bracco, L., Bollen, and Stamm. (2008). Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing. Hum Mol Genet, 52-70 • WP14 the PP1 inhibitor cantaridin and tautomycin regulates alternative splicing and can be a therapeutic agent against spinal muscular atrophy, published in Novoyatleva, T., Heinrich, B., Tang, Y., Benderska, N., Butchbach, M. E., Lorson, C. L., Lorson, M. A., Ben-Dov, C., Fehlbaum, P., Bracco, L., Bollen, and Stamm. (2008). Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing. Hum Mol Genet, 52-70 WP15 See for WP8 and 10: development of array assays WP22 Participation in daily management activities b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 30.304,08 consumables 15.643,52 training 0,.00 equipment 10.125,68

travel 3.763,68 = total direct costs + overhead 11.967,39 = total eligible costs 71.804,35 EC contribution

2006-2007

percentage spent % remaining budget

152

Major cost items • Research Personnel name wp Nataliya Benderska 6,8,10 502 12,566.11 Amit Khanna 1,2,4,14 502 12,566.00 Consumables Tissue culture material (WP10) 5800,00Euro oligonucleotides (WP10) 5000,00 Euro Enzymes (WP8, 14) 4500,00 Euro Laboratory supplies (WP 6, 8, 10, 14, 15) 4184,17 Euro Travel Participation at the EURASENT activities in Oxford and Trieste. The workshop in Oxford, initiated by Chris Smith was to determine which Array system should be used by the consortium. The informal meeting in Trieste was to discuss the role of alternative splicing in disease. • Integration (11.306,25 for upkeep and maintenance of web server, the fees include hosting, backup, reinstallation from backups, viral scans, viral clearance) • Dissemination (na) • Management Personnel Dominique Olbert Wp 22 229 hours 5,742 euro Collection of information from members

153

Participant 4 – Göran Akusjärvi a) Work performed during the period • WP2

Contributed cells and plasmids WP7

Characterized the RNA binding property of the adenovirus splicing factor L4-33K. Constructed substrate RNAs to further characterize the function of L4-33K

• WP9 Established optimal condition for MS2-based affinity purification of spliceosomes from adenovirus. Initiated large-scale purification.

• WP10 The significance of an RS repeat in L4-33K has been tested with respect to phosphorylation and function. The effect of protein kinase A on E1A alternative splicing has been tested.

• WP12 Characterized the function of two alternatively spliced mRNAs encoded by the adenovirus L4 transcription unit, L4-33K, and L4-22K.

• WP13 The work related to this package now done in the context of WP12 • WP18 Initiated the recruitment of an Assistant Professor in RNA and infectious disease. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 43867,23 consumables 7009,45 training equipment

travel 300,06 = total direct costs 51176,74 + overhead 10235,34 = total eligible costs 61412,08 EC contribution

2006-2007

percentage spent % remaining budget

154

Major cost items • Research Personnel Heidi Törmänen [WP 9, 10] 1782 hours 18 969,41 Euro Anette Carlsson [WP 7, 10, 12] 1023 hours 24 897,82 Euro Consumables Oligonucleotides 120,43 Euro Chemicals 1301,13 Euro Enzymes 873,20 Euro Laboratory supplies 4714,68 Euro Travel • Göran Akusjärvi 070201 300,06 Euro The research team working on EURASNET related projects consist of a total of five persons (full and part time workers). Of these the salaries for Heidi Törmänen (PhD student) and Anette Carlsson (technician) are paid from the EURASNET grant. The cost of consumables are mainly taken from other grants (primarily the Swedish Cancer Society and the Uppsala RNA Research Center)

155

Participant 5A/B – Peer Bork/Rolf Apweiler a) Work performed during the period • WP2 5A. Harrington, sharing code with EBI 5B. The EBI hosts the Alternative splicing and transcript diversity (ASTD) database. http://www.ebi.ac.uk/astd/ The data is publicly available and provides the consortium with a genome wide view of alternative splice events and full length transcripts for human, mouse and rat. The ASTD group is happy to incorporate datasets from consortium members that are publicly available and for which there is an interest and continued funding to maintain. • WP4 5A. Harrington, delivering code to EBI 5B. The ASTD database is maintained and updated in response to user feedback, data from consortium members and new publicly available genome wide data. • WP6 5A. Harrington developed a framework for the prediction of conserved alternative transcripts and identified some in as distant organisms as human and fly. suyama further identified as planned conserved regulatory sequences associated with alternative splicing. • WP8 5B. Genome wide splicing factor data generated from this work package can be visualized on the web site, one possibility is as a DAS track on the genome contig view. • WP16 5A. Bork/Harrington attended project meetings • WP17 5A. Harrington visiting Zavolan lab in Basel 5B. Bioinformatics is increasingly being used to improve our understanding of biology. The massive amount of information and bioinformatics tools must also be interlinked to enhance exploration and biologically meaningful interpretation. The EBI is Europe’s main provider of data resources for biologists, also providing tools for searching, retrieving and interpreting the data. The EBI has also a big commitment into “Training Bioinformatics” and has a tripartite User-Training Programme: One initiative is the roadshow, with more information please see here http://www.ebi.ac.uk/training/roadshow/ The second is hands on training for all EBI services within our new purpose-built IT training suite http://www.ebi.ac.uk/training/handson/ An elearning intiative is also being developed for researchers to be trained any time, anywhere, at their own pace.

156

• WP18 5A. Bork/Harrington PhD thesis preparation and defense 5B. In order to promote cooperation within Bioinformatics the EBI has a Visitors Programme. Visitors to the EBI include scientific collaborators, training of postgraduate students, users who need training in the EBI facilities, senior scientists who required a short sabbatical stay and Marie Curies fellowships. Please see here for more information http://www.ebi.ac.uk/training/Visitors_Programme/ The EBI also has a PhD programme as part of the EMBL International PhD Programme Please see here for more information http://www.ebi.ac.uk/training/Studentships/ • WP21 5A. Bork, talks at conferences, Harrington development of web tool 5B. The EBI’s Outreach team coordinates the communication of the scientific mission and activities of the EBI to the community. This includes a wide range of activities aimed at raising awareness of the EBI among potential users of the EBI’s data resources; our peers, our funders and the general public. For more information please see here: http://www.ebi.ac.uk/ott/ b) Major cost items with justification

Participant 5A Type of expenditure Budget total costs personnel 64.036,05 consumables 0 training 0 equipment 0

travel 301,54 = total direct costs 64.337,59 + overhead 12.867,51 = total eligible costs 77.205,11 EC contribution

2006-2007

percentage spent % remaining budget

Participant 5B Type of expenditure Budget total costs

personnel 78.951,12 consumables 64,61 training equipment

travel 3.071,93 = total direct costs 82.087,66 + overhead 16.417,53 = total eligible costs 98.505,19 EC contribution

2006-2007

percentage spent % remaining budget

157

Major cost items • Research Personnel 5A. Suyama Mikita 5.992,36 Euro Harrington Eoghan 8.508,11 Euro Arumugam Mani 22.966,92 Euro Yamada Takuji 26.568,66 Euro 5B. Vincent Le Texier 78.951,12 Euro Personnel costs are charged to the contract on a monthly basis and not by hours. • Integration Web-services – the EBI hosts the ASTD database and web-site • Dissemination

Poster and oral presentation at the Alternative splicing special interest group meeting (AS-SIG) that preceded ISMB 2007.

New release of the ASTD web-site.

158

Participant 6 – Gil Ast a) Work performed during the period • WP4 Group 6 (Ast) used several databases to create a user-friendly interface that allows users to search for splicing regulatory motifs in RNA sequences. WP6: The Ast’s group compiled a unified dataset of splicing regulatory sequences (Goren et al., 2006, Molecular Cell) and generated a website to search for all known splicing regulatory exonic binding sites (http://ast.bioinfo.tau.ac.il/ESR.htm) that is freely available. We also showed correlation between cancer and changes in alternative splicing pattern (Kim et al., 2007), and connection between phenotypic complexity and SNPs (Goren et al., 2007). • WP8 Group 6 (Ast) analyzed groups of PTB-regulated exons provided by Group 23 (Smith) for motif enrichment. • WP13 Group 6 (Ast) demonstrated AS of IKBKAP can be due to modulation of elongation rates. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 9626.91 consumables 24166.67 training equipment 711.62 other 3379.82

travel 1878.18 = total direct costs 39763.19 + overhead 7952.64 = total eligible costs 47715.84 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel

The main personnel cost is for part of scholarship of 6 PhD students for their relative time of research on EURANSET projects.

159

Asaf Levi [WP 6] 12 hours/week 1269.65 Euro Ron buchner [WP 6] 20 hours/week -576.57 Euro Hadas Keren [WP 6] 39 hours/week 3943.56 Euro Nurit Gal [WP 6] 12 hours/week 1290.3 Euro Noa Sela [WP 6] 30 hours/week 3098.03 Euro Schraga Schwartz [WP 6] 6 hours/week 601.92 Euro Consumables

Major consumables costs include sets of primers for the alternative splicing RT-PCR system, along with sequencing runs and associated reagents and chemicals.

Oligonucleotides 12,000 Euro Sequencing 6,466 Euro Chemicals 2,000 Euro Enzymes 2,000 Euro Laboratory supplies 2,000 Euro Travel • RNA meetings/AS Workshop France/visits 1,878 Euro Equipment Hardware and software for new website 711 Euro • Other Processional proofreading and publication costs 3379.82 Euro

160

Participant 7 – Prof. Baralle a) Work performed during the period • WP2: Collaboration with I. Eperon on CFTR and exchange of CFTR exon 9 plasmids

• WP4: Information for the ASD hosted at the EBI has been provided and/or updated as appropriate.

• WP7: With regards to further analysis of RNA secondary structures and their effects on splicing PID7 has continued to further characterize the splicing regulation of the ATM and CFTR pathological pseudoexon inclusions recently described in Buratti et al., NAR, 2007. Using a series of small antisense oligonucleotides we are currently mapping their ability to inhibit pseudoexon inclusion in both the ATM and CFTR systems. The long-range objective of this strategy is to obtain useful informations with regards to optimizing successful therapeutic strategies. In the short term, use of these oligos will also allow to further investigate other facets of pseudoexon inclusion such as splicing kinetics and spliceosomal complex formation.

With regards to mapping RNA-protein complexes on regulatory sequences PID7 has begun a project to characterize the RNA binding proteins that interact with one of the CERES elements within the exon 12 of the CFTR gene. CERES elements are short RNA splicing regulatory elements that, when mutated, can display either enhancer or silencer activities depending on the nucleotide that is altered. They have been originally identified in CFTR exon 12 (Pagani et al., 2002) because clinical studies identified within their sequence several missense mutations, which were subsequently shown to be also capable of affecting the splicing of exon 12. Using a pulldown analysis of wild-type and mutated CERES short RNAs followed by western blot assays against common RNA binding proteins of the SR and hnRNP protein families. Presently, we have identified SF2/ASF, SRp50 and hnRNP A1/A2 and C2 as the major interactors of this CERES sequence and are functionally validating this finding through a overexpression/knock-down approach.

Finally, with regards to the collaboration with the group of Prof. Biamonti in Pavia, the characterization of the silencer elements that bind to a ESS sequence within exon 12 of the Ron oncogene has been continued. Beside hnRNP A1 we have also found that the silencer element is also capable of binding specifically to hnRNP H and functional experiments are currently under way to define the importance of this interaction.

• WP12: PID7 has continued to study several regulatory elements in the genes of interest (CFTR, NF-1, HERG and ATM) from the point of view of their RNA-protein and RNA-RNA interactions. In parallel, PID7 is investigating the effect of knocking down interesting proteins such as TDP-43 a splicing factor involved in several neurodegenerative diseases.

Detailed information is available in the WP12 scientific report. • WP16: Organization of the first interdisciplinary focus meeting (IFM) on mis-splicing and

disease (F. Baralle and F. Pagani - ICGEB, Trieste, Italy) on January 17, 2007 in Cortina d’Ampezzo (Italy) and planning of the second IFM which will take place in Rome on May 31-June 5, 2008 to continue the discussions on RNA and disease with a more clinical approach. The organizers (Claudia Bagni, Francisco Baralle, Juan Valcarcel and Joel Richter) will follow the same pattern used in Cortina and preliminary details are already available at http://cwp.embo.org/cfs08-02/.

Detailed information is available in the WP12 scientific report. • WP17: As already indicated to the WP co-ordinator, Participant 7 did not access the

funding opportunities made available through the Staff Exchange and Training programme.

• WP18: Promotion of the PhD research skills development programme in place for all ICGEB PhD students, and OU registered students in particular, ensuring exposure to an international scenario through oral and poster scientific presentations at meetings and

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conferences (IFM Cortina and IFM Bariloche). Facilitating the incorporation of NoE members in the ICGEB PhD programme through the joint supervision and/or examination of OU PhD Students. Ian Eperon and Angus Lamond are the supervisors of two ICGEB-OU PhD students. Juan Valcarcel has been the examiner of one PhD Student.

• WP19: ICGEB is an international organization dedicated to advanced research and training in molecular biology and biotechnology, with special regards to the needs of the developing world.

Within this framework of activities, PID 7 has contributed to disseminate information and raise awareness of the importance of alternative splicing among the public by participating and co-organizing local and international scientific meetings, intensifying collaborations and providing advisory services to clinicians.

Detailed information is available in the WP12 scientific report. • WP21: As already reported in various occasions, Participant 7 contributes to foster the WP

objectives by enhancing existing links, promoting the establishment of new scientific collaborations, co-organizing a second IFM meeting in Rome (31 May-5 June, 2008 – see EMBO Conference Series, RNA and Disease) and finally, collaborating with G. Biamonti for a grant application submitted to AIRC for funding.

b) Major cost items with justification

Participant 7A Type of expenditure Budget total costs personnel 35,473.64 consumables Training – CORTINA 16,748.5 equipment

travel 20,985.74 = total direct costs 73,207.88 + overhead 14,641.57 = total eligible costs 87,849.46 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel RAPONI [WP 12]1 688 hours 10,400 Euro SKOKO [WP 12]2 168 hours 1,873.64 Euro MARCUCCI [WP 12]3 292 hours 6,000 Euro DE CONTI [WP 12]4 292 hours 6,000 Euro PASSONI [WP 12]5 656 hours 5,200 Euro MORETTI [WP 12]6 232 hours 6,000 Euro

1 Collaborative research on NF1 with group 32

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2 Production of splicing factors and analysis of their binding properties on slicing

3 Exploration of new splicing systems in Drosophila with a view to establish a screening methodology

4 Analyses of the HERG and BRCA1 mutations and their aberrant splicing

5 Establishment of a couple transcription/splicing system to study the dynamis of spliceosome assembly in normal and mutated genes

6 Exploration of new splicing systems in mouse with a view to establish a screening methodology

Consumables = 0 Travels:

Dr. Muro to San Carlos de Bariloche, Provincia di Río Negro, Argentina: partecipation to the meetings “Gene Expression and RNA Processing” and “Cell Biology, Signaling and Alternative Splicing - Eurasnet”

o Dr. Bhuvanagiri to Rome to obtain the UK Visa necessary to travel to a scientific meeting with her Open University supervisor, Dr. Claudio Alonso at the University of Cambridge.

Dr. Marcucci to San Carlos de Bariloche, Provincia di Río Negro, Argentina: partecipation to the meetings “Gene Expression and RNA Processing” and “Cell Biology, Signaling and Alternative Splicing - Eurasnet”

Dr. De Conti to San Carlos de Bariloche, Provincia di Río Negro, Argentina: participation to the meetings “Gene Expression and RNA Processing” and “Cell Biology, Signaling and Alternative Splicing - Eurasnet”

Participation of the Molecular Pathology Group to the 1st Embo Conference Series on RNA and Diseases and WP12 Meeting “IFM on Mis-Splicing and Diseases” in Cortina (Italy)

• Integration WP12 Meeting “IFM on Mis-Splicing and Diseases” in Cortina = 16,748.50 Euro*

*Travel and accommodation costs of invited speakers and other costs relevant to the organization of the IFM

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Participant 8 – Andrea Barta a) Work performed during the period • WP2 The plant alternative splicing community of the EURASNET has created an alternative splicing platform for investigating alternative splicing events in plants by a RT-PCR panel. This panel is now shared between the three plant groups (JB, AB, AJ) and has lead already to interesting results, part of which are published. The protocol has been made available to the member of this network. In addition, plasmids of plant splicing factors within a range of different vectors and different tags have been deposited in a resource data base. • WP4 The alternative splicing panel which is based on 96 known splicing events was used to test these splicing events under different environmental conditions and in various mutant Arabidopsis line. The data were statistically verified and a database for better storage and accessibility of the alternative splicing data has been started to be build. • WP8 The RT-PCR alternative splicing panel for plants has been expanded from the initial 96 AS events to 384. . The AS events have been selected from databases and focused on genes encoding RNA-interacting proteins, transcription factors and genes involved in signalling and stress responses. Transgenic over-expression lines and mutant lines of transacting factors such as SR proteins had been prepared and checked genetically before being analysed. The results of these experiments showed changing AS events in genes which are in pathways which were suspected to be effected by AS through the plant phenotype. Specifically, RAD-1 changed splicing pattern by overexpression of atRSp31 explaining the higher sensitivity of this mutant to UV light. In addition, similar patterns of changes in AS among subsets of genes have been identified and may indicate combinatorial control of AS. • WP9 We have performed yeast two-hybrid screens with plant SR proteins to screen for interaction partners. Interestingly, we found several kinases binding to SR proteins, in particular the SRPK –kinases. We could show that Arabidopsis has four members of this kinase family belonging to two different subfamilies. Characterization of these various kinases regarding their expression pattern and activity towards SR proteins is in progress. • WP11 In this work package alternative splicing of SR proteins in plants have been analysed and it was found that in the plant specific SR protein subfamilies these alternative splicing events are evolutionary highly conserved. In addition, the sequences around the alternative splice sites are conserved on the nucleotide level. Now knock-out mutants of the RSp and RSZ protein families have been isolated and characterized on the molecular level. Double knock-out mutants of the RSZ family have been created and are currently characterized. • WP19 Webpage for science and society issues. On this webpage alternative splicing in general and special mechanisms and topics with the focus on human disease and health are presented in a form comprehensible to the layman. (topics are splicing in general; alternative splicing and mechanisms; impact of alternative splicing on neurological diseases). Outstanding findings and publications of the NoE members are presented and explained. The EURASNET webpage is currently reorganized. It will be rearranged in a way that people visiting the webpage can specifically select for the section of their interest. A link will lead laymen to another path than scientists, medical doctors or teachers. Rearrangement of the EURASNET webpage is ongoing. Training workshop on public understanding of science.

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A media workshop with the aim to provide media training for network members is currently organized. In the framework of the First International EURASNET Conference on Alternative Splicing (23rd -25th May 2008 in Krakow) this media traning will take place. Collection of teaching material about RNA biology. A variety of slides and information leaflets are available on the EURASNET webpage in the “splicing info” domain. Each teaching block offers tables of content and short introductions for easier orientation. The information content reaches from introduction in the biochemistry of RNA in general to special topics of complex RNA biology and metabolism – including alternative splicing and also applications of RNA molecules in modern medical treatment. Addition of information to the EURASNET webpage (splicing info domain) is ongoing b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 37.038,39 consumables 876,05 training 1.205,00 equipment 369,58

travel 2.659,22 = total direct costs 42.148,24 + overhead 8.429,65 = total eligible costs 50.577,89 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Claudia Panuschka. [WP 19] 20h/m/5month: 30h/m/4month 23.962,49 Euro Zdravko Lorkovic [WP 11] 40h/month/3month 13.075,90 Euro Consumables RNA isolation kit 587.00 Euro Enzymes 289,05 Euro Travel 2.659,22 • Yearly Eurasnet Meeting Ile de Bendor for Panuschka, Barta, Kalyna • Workshop Meeting in Berlin for Panuschka, Barta

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Participant 9 – Jean Beggs a) Work performed during the period • WP2 Sharing resources The Beggs lab contributed rabbit anti-yeast Prp8p antibodies. • WP3 YIP As a member of the Steering Committee, J. Beggs participated fully in the YIP selection. • WP5 Durability • WP8 Genome-wide analyses J. Beggs performed many more microarray analyses of splicing in yeast. • WP9 Spliceosome complexity J. Beggs demonstrated that Prp8p, Snu114p and Cwc21p have direct physical interactions, and pinpointed specific amino acid residues that make contact in regions of Cwc21p and Prp8p that are highly conserved from yeast to human. J. Beggs showed that human SRm300, an SR protein that is implicated in splicing regulation in humans, is a functional homologue of Cwc21p. Characterisation of Brr2-containing complexes is underway. • WP12 Splicing and Disease J. Beggs published the analysis of the role of Prp8 in U5 snRNP biogenesis and retinitis pigmentosa: Boon, K.L., Grainger, R.J., Ehsani, P., Barrass, J.D., Auchynnikava, T., Inglehearne, C. and Beggs, J.D. (2007) prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nature Struc. Mol. Biol. 14:1077-83. • WP13 Co-transcriptional J. Beggs showed that the yeast prp45-113 splicing mutant apparently causes a defect in transcription elongation, and found very interesting synthetic genetic interactions with genes encoding chromatin remodeling factors. • WP16 Meetings J. Beggs organized the workshop “High Throughput Approaches in Biology” in Edinburgh, and I am involved with organization of the International EURASNET Conference. I participated in the IFM on Splicing and Disease in Cortina and in 2nd EURASNET meeting in Ile de Bendor and Steve Innocente in my group participated in the 2nd EURASNET meeting in Ile de Bendor and the IFM in Bariloche. • WP17 Staff exchange Alan Urban from Christiane Branlant’s laboratory visited the Beggs lab to perform microarray analyses with yeast RNA. • WP18 Career dev J. Beggs acted as mentor to 5 younger female staff in her Institute, and participated as a mentor at the Mentoring Lunch at the 2007 RNA Society Meeting in Madison. • WP21 Outreach J. Beggs organized an intranet meeting at the RNA UK meeting in January 2008 to discuss the future of RNA UK and whether there is interest in starting a “Splicing UK” meeting. • WP22 Management

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J. Beggs has had frequent contact with Reinhard Rauhut regarding EURASNET business, especially about issues relating to reporting and meeting organization.

b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 9583.64 consumables 32262.47 training 0 equipment 0

travel 2136.06 = total direct costs + overhead 8796.43 = total eligible costs 52,778.60 EC contribution

2006-2007 71,669.72

percentage spent 39.82% remaining budget 108,330.28

Major cost items • Research Personnel N.Barkas [WP 13] 874 hours 9,583.64 Euro

Salary for N. Barkas who worked for 5 months on the Prp45p project (WP13) Consumables Oligonucleotides 3,142.14 Euro Chemicals 6,499.12 Euro Enzymes 5,301.12 Euro Antibodies 6,753.47 Euro Laboratory supplies 10,566.62 Euro Total: 32,262.47 Euro DNA oligonucleotides are used as probes and primers for microarray analyses and to perform quantitative (Real Time) PCR to assess RNApol II and splicing factor recruitment in prp45 mutant strains. Chemicals includes Cy-dyes for microarrays, Protein A Sepharose and IgG agarose for immunoprecipitations, and culture media necessary to grow yeast and bacterial cell cultures Enzymes such as reverse transcriptase, Taq polymerase and specific RNA inhibitors are necessary to perform PCR analysis. Antibodies were raised against Prp45p and Prp8p; commercial antibodies were purchased for ChIP analyses and secondary antibodies for western blotting. Laboratory supplies include essential plasticware, such as pipette tips, tubes, disposable gloves, Petri plates, microwell plates and columns.

Travel

Accommodation costs for J. Beggs to give a talk at the IFM on Splicing and Disease in Cortina: 573.05 Euro

Travel and accommodation for S. Innocente to give a talk at the IFM in Bariloche: 1,158.66 Euro

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Total travel: = 2136.06 Euro Participant 10A – Giuseppe Biamonti a) Work performed during the period • WP2 Sharing Resources, Technology and Reliable Protocols – Ian Eperon 10A provided a protocol for the Northern blot analysis of small RNA. 10A developed a clone of HeLa cells for the site-specific integration of ectopic DNA sequences using the Cre/Lox technology. This system allows a rapid selection of recombinant clones. • WP7 Molecular Characterization of Splicing Substrates – Christiane Branlant. 10A developed a HeLa cell clone that expresses SRp30c fused to the Tap-Tag. This clone provides the opportunity to study RNP complexes comprising SRp30c. It will be applied to the analysis of complexes assembled on SatIII RNAs. The recombinant protein has the same sub-nuclear distribution of endogenous SR factors and is present in speckles in unstressed cells while is recruited to nSBs after heat shock. • WP8 Genome-wide Analyses of Splicing Regulation – Chris Smith 10A in collaboration with Juan Valcarcel used splicing–sensitive micro-arrays to study changes in splicing profile in an in vitro model of Epithelial to Mesenchymal Transition (EMT) • WP10 Post-translational Modification and Dynamic Regulation – Javier Cáceres 10A identified two signaling pathways, identified by ERK and JNK kinases, that are activated during in vitro EMT in poorly metastatic colon adenocarcinoma SW480 cells. Inhibition of ERK partially corrects the splicing profile of the Ron oncogene which is directly involved in EMT • WP12 Mis-splicing and Disease – Francisco Baralle 10A Unproductive splicing controls the expression of SF2/ASF. We found that unproductive splicing of SF2/ASF transcripts occurs in human cancers and during EMT. In SW480 cells this splicing event is controlled by the ERK signaling pathway. • WP13 Co-transcriptional Mechanisms of Alternative Splicing – Karla Neugebauer 10A developed a clone of HeLa cells for the site-specific integration of ectopic DNA sequences using the Cre/Lox technology. This will be exploited to study the effect of chromatin organization on splicing profiles. 10A has also found that splicing factors are co-trascriptionally recruited to sites of transcription of SatIII non-coding RNAs. They also found that SatIII RNAs are detectable at sites of transcription longer than splicing factors (more than 48 h vs. les then 10 h from heat shock). • WP17 Staff Exchange and Training – Angela Krämer A PhD student from 10A spent two weeks in Dr. Valcarcel’s lab to study changes of alternative splicing during EMT by means of splicing-sensitive arrays. • WP18 Career Development – Jørgen Kjems

WP19 Public Understanding of RNA Biology – Andrea Barta 10A gave a public seminar about alternative splicing and disease in front of the Italian Minister of Health • WP21 Reachout to the Broader RNA Community – John Brown

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b) Major cost items with justification

Participant 10A Type of expenditure budget total costs personnel 26.492,74 consumables 30.056,40 training equipment 722,80

travel 1.209,66 = total direct costs 58.481,60 + overhead 23.207,66 = total eligible costs 81689,26 EC contribution

2006-2007

percentage spent % remaining budget

Participant 10B Type of expenditure total costs personnel 60.305,15 consumables training equipment

travel = total direct costs 60.305,15 + overhead 52.827,31 = total eligible costs 113.132,46 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Giuseppe Biamonti [WP 2] 61,5 hours 4.492,22 € Fabio Cobianchi [WP 7] 61,5 hours 2.793,36 €

Fiorenzo Peverali [WP 8] 61,5 hours 1.986,93 € Fabio Cobianchi [WP 10] 61,5 hours 2.793,36 € Claudia Ghigna [WP 10] 61,5 hours 1.986,93 € Claudia Ghigna [WP 12] 184,5 hours 5.960,79 €

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Fiorenzo Peverali [WP 13] 61,5 hours 1.986,93 € Giuseppe Biamonti [WP 17] 61,5 hours 4.492,22 € Total 26.492,74

Consumables Cell culture 2506,64 € Plastic 8.335,18 € Reagents for Mol. Biol 7560,46 € Sequencing 198,90 € RT-PCR 5.591,25 € Antibodies 2.188,97 € Confocal microscopy charges 2500 € Thermalcycler 722,80 € Travel Most of the work entails the analysis of RNAs extracted from cells at different growing conditions. This accounts for cell culture costs (media, antibiotics, Petri dishes and pipettes). RNA analysis requires: RNA extraction kits, RT-PCR reagents and plastic. Sequencing of some RT-PCR products is also required. Immunofluorescence experiment to follow EMT require antibodies, confocal microscopy. A thermalcycler bought the previous year is routinely used for RT-PCR analysis Giuseppe Biamonti superintended to the development of protocols. Analysis of the results of the splicing-sensitive microarrays. Fabio Cobianchi Development of the HeLa cell clone expressing SRp30c fused to the Tap Tag and analysis of signal transduction pathway during in vitro EMT Claudia Ghigna Analysis of signal transduction pathway during in vitro EMT. Analysis of the splicing profiles of Ron, SF2/ASf and additional genes for splicing factors during in vitro EMT. Validation of the experimental system Fiorenzo Peverali Production of RNAs from SW480 cells grown at different density. Validation of the cell system. RT-PCR analysis. Development of the Cre/Lox system in HeLa cells• •

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Participant 10B – Glauco Tocchini-Valentini a) Work performed during the period • WP2 10B participated in WP-specific networking activities • WP8 10B developed novel applications of the original cis- and trans- Archaea-express technology for analysis of alternative splicing-specific, using yeast (S. cerevisiae) and mouse cultured cell models. • WP11 10B proceeded with experiments for application of the original cis- and trans- Archaea-express technology to the identification of various RNA spliced isoforms and their selective inactivation or modification. • WP14 10B carried out experimental tests for the development of novel cellular assays of inhibitors/modulators of alternative mRNA splicing, using yeast (S. cerevisiae) and mouse cultured cell models. • WP15 10B constructed and used novel plasmids for stable expression of tagged mRNA species, for studies on alternative splicing and targeted, post-transcriptional RNA modification, based on the original cis- and trans- Archaea-express technology • WP17 10B participated in WP-specific management activities. • WP18 10B participated in WP-specific management activities • WP20 10B participated in WP-specific management activities b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 60,305.15 consumables 0 training 0 equipment 0

travel 0 = total direct costs 60,305.15 + overhead 0 = total eligible costs 60,305.15 EC contribution

2006-2007

percentage spent % remaining budget

171

Major cost items • Research Personnel

WP name person-months

person-hours

cost (Euro)

2

Glauco Tocchini Valentini Research Director 1 135 6,287.61

8 Sabrina Putti Researcher 5 675 11,188.50

11 Giancarlo Deidda Staff researcher 3 405 13,005.35

11 Giuseppe Di Franco Senior Technician 1 135 3,142.94

14 Chiara Di Pietro Researcher 5 675 8,490.85

15 C. Di Pietro Researcher 5 675 8,490.85

17 C. Di Pietro Researcher 1 135 1,698.17

18 Serena Gastaldi Staff researcher 1 135 4,000.44

20 S. Gastaldi Staff researcher 1 135 4,000.44

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Participant 11 – Albrecht Bindereif a) Work performed during the period • WP2 Added our splicing constructs to list of reagents. • WP8

The splicing regulator hnRNP L was downregulated by RNAi in cell culture, RNA was prepared and analyzed by splice-sensitive exonarrays from Affymetrix. Data analysis was established and resulted in the identification of new modes of splicing regulation by hnRNP L and new targets. Alternative splicing effects on new targets were validated by quantitative RT-

PCR analysis. Publication: Hung, L.-H., Heiner, M., Hui, J., Schreiner, S., Benes, V., and Bindereif, A. (2008). Diverse roles of hnRNP L in mammalian mRNA processing: a combined microarray and RNAi analysis. RNA 14, 284-296.

• WP12 Minigene derivatives of new hnRNP L targets were constructed, as well as mutant derivatives thereof, and studied by transient transfection in mammalian cells, followed by RT-PCR assays. Quantitation was done by real-time PCR studies (publications: see above under WP8, and in addition, Lorenz et al. (2007): Lorenz, M., Hewing, B., Hui, J., Zepp, A., Baumann, G., Bindereif, A., Stangl, V., and Stangl, K. (2007). Alternative splicing in intron 13 of the human eNOS gene: a potential mechanism for regulating eNOS activity. FASEB J. 21, 1556-1564. (This latter publication is a collaboration with clinicians (Stangl, Stangl, Baumann; cardiologists at the Charité Berlin; from my lab: Hui and Bindereif), where we have contributed special splicing expertise and experiments. It focusses on alternative splicing of the human eNOS gene, a candidate gene for coronary artery disease).

• WP19 Contributed a short summary on our alternative splicing interests and activities for GenomXpress (in German), a publication, which is intended to make projects funded by the German Genome Project (NGFN-II) understandable for the general public.

b) Major cost items with justification

Participant 11 Type of expenditure Budget total costs personnel 30,941.24 consumables 2,134.49 training equipment

travel = total direct costs 33,075.73 + overhead 6,615.15 = total eligible costs 39,690.88 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Lee-Hsueh Hung [WP 8, 12, 19] 720 hours 20,839 Euro Silke Schreiner [WP 8, 12] 1,080 hours 10,101 Euro Consumables Literature 64 Euro Sequencing 2,070 Euro

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Participant 12A – Jamal Tazi a) Work performed during the period • WP2 Distributed 10 vectors and 6 Drosophila strains to other partners. • WP9 Optimization of conditions for the in vitro assembly of RNP complexes on the wild type and mutated LMNA sequence, and for their affinity purification. • WP10 Identification based on a genetic screen in Drosophila that Topo I is able to rescue the phenotypes induced by B52 overexpression. Topo I is demonstrated to phosphorylate SR proteins during drosophila development and is perfectly co-localizing with these factors on polythene chromosomes. • WP11 Performed validation of microarray data from Drosophila over expressing SR protein splicing facrtors. Identification of candidate gene involved in the biogenesis of neurones and the development of the visual system.

• WP12 Identification and characterization of splicing factors implicated in the deregulated control of splicing of mutated LMNA mRNA in the case of Hutchinson-Gilford progeria syndrome (HGPS). Establish human fibroblasts from aged individual in order to determine the expression of splicing factors involved in the HGPS Characterisation of a humanized mouse model containing knock in of the human LMNA gene which, harbours the HGPS mutation in exon 11. • WP14 Managing Workpackage 14. Identification and test of efficacy of lead molecules targeting SR proteins that:

- inhibit HIV1 replication - Block cell motility of metastatic cell lines. Test of these molecules in a mouse model of breast cancer. - Allow skipping of mutated exon 51 of Dystrophin gene in DMD. Test of the efficacy of the molecules

in mouse model of DMD the Mdx mice. Design, synthesis and screen analogues of lead molecules for maximum activity and minimum cytotoxic activity. • WP16 Organizer of La Grande Motte EURASNET IFM in 2007. Organizer of the EURASNET reporting meeting in Iles de Bendor 2007. • WP19 Several interviews to local (6), national (7) and international media (2) about molecules that inhibit splicing and thereby block HIV-1 multipilcation. Presented EURASNET-related material at 6 meetings and 10 seminars. • WP20 Collaboration with Sigma Aldrich and Affymetrix for the organization of meetings. Negotiating with industrials and clinicians the development of drugs that inhibit HIV-1 multiplication.

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b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 51.695,15 consumables 8507.08 training equipment

travel 5855.09 Audit certificate = total direct costs 66.057,32 + overhead 13.211,46 = total eligible costs 79.268,78 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel VAN TRAN BA Christophe WP12-14 130 hours 1834.19 Euro FERNANDO Céline WP10 130 hours 2085.10 Euro ZEKRI Latifa WP9-12-14 1040 hours 25912.58 Euro DE TOLEDO Marion WP9-14 910 hours 21863.28 Euro TAZI Jamal WP2 -12-14 1560 hours 23558.73 Euro Consumables Oligonucleotides 136 Euro Chemicals 509 Euro Enzymes 0 Euro Laboratory supplies 0 Euro Travel • meeting 1723 Euro • Integration IFMs 7939 Euro • Dissemination

annual meetings 4053 Euro

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Participant 12B – Bertrand Séraphin a) Work performed during the period • WP2 We sent plasmid constructs suitable for Gateway cloning to Partner 3. • WP9

- RES complex: We have engaged in a structural analysis and we are trying to correlate structure and function, including the choice of alternative splice sites. -EJC: We tested whether an EJC recruited after the splicing of an intron could have a role in modulating alternative splicing of a (slower-splicing) nearby intron through recruitment of specific factors. We got indications that this could be true but we have not yet been able to demonstrate that this is really a direct result of the EJC formed on the first intron. - Snu30: We completed the work on human and yeast Snu30 that had begun earlier. We demonstrated that Snu30 affect alternative 5' splice site selection. (See Puig O, Bragado-Nilsson E, Koski T, Séraphin B. The U1 snRNP-associated factor Luc7p affects 5' splice site selection in yeast and human. Nucleic Acids Res. 2007;35(17):5874-85. Epub 2007 Aug 28).

• WP10

- SF1/BBP: we have been analysing the role of its phosphorylation in collaboration with Angela Krämer (partner 21). We obtained interesting preliminary results suggesting a dominant negative effect but after more work this turned out to be an artefact.

• WP13

- We collaborated with partner 1b on the role of the various domains of Mud2 in co-transcriptional spliceosome formation. A collaborative manuscript that includes these results is in preparation.

• WP15

- We worked on improving the TAP method both for complex retrieval from yeast and mammalian cells or for complex assembly analysis. - We performed a feasibility test to check whether mutagenesis could be used to identify protein interaction surfaces. This will be pursued by more extensive analyses of this strategy.

b) Major cost items with justification

Participant 12B Type of expenditure Budget total costs personnel 125 391,66 consumables 5 631,99 training 0 equipment 0

travel 2,80 = total direct costs 131 026,45

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+ overhead 26 205,29 = total eligible costs 157 231,74 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel B. Séraphin* [WP 2, 9, 10, 13, 15] 676 hours 45 218,96 Euro H. Le Hir* [WP 9, 15] 1565 hours 61 935,99 Euro C. Faux* [WP 9, 10, 13, 15] 118 hours 2 505,36 Euro B. Marchadier** [WP 9,15] 1565hours 11 250 Euro C. Barrandon*** [WP 9, 15] 264 hours 7481,35 Euro * CNRS personnel (due to cost system) ** Ph.D student (on independent fellowship but indicated as part of the CNRS funding due to our cost system) *** Post-doc on EURASNET funding (started Nov. 2007) Consumables Oligonucleotides 2387,69 Euro Laboratory supplies 982,80 Euro Consumables for lab experiments. The major fraction coming from the CNRS budget cannot be reported by the CNRS administration. Travel • Travel to Bendor 2,80 Euro

EURASNET annual meeting.

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Participant 12C – Christiane Branlant a) Work performed during the period •WP2 Construction of yeast strains that produce U2 snRNAs partially or totally depleted of their pseudouridylations. These strains were used by partner 9 for DNA ships assays. •WP7 In collaboration with Partner 12a, study on the 2D structure of the WT and mutated pre-lamin A pre-mRNA, to test why a 5’ cryptic site is used in Progeria. In collaboration with partner 25, study on RNA-protein interactions at several HIV-1 RNA splicing sites and their regulatory elements and at the two extremities of the human troponin T exon 5 that is included in Type 1 Muscular Dystrophy. Investigation on the MBNL1 RNA binding properties, by SELEX and triple-hybrid approaches. •WP9 Identification by RNP purification and mass spectrometry of HeLa cell nuclear proteins which are capable to bind HIV-1 RNA regions containing splicing sites and their regulatory elements, the Rous sarcoma virus NRS element, RNA containing repeated CUG motifs which are implicated in Type 1 Muscular Dystrophy. •WP14 Study of the effect on HIV-1 splicing of an indole derivative that blocks ASF/SF2 activation and blocks HIV-1 multiplication (collaboration with partner 12a) •WP17 We settled two new collaborations in the frame of the network: one on the splicing defect responsible for progeria with a partner who Partner 12a, the other one with a partner partner 9 on the effect of pseudouridylations in U2 snRNA on splicing efficiency in yeast. •WP18 In its national and local responsibilities, C Branlant is giving advises to several young scientists. We are training several PhD students in the Lab and try to help them in developing their career at the best level. Two of them went as post-doctoral fellows in teams of the network: partner 16 and YIP 29. We are teaching to Master 1 and 2 students, receive them for stay in the Lab and try to motivate them to become scientists and in particular RNA specialists. •WP20 We characterized the action of chemical compounds against HIV-1 RNA splicing, we are the reference laboratory for quality tests of the nuclear extracts commercialized by the CIL Biotech company (Belgium). We are testing the effect of various HeLa cell growth conditions on the quality of splicing extracts. •WP21 C Branlant is member of numerous French and international comities deciding of scientific orientations and explains the importance of the RNA field in particular metabolism and splicing. Twice a year, we organize open days at the university and are explaining the importance of RNA splicing for gene expression and diseases to many people who visit the Lab. b) Major cost items with justification

Participant 12c Type of expenditure Budget total costs personnel 92.996,28 consumables 5.786,30 training equipment

travel 5.220,09 Audit certificate = total direct costs + overhead 20.800,54 = total eligible costs 124 803,21 EC contribution

2006-2007

percentage spent % remaining budget

178

Major cost items • Research Personnel Christiane Branlant (1) [WP 7, 9, 14, 18, 20, 21] 234 hours 17 074,78 Euro Iouri Motorine (2) [WP 2, 9, 17, 20] 147 hours 8 389,29 Euro

Athanase Visvikis (2) [WP 7, 9] 100 hours 5 707,00 Euro Aileen Bar (3) [WP 7, 9] 205 hours 2 978,65 Euro Jean-Michel Saliou (3) [WP 7] 250 hours 3 632,50 Euro Lilia Ayadi-Ben Mena (1) [WP 2, 7, 14, 20] 547 hours 15 941,88 Euro Christophe Charron (1) [WP 7, 9, 14] 115 hours 3 795,02 Euro Fabrice Leclerc (1) [WP 14] 469 hours 18 339,51 Euro Xavier Manival (1) [WP 7, 9, 14] 357 hours 12 932,08 Euro Severine Massenet (1) [WP 2, 7] 116 hours 4 205,57 Euro

(1) CNRS personnel (due to cost system) (2) University Henri Poincaré (due to cost system) (3) Ph. D student ( on independent fellowship but indicated as part of the UHP funding due to our cost

system) Consumables Chemicals 212,00 Euro Enzymes 4 534,30 Euro Laboratory supplies 1 040,00 Euro Travel • Travel to Cortina d’Ampezzo 1 650,41 Euro Congress “pre mRNA processing and disease” • Travel to Bendor 934,85 Euro

Eurasnet annual meeting • Travel to Edinburg 2 407,01 Euro

Eurasnet collaboration • Travel to Montpellier 210,35 Euro Eurasnet work reunion • Travel to Paris 17,47 Euro Scientific Reunion

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Participant 12D – Edouard Bertrand a) Work performed during the period • WP2

Exchange of microscopic slides, plasmids and protocols. • WP10

-Measurement of snRNP recruitment at the transcription site of an HIV-1 reporter gene. -Measurement of the dynamic of U1A, U1-70K, and SF2/ASF at the transcription site of an HIV-1 reporter gene, bu FRAP. -Construction reporter cell lines expressing an MS2-tagged MINX pre-mRNA. -Pilot expriment

• WP13

-Estimation of the transcription rate of RNA polymerase II on an HIV-1 reporter gene by the MS2 FRAP assay, and by FRAP of RNA polymerase II. -Measurement of the effects of UV light on the transcription rate of RNA polymerase II.

• WP 16

-Participation to meetings. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 100.066,22 consumables 190.22 training equipment

travel 233.05 Audit certificate = total direct costs 100.489,49 + overhead 20.097,89 = total eligible costs 120.587,38 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel RAGE Florence [WP 10] 780 hours 35295 Euro BERTRAND Edouard [WP 10,13] 458 hours 20386 Euro BOIREAU Stéphanie [WP 2, 10, 13, 16] 1560 hours 39655 Euro MOLLE Dorothée [WP13] 450 hours 4730 Euro Travel

meeting 233 Euro

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Research Personnel: Generating the MS2 constructs and cell line is time-consuming and required a important effort (about 2 months for a single cell line). Some FRAP experiments are rapid (one data set/week) but require extensive testing of experimental conditions and many repetitions. The MS2 FRAP experiments are more time-consuming (one data set/month). Stéphanie Boireau performed most of the imaging experiments reported in WP10 and WP13, and was supervised by Edouard Bertrand. Stéphanie Boireau, Florence Rage, Dorothée Molle, Edouard Bertrand generated the constructs and cell lines required for these experiments.

181

Participant 13 – Daniel Schümperli a) Work performed during the period WP5

• The participant formed a group of researchers (EURASNET participants and others) interested in therapeutic methods to modulate splicing to discuss about future funding. The discussions should become more concrete and potentially lead to one or more applications in 2008 and 2009.

WP12 WP14

• The group continued to work on the inhibition of HIV-1 replication by short RNAs (siRNAs that induce RNA degradation and U7 snRNAs that modulate splicing), in particular by looking at viral replication in primary human T-lymphocutes.

• Moreover the group has developed U7-based methods to induce exon inclusion in survival of motoneuron 2 gene (SMN2), which could be used in gene therapy for spinal muscular atrophy. Regarding this issue they now have very preliminary evidence that this approach works in vivo in a very severe mouse model for SMA where symptoms are clearly reduced and survival of the animals is prolonged.

• Moreover, they have made significant progress towards engineering inducible U7 snRNA cassettes.

Publications: 1. Asparuhova, M.B., Marti, G., Liu, S., Serhan F., Trono, D. and Schümperli, D. (2007) Inhibition

of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping. J. Gene Med. 9, 323-334.

2. Marquis, J., Meyer, K., Angehrn, L., Kämpfer S.S., Rothen-Rutishauser, B. and Schümperli, D. (2007) Spinal Muscular Atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence. Mol. Therapy 15, 323-334.

3. Scholl, R., Marquis, J., Meyer, K. and Schümperli1, D. (2007) Spinal Muscular Atrophy: position and functional importance of the branch site preceding SMN exon 7. RNA Biology 4, 34-37.

WP18

• Prof. Schümperli entertains a mentoring group of 10-12 people (students from the science and medical faculties of all levels) at the University of Bern.

• Moreover, he tries to send my students and postdocs to other labs and courses whenever possible. One of his PhD students gave a talk in Trieste in a workshop/course arranged by F. Baralle. His postdoc Julien Marquis gave a talk in Cortina and has been at several discussions with people from one of our sponsors, the Association Française contre les Myopathies (AFM). Finally another student has been in M. Carmo Fonseca's lab for a two courses on imaging/confocal microscopy; yet another student has been in Göttingen as part of a collaboration with R Lührmann's and H Urlaub's groups.

WP20

• The participant has made steps to obtain a patent for a a novel U7-based tool to enhance exon 7 inclusion in SMN2, a potential therapeutic target gene for Spinal Muscular Atrophy (SMA). The invention cannot be patented by itself since data on the tool have already been published. However, the idea is to try to obtain a patent in collaboration with the group of Dr. Martine Barkats at Genethon, Evry, France who are developing vectors capable of delivering the tool to motor neurons. It is not yet certain whether this strategy will lead to an accepted patent.

• Additionally participant 13 is collaborating with the groups of Prof. Christian Leumann, Department of Chemistry and Biochemistry, University of Bern, and that of Dr. Luis Garcia, Institut de Myologie Paris, France, on tricyclo-DNA applications to exon skipping in Duchenne Muscular Dystrophy (DMD). The tricyclo-DNA chemistry itself is no longer patentable, again for reasons of prior disclosure, but the collaborating groups hope to obtain a patent for the special application of their technology to DMD.

182

WP21

• Organisation of the Swiss RNA workshop 07 on September 14 2007 in Bern (jointly with Angela Krämer [EURASNET participant 21] and Oliver Mühlemann). Several talks and poster presentations, some of them from EURASNET member laboratories, focused on aspects of alternative splicing): Talks by Jernej Ule (CLIP) and Dominguez/Allain; Posters by Choleza/Krämer, Marquis/Schümperli, Tanackovic/Rivolta, von Känel.

• Main collaborations: Prof. Didier Trono, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland (lentiviral vectors, mouse transgenesis, HIV multiplication assays and reagents) Dr. Martine Barkats, Généthon-CNRS FRE 3018, Evry, France (AAV vectors, somatic gene transfer) Dr. Barbara Rothen-Rutishauser, Institute of Anatomy, University of Bern, Switzerland (Confocal microscopy) Prof. Christian Leumann, Department of Chemistry and Biochemistry, University of Bern, Switzerland (Oligonucleotide chemistry)

• Additionally, the participant 13 entertains many contacts with labs in France that are sponsored or involved in the organisation of AFM (Association Française contre les Myopathies) where there is a lot of interest in exon skipping or inclusion therapies.

b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel consumables 8,480 training equipment

travel = total direct costs 8,480 + overhead = total eligible costs EC contribution

2006-2007

percentage spent % remaining budget

Note that the University of Bern did not allow us to use all the planned budget, as the money for 2007 has not been transferred from the EU to us. In fact we would have spent all of it and more if we could have. Major cost items • Research Consumables Oligonucleotides 1,480 Euro Chemicals 1,000 Euro Enzymes 3,000 Euro

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Laboratory supplies 3,000 Euro (rough estimates) c) Main activities planned for the coming year:

• Our group will further exploit the mouse SMA model mentioned above to: (i) determine the time point when SMN production is critical and requires therapeutic boosting; (ii) study the ability of mutant SMN to counteract the SMA phenotype; (iii) study the role of SMN in motoneurons; (iv) develop ways to deliver the therapeutic U7 transgene to motoneurons.

• We plan to develop a system whereby U7 snRNA expression can be induced or turned off by cre-lox recombination

• We will further improve our U7-based exon skipping strategy. • Concerning the analysis of the role of PTB in pseudoexon splicing, we have just started a study to

look at whether PTB-nPTB or other neuron-specific splicing regulators affect the splicing of agrin, a protein important for synapse establishment and/or maintenance.

184

Participant 14 – John W. S. Brown a) Work performed during the period • WP1

During the reporting period the EURASNT management contacted me about taking over the running of the EURASNET website and creating a new dynamic website targeting specific groups of potential end-users. Following detailed discussions of the specifications of the new website a team of people have been assembled to generate and maintain the new website. The team includes a part-time post-doctoral scientists, the Public Understanding of Science officer and Communications and IT personnel from the host organization (SCRI). Work on the new website is progressing.

• WP6

A collaboration has been set up with Mihaela Zavolan to identify potential mRNA targets of novel, “orphan” snoRNAs from plants.

• WP8

The three plant labs (Barta, Brown, Jarmolowski) have made significant progress on expanding the alternative splicing RT-PCR panel for examining AS in Arabidopsis. The panel is being used to analyse a range of mutants in splicing factors and other genes involved in mRNA biogenesis. One of the important results from these initial analyses is the detection of many novel RT-PCR products which when characterized represent new AS events. This points to the far greater prevalence of AS in plants that current bioinformatic analyses predict. This is a strong collaboration among the three plant labs which has already involved post-doc and PhD student exchanges, which will continue over the next two years or so. In addition, a number of other plant labs in Europe wish to utilize the system and collaborations are being established which demonstrate activities in translating the EURASNET sponsored research to the wider RNA community.

• WP11

We are continuing to prepare over-expression lines of the Arabidopsis PTB-like protein isoform genes, PTBL1and PTBL3 have been prepared and are currently being transformed into Arabidopsis. Putative knock-out mutants have been identified and are being characterized genetically. Once prepared these mutant and over-expression lines will be analysed on the Arabidopsis AS RT-PCR panel.

• WP21

I manage the WP21 workpackage - Broader outreach to the wider RNA community. In the reporting period we have built good relations with WP19 – Public Understanding of Science and have taken over WP1 – the EURASNET website. There is clearly some overlap between the activities of these WPs and it makes sense for them to interact efficiently, co-ordinate activities, share some tasks and remove redundancy. One example is that we have taken measures to capture the wide range of activities in terms of contacts with the wider RNA community which EURASNET members are engaged in.

We have continued to expand the list of European RNA researchers to be able to target information about alternative splicing and EURASNET activities (meetings, workshops etc). Now that the second round of YIPs has been confirmed, we will produce a new poster for the network.

b) Major cost items with justification

185

Participant 14 Type of expenditure Budget total costs personnel 19,061.94 consumables 30,172.19 training 0.00 equipment 4,647.49

travel 2,146.40 = total direct costs 56,028.02 + overhead 10,060.06 = total eligible costs 66,088.08 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel

The main personnel cost is for a part-time post-doctoral scientist (Dominka Lewandowska). Towards the end of the reporting period some personnel time from the website team has accrued with the take-over of the website (WP1) and Dr. Lewandowska will become full-time from February 2008.

Dr. Dominika Lewandowska [WP 8/11/21] 20 hours/week 11,274 Euro Consumables

Major consumables costs include sets of fluorescently labelled primers for the alternative splicing RT-PCR system, along with sequencing runs and associated reagents and chemicals.

Oligonucleotides 12,000 Euro Sequencing 4,000 Euro Chemicals 2,000 Euro Enzymes 2,000 Euro Laboratory supplies 2,000 Euro Travel • RNA meetings/AS Workshop France/visits 2,000 Euro Equipment Hardware and software for new website 4,700 Euro • Integration Initial design and development of new website 8,000 Euro • Dissemination RNA meetings/AS Workshop France

186

Participant 15 – Javier F. Caceres a) Work performed during the period • WP8

We have starting using the CLIP protocol (Cross-linking and immunoprecipitation) to identify RNA targets of SR family proteins and hnRNP proteins

• WP10 I am the lead participant in WP10. In collaboration with Angus Lamond’s lab we have used FRET/FLIM microscopy to study the interaction between SR proteins and splicing components that are bound at the 5’ or 3’ splice site. FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM) allowed the mapping of these interactions to specific sites in the nucleus. This has resulted in a joint EURASNET-member publication (Ellis et al., 2008).

b) Major cost items with justification

Participant 15 Type of expenditure Budget total costs personnel 8704.67 Consumables & Other 14890.14 training equipment

travel 5884.23 = total direct costs 29,479.04 + overhead 5,895.81 = total eligible costs 35,374.85 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel : 8704.67 Jonathan Ellis. [WP 10] 2 months/ Ph.D. extension

Stephanie Hartz [WP 10] 2 months/Summer studentship

Consumables: 14,890.14

187

Oligonucleotides 2,639.Euro Chemicals 2,981Euro Enzymes 2,978Euro Laboratory supplies 6,292.14 Euro

Travel: 5,884.23 (some are itemized below) RNA Society meeting 2008

Javier Caceres: 989.88 Keystone RNAi meeting

Gracjan Michlewski: 1,010 Javier Caceres: 1,253.02

Eurasnet Reporting meeting/Berlin, January 2008/ Javier Caceres: 273.20

IMF “"Cell biology, signalling and alternative splicing", Bariloche Dasa Longman: 941.98 Eukaryotic mRNA processing meeting Javier Caceres: 597.40

188

Participant 16 – Maria Carmo-Fonseca a) Work performed during 2007 • WP2: shared plasmids, antibodies and protocols • WP3: participated in the identification of candidates and selection procedures • WP5: Preparation of information package; preparation of application to FP7 • WP9: Modeling intranuclear trafficking kinetics of spliceosomal proteins • WP10: FRAP and FRET analysis of splicing factor interactions and kinetics in the living cell nucleus • WP11: Microarray analysis of tissue-specific expression profiles of splicing factors • WP13: Role of the CTD of RNA pol II on pre-mRNA splicing and release from the transcription site • WP16: participated in the planning of conferences, workshops and meetings • WP17: contributed to training through staff exchange • WP18: participated in career development actions • WP21: participated in outreach activities • WP22: contributed to Network management b) Major cost items with justification

Participant 16 Type of expenditure Budget total costs personnel 36,752.00 consumables 12,480.61 training equipment

travel 10,310.79 other (the rest) = total direct costs

59,543.40

+ overhead 11,908.68

= total eligible costs 71,452.08 EC contribution

2006-2007

percentage spent % remaining budget

189

Major cost items • Research Personnel Name WP Person-months Euros Inês Mollet 11 12 16,954.50 Houda Hallay 9 11 16,445.00 Joana Borlido 11 2 1,490.00 José Nuno Pereira 11 3 1,862.50 Consumables Laboratory supplies, chemicals, reagents for molecular biology: 12,480.61 Euro Travel Name Date Place Event Natalia Kozlova Jan 14-17, 2007 Cortina, I EMBO pre-mRNA processing and disease Marco Campinho Feb 25-27, 2007 London, UK Training visit lab Masa Tada Ana Grosso Mar 9-10, 2007 Coimbra, PT Workshop Statistics in Genomics & Proteomics M. Carmo-Fonseca Ap 14-16, 2007 Ile Bendor, F EURASNET Annual Meeting M. Carmo-Fonseca May 28-Jun 5 Madison, USA XII RNA Meeting Ana Grosso May 28-Jun 5 Madison, USA XII RNA Meeting Inês Mollet Jul 18-25, 2007 Vienna, A Alternative Splicing-Special Interest Group Meeting Ana Grosso Jul 18-25, 2007 Vienna, A Alternative Splicing-Special Interest Group Meeting Ana Grosso Aug -28 NY, USA Cold Spring Harbor Laboratory Meeting: Eukaryotic

mRNA Processing Marco Campinho Aug 26-Sep 1 Helsinkia, FL EMBO Practical course Ana Grosso Nov 25-28 Barcelona, E BioMed Workshop – Bioinformatics Resource Days M. Carmo-Fonseca Nov 25- Dec 1 Bariloche

(Arg)

Benjamin Blencowe (Canada)

Jan 28-30, 2007 Lisboa Lab visit, seminar and discussions with PhD students

Manuel Santos (PT)

Jan 29-30, 2007 Lisboa Lab visit, seminar and discussions with PhD students

Juan Valcarcel (E) Feb 13-14, 2007 Lisboa Opponent in PhD viva (Luis Mendes Soares) • Dissemination & Training Natalia Kozlova, Marc Schneider, Reinhard Lührmann and Maria Carmo-

Fonseca. 14th January-17th January 2007 – 1st EMBO Meeting on pre-mRNA processing and disease held in Cortina d´Ampezzo, BL, Italy – Poster presentation entitled “AG-dependent and AG-independent variants of IgM form distinct commitment complexes.”

Borlido, J., Martins, S., Mollet, I. and Carmo-Fonseca, M. 14th January-17th

January 2007 – 1st EMBO Meeting on pre-mRNA processing and disease held in Cortina d´Ampezzo, BL, Italy – Poster presentation entitled “ RT-PCR validation of

190

U2AF35 and U2AF35-like alternatively spliced isoforms predicted through bioinformatics strategies.”

Carmo-Fonseca, M. Tissue specific differences in splicing factor expression levels.

EMBO Conference “pre-mRNA processing and disease” Cortina d’Ampezo, Italy, January 14-17, 2007.

Marco Campinho - 16-28 July 2007 - EMBO Practical course on Animal Models for Development, Physiology and disease. Sheffield, UK.

Marco Campinho - 27-31 August 2007 - EMBO Practical course in Genome-

wide SNP association studies. Helsinki, Finland

Ana Rita Grosso - November 26 th to 28 th , 2007 – Barcelona BioMed Workshop – Bioinformatics Resource Days , organised by the Institute for Research in Biomedicine (IRB Barcelona) and the Barcelona Supercomputing Center (BSC), Barcelona (Spain).

Ana Rita Grosso. March 9 th to 10 th , 2007 – Presentation of the talk “Identification of tissue-specific splicing related factor signatures” in Workshop on Statistics in Genomics and Proteomics Follow-up Meeting” in Coimbra (Portugal). Natalia Kozlova, Marc Schneider, Reinhard L ührmann and Maria Carmo- Fonseca. 14-18 April 2007 EURASNET annual meeting 2007, Ile de Bendor, France - Poster presentation entitled “AG-dependent and AG-independent variants of IgM form distinct commitment complexes.” Ana Rita Grosso. May 29 th to June 3 rd , 2007– Presentation of talk “ Identification of tissue-specific splicing-related gene signatures ” in XII RNA Meeting in Madison (USA) Ana Rita Grosso. July 19 th to 25 th , 2007– Presentation of poster “Identification of tissue–specific splicing-related gene signatures” in Alternative Splicing-Special Interest Group Meeting of the 15 th Annual International Conference on Intelligent Systems for Molecular Biology. Ana Rita Grosso. August 22sd to 26 th , 2007– Presentation of poster “Identification of tissue–specific splicing-related gene signatures” in Meeting Eukaryotic mRNA Processing in Cold Spring Harbor Laboratory, NY (USA).

PhD Thesis: Luís Manuel Mendes Soares (2007) “Role of DEK in 3’ splice site definition by U2AF” Faculdade de Medicina da Universidade de Lisboa. Supervisors: Juan Valcarcel and M. Carmo-Fonseca.

191

Participant 17 – I.C. Eperon a) Work performed during the period

• WP7: development of single molecule FRET assays of RNA conformation in nuclear extract, single molecule detection of binding by fluorescently-labelled splicing proteins to pre-mRNA in splicing reactions, cloning of some splicing factors in expression vectors. • WP12: testing effects on recovery of SMN2 splicing when TOES oligonucleotide is altered in respect of its tail length, site of annealing, tail sequence and chemistry; analysis of splicing in vitro and in patient fibroblasts; analysis of uptake and persistence by FRET begun. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 43389.22 consumables 5539.92 training 0 equipment 0

travel 0 = total direct costs 48929.14 + overhead 9785.84 = total eligible costs 58714.98 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel D. Cherny [WP 7] 416.25 hours 16644.63 Euro L.P. Eperon [WP 7] 666 hours 21584.90 Euro

A. Neve [WP 7] 213 hours 2380.45 Euro G.E. Eperon [WP 7] 252 hours 2779.24 Euro

Figures include National Insurance contribution paid by university to government, and do not represent amount received by employee. Number of hours worked by DC and LPE, who were paid monthly and not hourly, is after subtraction of normal holidays. Consumables Oligonucleotides 3713.80 Euro Chemicals 1305.81 Euro Enzymes 520.30 Euro Laboratory supplies 0 Euro

192

Personnel: Dr Cherny was working on developing the TIRF microscopy for single molecule analysis of protein binding in nuclear extracts (3 months); Dr Eperon was engaged part-time in PCR amplification of cDNA for splicing factors, sequencing, cloning and expression; Miss Neve was employed to optimize methods of making nuclear extracts from transfected cells; Mr G. Eperon was engaged to make mutations in a series of substrates containing putative hnRNP A1 high affinity sites and to do splicing assays; he also tested the ability of confocal microscopy to visualize low numbers of molecules in ultra-small wells (several femtolitres) with a view to extending the method to sub-femtolitre chambers in future. Consumables: Standard oligonucleotides were bought for mutagenesis, PCR and sequencing. Modified oligonucleotides were bought for single molecule experiments to reduce degradation and to provide fluorescent signals. Enzymes were bought for cloning experiments. Chemicals included cDNA clones (PCR templates for expression cloning) and an antibody to check expression.

193

Participant 18 – Artur Jarmolowski a) Work performed during the period • WP8

All three plant groups participated in the selection of genes/AS events to expand the AS RT-PCR panel to contain 384 events. The AS events were selected from transcript information in the Alternative Splicing in Plants (ASIP) database and focused on genes encoding RNA-interacting proteins, transcription factors, splicing factors and genes involved in stress responses and signaling. The full 384 AS RT-PCR panel has been used to analyse mutants in the nuclear cap-binding proteins, cbp20, cbp80, and the double mutant, cbp20cbp80. Of the alternative splicing events which change significantly in the mutants, many are found in the first intron of the transcript consistent with a role of the CBC in splicing. In addition, an initial experiment with a mutant in the microRNA processing pathway, hyl1, which affects the processing of around 25% of pri-miRNAs, has also identified changes in AS in known miRNA targets and in other genes. • WP17 Dorota Raczynska, Adam Ciesiolka and Lukasz Szewc visited the Scottish Crop Research Institute in Dundee (John Brown group) to use the plant alternative splicing RT-PCR platform. • WP19 We participated in organization of the X Festival of Art and Science, Poznan, Poland, October 2007. Artur Jarmolowski gave a general lecture about RNA.

• WP21

The group co-organized the Plant Alternative Splicing Workshop, Carry-le-Rouet, France, May 2007, and the Summer School of Bioinformatics, Poznan, Poland, July 2007.

b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 0 consumables 21.872,52 training 0 equipment 0

travel 8.157,13 = total direct costs 30.029,65 + overhead 6.005,93 = total eligible costs 36.035,58 EC contribution

2006-2007

percentage spent % remaining budget

194

Major cost items • Research Consumables Oligonucleotides 2.853,23Euro Chemicals 6.366,31 Euros Enzymes 1.732,04 Euro Laboratory supplies 10.290,93 Euro Travel • Dundee 5.554,38 Euro • Marseille 2.494,72 Euro • Krakow 108,03 Euro All funds were spent on research, namely on consumables and travels. Consumables included chemicals, restriction and modification enzymes, reagents for RNA isolation, reverse transcription and PCR amplification, labeled oligonucletides for the AS RT-PCR panel, plastic dispensable materials. Travels included trips to Dundee, UK (meeting of all three plant groups), Marseille, France (Plant Alternative Splicing Workshop) and Krakow, Poland (organization of the First International EURASNET Conference on Alternative Splicing).

195

Participant 19 – Jørgen Kjems a) Work performed during the period • WP2 UAAR has provided list of reagents and lab protocols • WP7 UAAR has characterized splicing factors involved in alternative splicing of HIV-1 and MCAD • WP8 UAAR has performed genomic SELEX and isolated human cellular mRNA target that bind HIV-1 Rev with high affinity and characterized them as functional nuclear export substrates for Rev. • WP9 UAAR has performed a mass spec analysis of proteins binding to wild type and mutated MCAD and characterized a number of SR and hnRNP proteins that binds didfferentially. • WP12 UAAR has investigated the possibility of regulating exon 5 inclusion in MCAD using chemically modified oligonucleotides. • WP13 UAAR has characterized a novel functional interaction between U1 snRNP binding and transcriptional initiation. • WP17 UAAR has not had any student exchanges in this period. • WP18 UAAR has led this work package, given a seminar about career development at the Ile Bendor meeting and helped organizing the career development session at the Krakow meeting. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel EUR 16.533,36 consumables EUR 14.297,00 training equipment

travel EUR 3.643,38 = total direct costs EUR 34.473,74 + overhead EUR 6.894,74 = total eligible costs EUR 41.368,48 EC contribution

2006-2007

percentage spent % remaining budget

196

Major cost items • Research Personnel Maria Lauersen [WP 13] 600hours 16.533,36 Euro Consumables Oligonucleotides 2000 Euro Chemicals 4000 Euro Enzymes 4000 Euro Laboratory supplies 4297 Euro Travel 3.643,38•

197

Participant 20 – Alberto Kornblihtt a) Work performed during the period • WP2 We exchanged protocols and tools with the groups of Drs. Karla Neugebauer, Edouard Bertrand and Francisco Baralle. • WP13 - Help to define factors that regulate alternative splicing through the control of transcriptional elongation by RNA polymerase II. - In collaboration with Edouard Bertrand, determine that a mutant of RNA polymerase II that was previously shown to display slow elongation rates in vitro, behave slowly in vivo. This demonstration is very important because transcription by this mutant affacte alternative splicing patterns. - Study the effects of DNA damage caused by UV light on alternative splicing. • WP17 Dr. Paula Cramer, from our laboratory, visited the laboratory of Dr. Juan Valcárcel in Barcelona to perform microarrays to evaluate the global effects of inhibitors of transcriptional elongation on alternative splicing. • WP18 - Dr. Juan Pablo Fededa approved his PhD thesis in 2007. - • WP21 - Dr. Alberto Kornblihtt presented a keynote lecture at the Second Microsymposium on Small RNAs.

Vienna, Austria, 21-23 May 2007. The dynamic community of small RNAs got a comprehensive view of the importance of alternative splicing and its regulation.

b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 0 consumables 27.742,95 training 0 equipment 0

travel 2.705,100 Other (the rest) 0 = total direct costs 30.448,05 + overhead 0 = total eligible costs 30.448,05 EC contribution

2006-2007

percentage spent % remaining budget

198

Major cost items • Research Consumables Oligonucleotides 2.842,15 Euro Antibodies 6.103,00 Euro Chemicals 5.899,10 Euro Enzymes 4.801.10 Euro Laboratory supplies 8.097,60 Euro Total 27.742,95 Euro Plasticware, serum, culture media are necessary to study alternative splicing in cell cultures in vivo. Synthetic RNA oligonucleotides are necessary to knockdown the expression of specific proteins that regulate alternative splicing. Synthetic DNA oligonucleotides are used to perform radioactive or quantitative (Real Time) PCR to assess the variations in mRNA isoform patterns due to changes in alternative splicing. Enzymes such as reverse transcriptase, Taq polymerase and specific RNA inhibitors are necessary to permor PCR analysis. Travel 2.705,100 • Participation of several graduate students and post docs of our laboratory (Juan Fededa, Manuel Muñoz, Manuel de la Mata, Ignacio Schor, Ezequiel Petrillo, Mariano Alló, Paula Cramer) in the ICGEB meeting “Gene Expression and RNA processing” held in Bariloche, Argentina, 26-30 November 2007. • Integration Participation of several graduate students and post docs of our laboratory (Juan Fededa, Manuel Muñoz, Manuel de la Mata, Ignacio Schor, Ezequiel Petrillo, Mariano Alló, Paula Cramer) in the IFM “Cell Biology Signalling and Alternative splicing” held in Bariloche, Argentina, 26-30 November 2007. • Dissemination Trip of Dr. Paula Cramer, from our laboratory, to the laboratory of Dr. Juan Valcárcel in Barcelona to perform microarrays to evaluate the global effects of inhibitors of transcriptional elongation on alternative splicing. Seminars and talks in meeting where EURASNET was acknowledged - Instituto FIRC di Oncologia Molecolare and European Institute of Oncology, Milán, Italia. - EMBO Conference Series. "First meeting on pre-mRNA and disease". Cortina d'Ampezzo, Italia. - Howard Hughes Investigator’s meeting on RNA, Chevy Chase, MD, EEUU. - Workshop “Avaliação pós-genomica da expressão gênica em parasitas”, opening lecture.

Curitiba, Brasil - Second Microsymposium on Small RNAs. “Keynote Lecture”. Vienna, Austria. - FEBS Meeting “Molecular Machines”, Vienna, Austria. - "Eukaryotic mRNA Processing Meeting". Cold Spring Harbor, New York, EEUU. Discussion

leader. - Annual Meeting of the Sociedad de Bioquímica y Biología Molecular de Chile, opening lecture,

Termas de Chillán, Chile.

199

Participant 21 – Angela Krämer a) Work performed during the period • WP2

Antibodies and plasmids sent to three EURASNET labs • WP3

Participation at all steps of the election of new YIP members • WP8

Application of the CLIP method for the isolation of SF1 and U2AF65 targets in nuclear and cytoplasmic fractions of HeLa cells to distinguish between functions in the two cellular compartments Follow-up experiments to analyse the role of SF1 in alternative splicing Adaptation of the CLIP method for high-throughput sequencing

• WP11

Design and testing of siRNAs for knock-down of individual isoforms of SF1 Analysis of protein-protein interactions of SF1 isoforms with proteins binding to Pro-rich sequences Analysis of the nuclear localization of individual SF1 isoforms

• WP16

Co-organiser of the First International EURASNET Conference on Alternative Splicing” in Krakow, May 2008

• WP17

Coordination of staff exchange and training • WP18

External experts for two PhD theses in Geneva: Jamal Tazi (12a) and Karla Neugebauer (1b) • WP21

Co-organizer of the annual Swiss RNA Workshop in Bern, September 14, 2007, sponsored by the Swiss Committee for Molecular Biology (funded by the Swiss National Science Foundation), with ca. 100 participants, 12 short talks selected from submitted abstracts, 2 talks from invited speakers (Gunter Meister, Jernej Ule) and poster sessions. Presentation of results to the “RNA Club” at the University of California San Diego, February 9, 2007

• WP22

Participation in telephone conferences and discussions between management members at meetings

b) Major cost items with justification

200

Participant 1A Type of expenditure Budget total costs personnel 7398.30 consumables 27997.36 training equipment

travel -1357.76 = total direct costs 34037.90 + overhead 6807.58 = total eligible costs 40845.48 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Eva Bernabeu Dizin [WP 8] 320 hours 7398.30 Euro Consumables Oligonucleotides 999 Euro Chemicals 13534 Euro Enzymes 5850 Euro Laboratory supplies 5569 Euro Others 2048 Euro Identification of CLIP targets of SF1 and U2AF65 Mechanism of action of SF1 in alternative splicing Analysis of the function and localisation of SF1 isoforms

201

Participant 22 – Angus Lamond a) Work performed during the period WP2

Dr Laura Trinkle-Mulcahy from the Lamond group developed a quantitative procedure for reliably identifying protein interaction partners using mass spectrometry and SILAC. A detailed workflow and protocol for SILAC interaction experiments and data analysis was developed.

WP3 Angus Lamond has acted as a mentor to YIP Dr Edouard Bertrand (Montpellier). A collaboration was established between the Lamond and Bertrand groups to use SILAC MS methods to study protein-protein interactions. Dr Séverine Boulon from the Lamond group has carried out SILAC MS analyses for the Bertrand group.

WP10 David Lleres developed and applied an in vivo assay for detecting and quantifying protein-protein interactions between splicing factors using fluorescence live-time imaging microscopy and fluorescence resonance energy transfer (FLIM-FRET). David Lleres worked in collaboration with the group of Javier Caceres (Edinburgh) to apply FLIM-FRET to measure splicing factor interactions in vivo in human cells.

WP14

Andrea Pawellek evaluated alternative in vitro pre-mRNA splicing assay formats and tested custom made extracts and reagents suitable for use in high throughput screening of chemical compounds.

WP15

The identification of protein-protein interactions between splicing factors was investigated both in vitro, using quantitative SILAC MS (Dr Laura Trinkle-Mulcahy) and in vivo using FLIM-FRET (Dr David Lleres) and detailed protocols derived for these procedures.

WP17 Dr David Lleres (Lamond Group) trained visiting PhD student Jonathan Ellis (Caceres Group) in the use of FLIM-FRET and advanced fluorescence imaging.

WP18 Angus Lamond acted as a mentor to YIP Dr Edouard Bertrand (Montpellier) and participated in training postdoctoral researchers in the Lamond group and PhD student Jonathan Ellis (Caceres Group) in the use of advanced fluorescence imaging. A commercialization factfile describing steps involved in commercialization of research tools and formation of spin-out companies was prepared and sent to all participating groups. WP20

Dr Paul Ajuh prepared an updated commercialization factfile detailing the process of forming a spin-out company. Angus Lamond and Paul Ajuh founded Dundee Cell Products, a spin-out venture from the University of Dundee that manufactures and sells RNA splicing reagents and cell frations and extracts. Andrea Pawellek worked on the development of a new assay format suitable for screening HeLa cell splicing extracts in high throughput to identify chemical inhibitors. Multiple alternative assay formats were investigated and evaluated along with detailed comparison and evaluation of extracts and reagents suitable for use in the high throughput screen. Andrea Pawellek also constructed and characterised stable cell lines expressing separate red and green fluorescent proteins encoded from the same transcript in vector design that allows detection and splicing inhibition in vivo. Thus, active splicing results in expression of a red fluorescent protein while inhibiting splicing decreases the level of red protein and concomitantly increases the level of green protein. This provides an in vivo assay system suitable for screening chemical compound libraries directly in an automated fluorescence

202

screen. It also provides a secondary area for screening in vivo compounds identified as inhibiting splicing in vitro.

WP21

Angus Lamond has participated in regular interactions with European research groups outside of the Eurasnet network, particularly in the development of advanced quantitative mass spectrometry based proteomics techniques applied to study protein-protein interactions. Specifically interactions with the groups of Dr Matthias Mann (MPI, Munich), Dr Heinrich Leonhardt (University of Munich) and Dr Jens Andersen (University of Southern Denmark, Odense).

WP22 Angus Lamond has participated in the ongoing activities of the steering committee and management of the EURASNET network.

b) Major cost items with justification

Participant 22 Type of expenditure Budget total costs personnel 49,616.23 consumables 0 training 0 equipment 0

travel 2,279.62 = total direct costs 51,895.85 + overhead 10,379.17 = total eligible costs 62,275.02 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items

• Research Personnel Andrea Pawellek [WP 20] 1862 hours 49,616.23 Euro Travel

Annual Meeting, April 2007, Ile de Bandor, France RNA Society Annual Meeting, August 2007, New York, USA IFM, November-December 2007, Bariloche, Argentina

• Integration

IFM, November-December 2007, Bariloche, Argentina • Dissemination

Annual Meeting, April 2007, Ile de Bandor, France RNA Society Annual Meeting, August 2007, New York, USA

All funds requested were specifically to fund personnel and research activities in WP20 and for travel to participate in IFM in Bariloche and travel to Annual meeting.

203

Participant 23 – Chris Smith a) Work performed during the period • WP7 Characterized binding of MBNL proteins to regulatory elements in Tpm1 pre-mRNA. Characterized silencer and enhancer elements through a “nonsense exon” in Tpm1 pre-mRNA, and demonstrated a crucial role for hnRNP H and F as repressors of the nonsense exon. • WP8 WP leader. Carried out quantitative proteomic (2D-DiGE, BVA) analysis of HeLa cells knocked down for PTB and nPTB and identified 25 PTB-regulated alternative splicing events by this approach. Validated by RT-qPCR 38 PTB-regulated splicing events predicted from Affymetrix ExonArrays. Established that highest validation rates were obtained by an “Internal Data Analysis” pipeline developed by Affymetrix. Started to validate predictions generated by the analysis of PTB/nPTB knockdown samples using an in-house Affymetrix research array featuring junction probes. • WP11 Characterized regulatory network between the splicing regulators PTB, nPTB and ROD1. Carried out mutagenic analysis of the minimal repressor domain of PTB and established that residues on both the raver1 binding face, and the RNA binding surface of RRM2 are crucial, even when the domain is recruited artificially by MS2. • WP13 Completed quantitative proteomic analysis of consequences of Upf1 knockdown in HeLa cells. New examples of genes regulated by NMD due to alternative splicing, or upstream ORFs were identified. Found that proteasome inhibition might be essential to allow proteomic identification of examples of AS-NMD where truncated proteins would be produced if NMD substrate RNA is stabilized. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 21.350,64 consumables 10.515,88 training 0 equipment 0

travel 1.241,14 = total direct costs + overhead 6.621,53 = total eligible costs 39.729,19 EC contribution

2006-2007

percentage spent % remaining budget

204

Major cost items • Research Personnel: 21.350,64 Adele Gilmour [WP 8] 3 PM 12 204.02 Euro Nick McGlincy [WP 13] 3 PM 9 146.62 Euro Consumables: Oligonucleotides 3 680.56 Euro Chemicals 1 051.77 Euro Enzymes 3 154.76 Euro Laboratory supplies 2 628.79 Euro Travel 1 241,14

205

Participant 24 – Hermona Soreq a) Work performed during the period • WP8- Genome-wide Analyses of Splicing Regulation- Work was carried out as part of this work package looking at global splice patterns in cancer using affymetrix exon chips. Specific splice patterns were identified in human thymomas. • WP13 Mis-splicing and Disease- Work was carried out as part of this work package looking at alternative splicing in both Alzheimer’s disease as well as a mouse stressmodel. * WP18 Career Development- As part of this work package Professor Soreq gave a number of talks and workshops about career development for young researchers in Israel and abroad. • WP21 Reachout to the Broader RNA Community – As part of this work package research programs involving other RNA researchers who are not part of EURASNET was developed. b) Major cost items with justification

Participant 24 Type of expenditure Budget total costs personnel 25,056.00 consumables 17,071.81 training 0 equipment 0

travel 4,295.25 = total direct costs 152,422 + overhead 9,284.61 = total eligible costs 56,267.25 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Months Cost Deborah Toiber 1.5 3516.70 Rafi Shraga 2.42 6922.61 Yehudit Ben Shoshan 1.85 3666.28 Petra Pollins 3.6 5347.67 Tirosh Shapiro 1.1 1386.22 Yochai Wolff 0.85 1090.92 Chanan Mor 1.58 1998.95

206

Lior Kashani 0.9 1127.03 Consumables Chemicals 5890.00Euro “ Enzymes 7252.00 Euro “ Laboratory supplies 3929.81 Euro “ Travel 4,295.25 Personnel: The personnel were members of the laboratory who carried out the research involved in work packages 8 and 13. The personnel include both research associates as well as technical staff. The research associates carried out the experiments where as technical staff carried out routine laboratory support such as glassware washing, preparing reagents etc. Consumables: Consumables includes all reagents required for the experiments including expensive molecular biology reagents such as reverse transcriptase and RNAse inhibitors. Also included are kits for production of probes and RNA extraction. Other supplies include routine laboratory disposables such as gloves , plasticware test tubes etc. Travel: Travel was to attend the EURASNET meetings including the meeting in Edinburgh .

207

Participant 25 – James Stevenin a) Work performed during the period WP7 We are studying the molecular mechanisms of SC35 pre-mRNA alternative splicing. In the terminal SC35 exon, we identified binding sites for the splicing activator SC35 and for the repressors hnRNP H and TDP43, and showed their respective effects on splicing in vitro. We will try to confirm these results ex vivo and determine the secondary structure of this evolutionary conserved regulatory region (in collaboration with Christiane Branlant, partner 12c). WP12 We used specific extracts isolated from human cells overexpressing various SR proteins to analyse splicing of wild-type LMNA transcripts versus transcripts that are mutated in exon 11 in a HGP Progeria patient. We showed that individual SR proteins modify differently the choice between the wild-type exon 11 5' splice site and the cryptic 5' splice site which is activated in the patient This work is done in collaboration with Jamal Tazi (partner 12a). WP14 We validated our novel experimental system to study alternative splicing in vitro, using extracts from cells overexpressing specific SR proteins (see also WP12). More experiments are required to consolidate our first results before using this new system to test the effects of specific drugs against SR proteins, developed by Partner 12a. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 29737 consumables 12599.72 training 0 equipment 0

travel 429.28 = total direct costs 42766.00 + overhead 7698 = total eligible costs 50464.00 EC contribution

2006-2007

percentage spent % remaining budget

208

Major cost items • Research Personnel Natacha Dreumont [WP 7,12,14 ] 1128 hours 29737 Euro Consumables Oligonucleotides 474.88 Euro Cell Culture/Medium + serum 3727.75 Euro DNA Sequences 1056.00 Euro Polyclonal Antibodies 1550.00 Euro Laboratory supplies 5791.09 Euro Travel Stevenin James : 14-18.04.07 Mission to Bandor, France (Flight, Parking etc) 390.28 Euro Bourgeois Cyril : 14-18.04.07 Mission to Bandor, France (Parking) 39.00 Euro

Personnel: Natacha Dreumont took approximately 4 months maternity leave during 2007.

209

Participant 26 – Didier Auboeuf a) Work performed during the period • WP2 Exchange of protocols and technical expertise in molecular biology and analysis of micro-arrays. • WP8 Analysis of the effects of estradiol on gene expression regulation at the exon level using Affymetrix Exon Arrays, and development of bioinformatics tools that permit to analyze and to visualize the data from Affymetrix Exon Arrays in a user-friendly web interface. • WP10 Demonstration of the role of p68 in mRNA export. • WP13 Demonstration that co-transcriptional splicing is gene specific and depends on the level of the serine 5 phosphorylation of RNA polymerase II at the gene 5’ end. Also, demonstration that coupling between transcription and splicing may permit the efficient production of a subset of mRNAs in response to transcriptional stimuli. • WP21 Participation in local and national programs with the specific view of raising awareness about alternative splicing in cancer. b) Major cost items with justification

Participant 26 Type of expenditure Budget total costs personnel 2811,07 consumables 26159,33 training 0 equipment 0

travel 1843,12 = total direct costs 30813,52 + overhead 6162,70 = total eligible costs 36976,22 EC contribution

2006-2007

percentage spent % remaining budget

210

Major cost items • Research Personnel 2811.07 Euros Anaelle Sosthene was employed during 168 hours to develop specific tools used to analyze Affymetrix Exons Arrays (WP8) Consumables Oligonucleotides 2510.00 Euros Chemicals 3353.7 Euros Enzymes 14851.64 Euros Laboratory supplies 3564.09 Euros Travel: 1843.12 Euros

- EURASNET Affymetrix Exon Array Workshop, 14 Sep. 2007, Cambridge, UK

- Progress reports of EURASNET Member, 14-18 Apr. 2007, Ile de Bendor, France

211

Participant 27 – Davide Gabellini a) Work performed during the period • WP12 We found that FRG1 over-expression inhibits muscle differentiation of C2C12 in tissue culture. This cell culture model of FSHD is currently used to identify the molecular pathways affected by FRG1 over-expression and to evaluate possible therapeutic approaches. 2-hybrid and TAP-tag methods are currently used to identify FRG1 interactors. RNAs extracted from different muscles of WT or the FSHD mouse model and from control or FRG1 C2C12 cells is currently analyzed by splicing-sensitive microarrays to conduct genome-wide studies of aberrant splicing in FSHD. b) Major cost items with justification

Participant 27 Type of expenditure Budget total costs personnel 0 consumables 27.025,90 subcontracting 13.079,80 training 0 equipment 0

travel 885,55 = total direct costs 40.991,25 + overhead 5582,29 = total eligible costs 46.573,54 EC contribution

2006-2007 34.000,00

percentage spent 136,98% remaining budget -12.573,54

Major cost items • Research Consumables Chemicals 4.279,90 Euro Enzymes 12.453,45 Euro

Laboratory supplies 10.292,55 Euro

Travel

• 885,55 Euro for the participation to the EMBO workshop on Pre-mRNA Processing and Disease and the EURASNET workshop on Mis-splicing and Disease, 14-18 January 2007, in Cortina d'Ampezzo, Italy.

212

Our project deal with understanding the molecular mechanism at the basis of facioscapulohumeral muscular dystrophy (FSHD), one of the most important muscular dystrophy. We are also involved in developing possible therapeutic approaches. Transgenic mice are a fundamental aspect of our project since they are a faithful animal model of FSHD. Mice are a useful model for the characterization of complex cellular processes. One advantage relies on the ability to dissect biochemical reactions in vivo by the use of insertion of exogenous DNA (transgenic). This type of manipulation is particularly successful in mice and can provide an essential tool for the molecular analysis of biological functions. This specific advantage cannot be achieved with in vitro systems. Furthermore, mice represent the best model for studying slowly progressive disorders like muscular dystrophy. Our preliminary data show that FRG1 mice display a variety of features found in FSHD providing the first animal model of the disease. We propose a detailed study of these mice to provide novel insights into the pathogenesis of FSHD and evaluate therapeutic strategies. These studies are a crucial point for the delineation of the basis of FSHD pathogenesis and the development of an appropriate therapy for FHSD. These studies will not be possible without mice. Currently, the space available at the animal facility of Fondazione San Raffaele del Monte Tabor (HSR) is not sufficient to host all the animals required for the different research groups. A new animal facility in under construction, but it will not be available before 2009. Hence, it was necessary to identify an animal facility outside the Institution to host the animals that did not find a home at HSR. For this reason, HSR contacted the two companies providing laboratory animal support service available (Charles River Laboratories and Harlan Sprague Dawley, Inc.). Based on the best quality for the money, an agreement was reached with Charles River. As a result, HSR investigators can keep their animals at Charles River at the same cost as keeping them at HSR. When my group stated its activity at HSR, the HSR animal facility was full and we had no other option than hosting our animal model of FSHD at Charles River. It should be noted that Charles River is involved only in maintaining the animals and is not performing any aspect of our research project. In other words, the interaction with Charles River concern the production of a service necessary for the good implementation of the project but do not constitute a core element of the project itself. I retain sole responsibility for carrying out the actions and for compliance with the provision of the agreement. For these reasons, in Form C 13.079,80 Euro are claimed as Subcontracts (F) and represents the costs of maintaining our animal model of FSHD at Charles River.

213

Participant 28 – Mihaela Zavolan a) Work performed during the period • WP6 In this work package we are following two directions of research: 1. characterization of the role of snoRNAs in alternative splicing and 2. analysis of gene expression changes brought about by microRNAs. For the first subproject, during the current reporting period we have trained a model based on known rRNA – snoRNA interactions and we applied it to predict binding sites for snoRNAs in human, mouse and Arabidopsis thaliana. For the second subproject, we have updated predictions of miRNA target sites in the 3’ UTRs of human, mouse, C. elegans, and D. melanogaster, uploaded these on a public web server, and incorporated them into the ASTD database of EBI. • WP8 Within this work package (Genome-wide analyses of splicing regulation) we have completed an analysis of human and mouse cassette exons, and we have uncovered many such exons whose inclusion in the transcript depends on the promoter that was used to initiate the corresponding transcript. b) Major cost items with justification

Participant 1A Type of expenditure Budget total costs personnel 28275.18 consumables training equipment

travel = total direct costs + overhead 5655.36 = total eligible costs 33930.54 EC contribution

2006-2007

percentage spent % remaining budget

Major cost items • Research Personnel Piotr Balwierz [WP 6] 1345 hours 26409.08 Euro Jean Hausser [WP 6] 95 hours 1865.33 Euro

214

Joint Programme of Activities

Integrating activities1

WP1 6 3

WP2 0.5 0.5 1 1 0.5 1 1 1 1 1 1 1 0.5 1 0.5 1.43 0.5 1 1 1 1 2 1.5 1 0.5 1 1 1 0.5 1 1 WP3 4 1 1 1 1 1 1

WP4 1 1 1 7 1 1 4 1 0.5

WP5 0.5 1 6

Jointly executed research activities3

WP6 3 17 28 2.5

WP7 4.5 6 6 4.91 26 4 18 6

WP8 12 2 3.5 7 2 4 6 5 30 12 8 9 2 12 32 6 6

WP9 60 11 2 6 4.28 2.84 18 2 WP10 20 1 12 6 4.44 22 20 32 16 6 10 6

WP11 1 32 2 4 1 10 2 18 14 12

WP12 5 30 3 6 6 0.9 20 5 6 6 12.5

WP13 48 7 16 6 2.5 5 13 6 8 12 3.5 12

WP14 1 1 5 16,13

8.93 30 5 7 6

WP15 3 24 11 1 5 3 6

Spreading of Excellence activities3

WP16 2 0.5 0.5 1 2.5 0.25 1 0.5 0.5 1 1 WP17 0.5 0.5 1 0.5 0.3 0.2 0.5 0.5 1 0.17 0.5 0.2 0.5 1 0.5 2 0.5 1 0.5 0.5 0.5

WP18 0.5 1 0.5 0.5 0.5 0.1 0.3 0.2 0.5 1 0.17 2 0.2 0.25 0.5 0.5 0.5 0.2 0.5 0.5 0.5

WP19 0.3 6 0.5 0.5 0.5 4

WP20 0.5 1 0.67 0.5 27

WP21 1 0.5 0.5 0.5 0.5 0.1 0.3 0.3 0.5 0.37 1 2 0.25 0.5 1 0.5 0.5 1 0.5 0.2 0.5 0.5 0.5

TOTAL JPA

92.5 60 25 50 17 34 21 12 45.5 39.8 17.8 33.5 32.5 23 37 26.75

29 19.49

25.75

56 27.9 44.5 75 37.5 14 26.5 9.5 31.5 51.5 78 13.9 20.5 25 15 11

Consortium Management Activities WP22 Management

24 10 6 0.5 1 0.5 1 0.5 0.5

TOTAL per pp

116.5

60 25 60 23 34 21 12 45.5 39.8 17.8 34 32.5 23 37 26.75

29 19.49

25.75

57 27.9 45 76 37.5 14 26.5 9.5 32 52 78 13.9 20.5 25 15 11

11 PERSON-MONTH ANALYSIS FOR THE REPORTING PERIOD 2007

MPG

1a

MPG

1b

MPG

1c

CR

G

UER

LN

UU

EMB

L5a

EMB

L5b

TAU

ICG

EB

MU

W

UED

IN

CN

R 1

0a

CN

R 1

0b

LUG

CN

RS

12a

CN

RS

12b

CN

RS

12c

CN

RS

12d

UN

IBE

SCR

I

MR

C

IMM

Uni

leic

AM

U

UA

AR

FCEN

-U

BA

UN

IGE

UN

IVD

UN

UC

AM

- D

BIO

C

HU

JI

CER

BM

-G

IE

IN

SER

M

FCSR

ISB

-SIB

215

2 Page 1/1

Contractor AC Third party(ies)

FC/FCF Third

party(ies)Contractor AC Third

party(ies)

FC/FCF Third

party(ies)Contractor AC Third

party(ies)

FC/FCF Third

party(ies)Contractor AC Third

party(ies)

FC/FCF Third

party(ies)

Direct eligible costs 246.472,00 0,00 77.156,27 0,00 246.472,00 0,00 0,00

of which direct eligible costs of subcontracting 1.650,00 1.650,00 1.650,00 0,00 0,00

Indirect eligible costs 49.019,40 0,00 15.101,25 0,00 49.019,40 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 295.491,40 0,00 0,00 92.257,52 0,00 0,00 295.491,40 0,00 0,00

Direct eligible costs 49.911,51 0,00 18.327,40 0,00 49.911,51 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 9.982,30 0,00 3.665,48 0,00 9.982,30 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 59.893,81 0,00 0,00 21.992,88 0,00 0,00 59.893,81 0,00 0,00

Direct eligible costs 59.836,96 0,00 14.965,00 59.836,96 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 11.967,39 0,00 2.993,00 11.967,39 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 71.804,35 0,00 0,00 17.958,00 0,00 0,00 71.804,35 0,00 0,00

Direct eligible costs 51.176,74 0,00 51.176,74 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 10.235,34 0,00 10.235,34 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 61.412,08 0,00 0,00 0,00 0,00 0,00 61.412,08 0,00 0,00

Direct eligible costs 146.425,25 0,00 146.425,25 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 29.285,05 0,00 29.285,05 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 175.710,30 0,00 0,00 0,00 0,00 0,00 175.710,30 0,00 0,00

Direct eligible costs 39.763,20 0,00 39.763,20 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 7.952,64 0,00 7.952,64 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 47.715,84 0,00 0,00 0,00 0,00 0,00 47.715,84 0,00 0,00

Direct eligible costs 73.207,88 0,00 73.207,88 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 14.641,58 0,00 14.641,58 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 87.849,46 0,00 0,00 0,00 0,00 0,00 87.849,46 0,00 0,00

0,007 ICGEB AC 0,00

6 TAU AC 0,00 0,00

0,00 0,00

0,00 0,00

31.12.2007Type of activities

Total eligible costs(A) ReceiptsJoint Programme of Activities

(A)

Summary Financial ReportEURASNET Contract N° 518238Project Title (or Acronym)NoEType of Instrument

AC

AC

4 UU

5 EMBL

Contractor n°

of whichManagement of the consortium

3 UERLN

Eligible costs(in €)

Cost model used

Organisation Short Name

AC1 MPG

Reporting period number From (dd/mm/yyyy) 01.Jan To (dd/mm/yyyy)

AC

AC

2 CRG

0,00 0,00

0,00 0,00

0,00 0,00

216

Direct eligible costs 42.148,24 0,00 42.148,24 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 8.429,65 0,00 8.429,65 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 50.577,89 0,00 0,00 0,00 0,00 0,00 50.577,89 0,00 0,00

Direct eligible costs 43.982,17 0,00 43.982,17 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 8.796,43 0,00 8.796,43 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 52.778,60 0,00 0,00 0,00 0,00 0,00 52.778,60 0,00 0,00

Direct eligible costs 118.786,75 0,00 118.786,75 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 75.479,46 0,00 75.479,46 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 194.266,21 0,00 0,00 0,00 0,00 0,00 194.266,21 0,00 0,00

Direct eligible costs 33.075,73 0,00 33.075,73 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 6.615,15 0,00 6.615,15 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 39.690,88 0,00 0,00 0,00 0,00 0,00 39.690,88 0,00 0,00

Direct eligible costs 376.138,09 25.437,84 376.138,09 0,00 25.437,84

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 75.227,62 5.087,57 75.227,62 0,00 5.087,57

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 451.365,71 0,00 30.525,41 0,00 0,00 0,00 451.365,71 0,00 30.525,41

Direct eligible costs 8.480,00 0,00 8.480,00 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 0,00 0,00 0,00 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 8.480,00 0,00 0,00 0,00 0,00 0,00 8.480,00 0,00 0,00

Direct eligible costs 56.028,02 0,00 56.028,02 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 10.060,06 0,00 10.060,06 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 66.088,08 0,00 0,00 0,00 0,00 0,00 66.088,08 0,00 0,00

Direct eligible costs 29.479,04 0,00 29.479,04 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 5.895,81 5.895,81 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 35.374,85 0,00 0,00 0,00 0,00 0,00 35.374,85 0,00 0,00

15 MRC FC 0,00

0,00

0,00

14 SCRI 0,00 0,00AC

13 UNIBE AC 0,00

0,00

11 LUG

12 CNRS FCF 0,00

AC 0,00

AC 0,00

0,00

0,00

0,0010 CNR FC 0,00

9 UEDIN

8 MUW AC 0,00 0,00

217

Direct eligible costs 59.543,40 0,00 59.543,40 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 11.908,68 0,00 11.908,68 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 71.452,08 0,00 0,00 0,00 0,00 0,00 71.452,08 0,00 0,00

Direct eligible costs 48.929,15 0,00 48.929,15 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 9.785,83 0,00 9.785,83 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 58.714,98 0,00 0,00 0,00 0,00 0,00 58.714,98 0,00 0,00

Direct eligible costs 30.029,65 0,00 30.029,65 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 6.005,93 0,00 6.005,93 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 36.035,58 0,00 0,00 0,00 0,00 0,00 36.035,58 0,00 0,00

Direct eligible costs 34.473,75 0,00 34.473,75 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 6.894,75 0,00 6.894,75 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 41.368,50 0,00 0,00 0,00 0,00 0,00 41.368,50 0,00 0,00

Direct eligible costs 25.373,38 0,00 25.373,38 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 5.074,67 0,00 5.074,67 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 30.448,05 0,00 0,00 0,00 0,00 0,00 30.448,05 0,00 0,00

Direct eligible costs 34.037,90 0,00 34.037,90 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 6.807,58 0,00 6.807,58 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 40.845,48 0,00 0,00 0,00 0,00 0,00 40.845,48 0,00 0,00

Direct eligible costs 51.895,85 0,00 51.895,85 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 10.379,17 0,00 10.379,17 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 62.275,02 0,00 0,00 0,00 0,00 0,00 62.275,02 0,00 0,00

Direct eligible costs 33.107,66 0,00 33.107,66 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 6.621,53 0,00 6.621,53 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 39.729,19 0,00 0,00 0,00 0,00 0,00 39.729,19 0,00 0,00

23 UCAM-DBIOC AC 0,00

0,00

0,00

0,00

22 UNIVDUN AC 0,00

21 UNIGE AC 0,00

0,00

19 UAAR

20 FCEN-UBA AC 0,00

AC 0,00

0,00

0,00

0,00

18 AMU AC 0,00

17 UNILEIC AC 0,00

0,0016 IMM AC 0,00

218

Direct eligible costs 46.423,06 0,00 46.423,06 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 9.284,61 0,00 9.284,61 0,00 0,00

Adjustment on previous period(s) 559,58 559,58 0,00 0,00

Total eligible costs 56.267,25 0,00 0,00 0,00 0,00 0,00 56.267,25 0,00 0,00

Direct eligible costs 42.766,00 0,00 42.766,00 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 7.698,00 0,00 7.698,00 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 50.464,00 0,00 0,00 0,00 0,00 0,00 50.464,00 0,00 0,00

Direct eligible costs 30.813,52 0,00 30.813,52 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 6.162,70 0,00 6.162,70 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 36.976,22 0,00 0,00 0,00 0,00 0,00 36.976,22 0,00 0,00

Direct eligible costs 40.991,25 0,00 40.991,25 0,00 0,00

of which direct eligible costs of subcontracting 13.079,80 13.079,80 0,00 0,00

Indirect eligible costs 5.582,29 0,00 5.582,29 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 46.573,54 0,00 0,00 0,00 0,00 0,00 46.573,54 0,00 0,00

Direct eligible costs 28.275,18 0,00 28.275,18 0,00 0,00

of which direct eligible costs of subcontracting 0,00 0,00 0,00

Indirect eligible costs 5.655,36 0,00 5.655,36 0,00 0,00

Adjustment on previous period(s) 0,00 0,00 0,00

Total eligible costs 33.930,54 0,00 0,00 0,00 0,00 0,00 33.930,54 0,00 0,00

2.303.579,89 0,00 30.525,41 132.208,40 0,00 0,00 2.303.579,89 0,00 30.525,41 0,00 0,00 0,00

2.303.579,89 0,00 30.525,41 132.208,40 0,00 0,00

0,00

27 FCSR

28 ISB-SIB AC 0,00

AC 0,00

0,00

0,00

0,00

26 INSERM FCF 0,00

25 IGMBC-GIE AC 0,00

0,0024 HUJI AC 0,00

2.334.105,30

Requested EC contribution for the reporting period (in €) taking into account receipts [=Periodic Invoice] 2.334.105,30

2.334.105,30 132.208,40 0,00Total eligible costs 2.334.105,30

Amount of the financial interests generated by the prefinancing 8.265,52

Requested EC contribution for the reporting period (in €) without taking into account receipts 2.334.105,30 132.208,40

219

13 REPORT ON THE DISTRIBUTION OF THE COMMUNITY'S CONTRIBUTION

From To From To From To From To From To From To From To1.01.2006 31.12.2006 1.01.2007 31.12.2007

Date Amount(A) Date Amount

(B) Date Amount(C) Date Amount

(D) Date Amount(E) Date Amount

(F) Date Amount(G) Date Amount

(H)31.12.2005 2.550.000,00 31.12.2006 0,00 2.550.000,00

Contractor n°

Organisation Short Name

Country Code Date(s) (5)

Amount(s)(A') (5)

Date(s) (5)Amount(s)

(B') (5)Date(s) (5)

Amount(s)(C') (5)

Date(s) (5)Amount(s)

(D') (5)Date(s) (5)

Amount(s)(E') (5)

Date(s) (5)Amount(s)

(F') (5)Date(s) (5)

Amount(s)(G') (5)

Date(s) (5)Amount(s)

(H') (5)

27.01.2006 102.000,00 102.000,0027.01.2006 91.800,00 91.800,0024.07.2006 34.000,00 34.000,004.10.2006 208.250,00 208.250,00

Total 436.050,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 436.050,0027.01.2006 51.000,00 51.000,0027.01.2006 38.767,00 38.767,0027.02.2006 66.025,69 66.025,69

0,00Total 155.792,69 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 155.792,6927.01.2006 51.000,00 4.06.2007 9.591,78 60.591,7827.01.2006 45.900,00 45.900,0027.01.2006 63.750,00 63.750,0027.01.2006 12.750,00 12.750,00

Total 173.400,00 Total 9.591,78 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 182.991,7827.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 102.000,00 102.000,00

0,000,000,00

Total 102.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 102.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 51.000,0020.06.2006 20.000,00 20.000,00

0,000,00

Total 71.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 71.000,0027.01.2006 51.000,00 14.05.2007 10.000,00 61.000,0027.01.2006 51.000,00 24.05.2007 3.000,00 54.000,00

0,000,00

Total 102.000,00 Total 13.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 115.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 102.000,00 102.000,00

0,000,000,00

Total 102.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 102.000,00

Page n° / 1

10 CNR IT

Report on the Distribution of the Community's contribution

EURASNET 518238Contract N°Type of Instrument NoE Project Title (or Acronym)

Part I

Part II

1 MPG

SE

6

Total (X)

DE

ES

TAU IL

5 EMBL EU

Reporting Period 6 Reporting Period 7

IT7 ICGEB

3 UERLN

4 UU

DE

Community's prefinancing (or payment) sent to the coordinator (1)

Distribution of the Community's prefinancing (or payment) between contractors according to the consortium decision(s) (4)

Total Amount(I) (3)

Final paymentReporting Period 6 (2) Reporting Period 7 (2)Reporting Period 2 (2) Reporting Period 3 (2) Reporting Period 4 (2) Reporting Period 5 (2)Reporting Period 1 (2)

Reporting Period 4Total Amount

(I') (6)

2 CRG

Reporting Period 5 Final paymentReporting Period 1 Reporting Period 2 Reporting Period 3

8 MUW AT

9 UEDIN UK

3

220

Contractor n°

Organisation Short Name

Country Code Date(s) (5)

Amount(s)(A') (5)

Date(s) (5)Amount(s)

(B') (5)Date(s) (5)

Amount(s)(C') (5)

Date(s) (5)Amount(s)

(D') (5)Date(s) (5)

Amount(s)(E') (5)

Date(s) (5)Amount(s)

(F') (5)Date(s) (5)

Amount(s)(G') (5)

Date(s) (5)Amount(s)

(H') (5)

27.01.2006 51.000,00 51.000,000,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 153.000,00 21.03.2007 20.000,00 173.000,0024.07.2006 34.000,00 34.000,00

0,000,00

Total 187.000,00 Total 20.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 207.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 25.04.2007 20.000,00 71.000,00

16.05.2007 60.000,00 60.000,000,000,00

Total 51.000,00 Total 80.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 131.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 11.01.2007 8.196,72 59.196,72

0,000,000,00

Total 51.000,00 Total 8.196,72 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 59.196,7227.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 11.01.2007 10.000,00 61.000,00

0,000,000,00

Total 51.000,00 Total 10.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 61.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 11.01.207 25.000,00 76.000,00

0,000,000,00

Total 51.000,00 Total 25.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 76.000,00

Page n° / 2

22 UNIVDUN UK

3

21 UNIGE CH

19 UAAR DK

20 FCEN-UBA AR

17 UNILEIC UK

18 AMU PL

16 IMM PT

Report on the Distribution of the Community's contribution

Type of Instrument NoE Project Title (or Acronym) EURASNET Contract N° 518238

Part II Distribution of the Community's prefinancing (or payment) between contractors according to the consortium decision(s) (4)

Reporting Period 7 Final paymentReporting Period 1 Reporting Period 2 Reporting Period 3 Reporting Period 4

15 MRC UK

12 CNRS FR

13 UNIBE CH

14 SCRI UK

Total Amount(I') (6)

11 LUG DE

Reporting Period 5 Reporting Period 6

221

Contractor n°

Organisation Short Name

Country Code Date(s) (5)

Amount(s)(A') (5)

Date(s) (5)Amount(s)

(B') (5)Date(s) (5)

Amount(s)(C') (5)

Date(s) (5)Amount(s)

(D') (5)Date(s) (5)

Amount(s)(E') (5)

Date(s) (5)Amount(s)

(F') (5)Date(s) (5)

Amount(s)(G') (5)

Date(s) (5)Amount(s)

(H') (5)

27.01.2006 51.000,00 51.000,0028.11.2006 16.000,00 16.000,008.12.2006 4.500,00 4.500,00

0,00Total 71.500,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 71.500,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0027.01.2006 51.000,00 51.000,00

0,000,000,00

Total 51.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 51.000,0021.07.2006 34.000,00 34.000,00

0,000,000,00

Total 34.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 34.000,0028.08.2006 34.000,00 34.000,00

0,000,000,00

Total 34.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 34.000,0021.07.2006 34.000,00 34.000,00

0,000,000,00

Total 34.000,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 34.000,000,000,000,000,00

Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 0,000,000,000,000,00

Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 0,00Total 2.318.742,69 Total 165.788,50 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 Total 0,00 2.484.531,19

Total Amount

65468,81

Explanatory notes Page n° / 3(1): To be filled in only by the coordinator. (2): Established in conformity with articles 4.2 and 6 of the contract.(3): (I) = (A) + (B) + (C) + (D) + (E) + (F) + (G) + (H) (4): To be filled in only by the coordinator. (5): Insert the dates (dd/mm/yyyy) and the amounts (x,xxx.xx €) transferred to a contractor (including the coordinator) for a reporting period. If there are more than one transfer to a contractor during a reporting period, identify each date and each relating transferred amount.(6): (I') = (A') + (B') + (C') + (D') + (E') + (F') + (G') + (H') (7): (Z) = (X) - (Y)(8): One the following persons : authorised contact person or first or second administrative official authorised to sign the contract, as mentioned in your Contract Preparation Form (Form A2b)

3

MANFRED MESSERSCHMIDT

0,00

I certify that the information set out in this(these) form(s) is accurate and correct and agreed by all contractors.

Name (8) Surname (8)Date

(dd/mm/yyyy) Signature of the administrative official authorised to commit the organisation of the coordinator (8)

0,00 0,00 0,00 0,00Community's prefinancing (or payment) not yet distributed between contractors (Z) (7)

231257,31 -165788,50 0,00

Part III Difference between Community's prefinancing (or payment) sent to the coordinator and Total Distribution of the Community's prefinancing (or payment) between contractors according to the consortium decision(s) (4)

Reporting Period 1 Reporting Period 2 Reporting Period 3 Reporting Period 4 Reporting Period 5 Reporting Period 6 Reporting Period 7 Final payment

30

Total (Y)

28 ISB-SIB CH

29

26 INSERM FR

27 FCSR IT

24 HUJI IL

25 IGMBC-GIE FR

Total Amount(I') (6)

23 UCAM-DBIOC UK

Part II Distribution of the Community's prefinancing (or payment) between contractors according to the consortium decision(s) (4)

Reporting Period 1 Reporting Period 2 Reporting Period 3 Reporting Period 4 Reporting Period 5 Reporting Period 6 Reporting Period 7 Final payment

Report on the Distribution of the Community's contribution

Type of Instrument NoE Project Title (or Acronym) EURASNET Contract N° 518238

222

14 SUMMARY EXPLANATION OF THE IMPACT OF MAJOR DEVIATIONS FROM COST AND PERSON MONTH BUDGET The EC payment for 2007 has not been received yet due to reporting problems for the first annual report 2006. The Coordinator therefore could not distribute the next payment of the research grants to participants. Only few transfers from the Coordinator's escrow account were made to pay for major network projects and the organization of meetings and workshops. These payments are listed in the justification of major cost items for participant 1a (coordinator). The fact that no payments have been made did not impair reseach. Only participant 13 (D. Schumperli) was affected. Due to strict financial regulations of the University of Berne he could only claim a very limited amount of costs. 15 JOINT PROGRAM OF ACTIVITIES (MONTH 25-42, JAN 2008-JUNE 2009) 15.1 THE FUTURE OF EURASNET RESEARCH: GOALS AND OBJECTIVES AFTER TWO YEARS Two years into the network, EURASNET thought it appropriate to reconsider its goals and perspectives, its approaches and structures. To this purpose the coordinator convened a PI-only session during the IFM on Biophysical Methods, Berlin January 2008. Taking into consideration achievements and deficits of the last two years, surveying the rapidly evolving field of alternative splicing with many new technologies and research options emerging, network members discussed where new avenues of research should be ventured and where outdated goals should be abandoned. For most work packages here follows a short summary of things achieved and how to continue in the future with new cutting edge deliverables, thus making the most profitable use of existing network structures and technologies. Explanations closer to the set of new deliverables can also be found in the new JPA for months 25-42 and the work package reports. These discussions will also secure to establish durable structures of scientific and policy-making interactions beyond the lifespan of the network. Work Package 1: The EURASNET Web site New content has been added to the website throughout the reporting period. This content includes information on diseases caused by mistakes in splicing or alternative splicing, educational material in the form of Powerpoint lectures for undergraduates and new database content including protocols. In addition, information on alternative splicing and its importance in human disease has prepared for the general public. Member profiles of the new YIPs appointed in 2007 have been added to the website. Finally, the various meetings and workshops organised by EURASNET have been advertised on the website giving access to information on registration, accommodation etc. The website contains databases of resources available across the network including protocols and reagents (plasmids, antibodies etc.). The current format for inputting such information relies on FilemakerPro software. Although allowing remote uploading of information both information input and retrieval is not ideal. Therefore, we are investigating other modes of presentation and retrieval of database content. A number of bugs have been removed and improvements made. A new user-friendly website is under development. During the reporting period the EURASNET Co-ordinator team decided to reorganize the management and running of the EURASNET website. The main objectives behind the re-organisation were:

223

• to change the overall look of the website to make it more attractive to target audiences, • to increase interactions of members via the website, • to enhance the information and utility of the website to target groups such as clinicians and clinical

labarotories and the general public, • to provide access to educational material for school and college students, and • to increase the efficiency of both information gathering from the members and updating of the website.

The reorganization will be achieved by hosting the website at the Scottish Crop Research Institute with

access to a range of specialized web, journalistic and IT skills, linking the activities of the main dissemination WPs (WP1 – Website; WP19 – Public Understanding of RNA Biology; and WP21 – Broader outreach to the wider RNA community); and forming a team of individuals from these WPs with responsibility for regular information gathering and production of content for target audiences. The Web-team will be managed by John Brown (Leader of WP1 and WP21) with Andrea Barta (Leader of WP19). The Web-team will consist of a scientific and information group made up of Dominika Lewandowska (a part-time webmaster located at SCRI), Claudia Panushka (the Public Understanding of Science officer for EURASNET - Vienna) and Lorraine Wakefield (SCRI – responsible for day-to-day running and production of content of the SCRI website who will commit time to the EURASNET website). The Web-team will have a technical group consisting of a part-time technical assistant with committed time and input from Robert McCreary (an SCRI IT/web specialist) with responsibility for technical specifications of the website, maintenance, backing up and archiving. The scientific and information group will have responsibility for contacting members of EURASNET for information such as articles, publications etc, and will liaise with target groups to design and produce content accessible to scientists, clinicians, the general public and students at different levels of education. These functions will not only ensure regular posting of information and a dynamic website but will also aid the EURASNET Management Group in monitoring and reporting on the progress of the network. The understanding of the importance of alternative splicing for the general public has been addressed in WP19 – Public Understanding of Science, who have produced a explanatory pamphlet and are currently preparing a brochure explaining alternative splicing. This production of this material has benefited from interactions with the University of Vienna PUS office. Educational material has also been obtained from EURASNET members and placed on the website. To address the content for clinicians working with patients with diseases associated with splicing or alternative splicing defects, we will communicate with appropriate EURASNET members and other clinicians to assess the type of information which would be most useful to this group and then prepare such information for the website. The website is an important tool for the members of EURASNET in terms of a repository for protocols and information on various resources such as plasmids, antibodies and oligonucleotides available across the network. Stefan Stamm is revising the way in which this information is made available and accessible to members. The website is also a point of focus for some of the deliverables of other WPs. For example, WP2 – sharing resources; WP5 – durability; WP16 – Conferences/meetings; WP18 – career development; WP20 – SMEs, as well as some of the scientific WPs. The Web-team will be the point of contact for these WPs to have their information put on the EURASNET website and will facilitate functions such as advertising of upcoming meetings, job opportunities etc.. The need for the network to increase interactions with clinicians was highlighted in the first year report. Similarly, the first year report pointed out the need to establish a structure for ensuring the regular gathering of information on activities by the members of EURASNET. To achieve these goals requires time and effort to prepare content specifically for target audiences and to make and follow up on regular requests for information. This is being addressed by changing the management of the website and establishing a team of individuals to co-ordinate the activities of WP1, WP19 and WP21. Work Package 2: Sharing Resources, Technology and Reliable Protocols Overall, the network was very successful in sharing resources and expertise. We will focus now on making this “tested expertise” available to the general public by a) creating tested internet interfaces that allow subgroups to collect protocols and b) by publishing our protocols.

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Work Package 4: The Alternative Splicing and Transcript Diversity (ASTD) Database In June 2007, the EBI announced a substantial reorganisation and consolidation in the management of biomolecular sequence information. This affected all groups within the sequence database team, including Integr8 and ASTD, and the EBI team working on Ensembl. The long-standing sequence database group led by Rolf Apweiler, which is responsible for EMBL-Bank (nucleotide sequences) and UniProt (protein sequences), is now merged with Ewan Birney’s group, which includes the EBI’s part of the Ensembl team. Ewan Birney manages Ensembl, which annotates vertebrate reference genomes, in collaboration with Tim Hubbard at the Wellcome Trust Sanger Institute.

The new team structure reflects the need to integrate DNA data from an increasing diversity of high-throughput methods, and will be jointly led by Apweiler and Birney, with Apweiler taking responsibility for proteins and functional information and Birney for nucleotides and sharing responsibility for other areas. The well-established global collaborations to collect and exchange nucleotide and protein sequence data will remain an essential part of the future operation.

The newly merged group is the Protein and Nucleotide Database Group (PANDA). It is a large group providing all the sequence resources at the EBI, from DNA through to protein and encompassing the associated databases, such as:

genome databases (eg, Ensembl <http://www.ebi.ac.uk/ensembl/>)

protein families (eg, InterPro <http://www.ebi.ac.uk/interpro/>)

protein function (eg, UniProtKB <http://www.ebi.ac.uk/uniprot/>)

proteomics (eg, IntAct <http://www.ebi.ac.uk/intact/>)

ontologies (eg, GOA <http://www.ebi.ac.uk/GOA/>)

pathways (eg, Reactome <http://www.reactome.org/>).

While PANDA has a large number of specific projects <http://www.ebi.ac.uk/panda/projects.html>, each with their own collaborators, data flows and distributions, there is considerable coordination of information across the entire PANDA group - for example, new DNA sequences deposited in the EMBL archive provide the foundation for genome databases, which in turn provides protein sequence for protein predictions; furthermore the curation of protein information informs the gene structure predictions on genomes and the association of genes to function. The requirement for extensive linking of both the underlying data and the coordination of web resources is the driving force behind having one overarching group.

The philosophy of the PANDA team is to capture, organise and interpret sequence-related data, providing all information in a variety of formats, including user friendly web sites. Wherever possible, we aim to collaborate with other groups worldwide with similar aims.

As a consequence of the reorganization, the PANDA group is moving towards a unified presentation of all the data, one web site allowing users to move from genome to transcriptome to proteome. Ensembl is to become the consolidated view, with the integration of sequence database resources Ensembl will grow enormously in terms of organism coverage and depth of annotations.

To achieve this augmentation of the transcript data in Ensembl, ASTD is to be integrated. ASTD will be providing the expertise to generate full length transcript sequences from the 5’ transcription start site to the 3’ poly(A) signal, annotation of each transcript for expression states using eVOC and MESH data and comprehensive splice event annotations. These are currently not available in Ensembl, so ASTD is able to provide new technology thereby increasing the knowledge within Ensembl.

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Future work for the ASTD team is a close examination and comparison of our gene-building pipeline with that from Ensembl. Extensive benchmarking of exon coordinates on the genome is being performed to assess the benefits and drawbacks of each method, so that the combined procedure will delineate the optimal gene model using all available data.

Within the new Ensembl Genomes structure, the ASTD data for the human, mouse and rat transcriptomes will be held within the chordate Ensembl section. The next species that will be targeted for development are within the non-chordate Ensembl Metazoa section, namely the twelve Drosophila genomes that have recently been made publicly available (Drosophila 12 Genomes Consortium, 2007). The inclusion of these species would be beneficial to the research community providing a similar ‘look and feel’ to each genome with extensive linking between the species. Comparative analysis of the genomes is performed at the DNA-DNA level (whole genome alignments and identification of constrained elements) and Protein-Protein level (determination of orthologs and paralogs within ‘genetrees’). The Ensembl web code allows the visualization of the comparative data linking related regions of the genomes and a comprehensive Perl Application Programme Interface (API) provides efficient access for script based data retrieval and processing.

Ensembl acts both as a Distributed Annotation System (DAS) server and client. This means that data from geographically distant servers can be displayed in the genome browser (e.g. the ContigView pages showing a region of the genome with feature tracks; Genes, similarities, microarray probes etc). When viewing a region of the genome the browser will send a query to the DAS servers requesting any features that map to the region being displayed. Users can define which DAS sources they would like to see and so the system is highly configurable. This system could be used to visualise data from consortium members in the context of their genome of interest. DAS tracks are a simple way of displaying data that can be used both ‘in house’ for private data or publicly by registering the DAS server for third-parties to access. Each group retains responsibility for the data and future updates. Setting up a DAS server is relatively straightforward and training by EBI personnel can be provided for this service.

A further database that is to be integrated into the Ensembl schema is Integr8. Currently the two databases have a very different emphasis on their chosen species: Ensembl are metazoan (Release 47 of October 2007 contains 35 species, with preliminary support for six additional species) and Integr8 are bacterial. So combining these resources immensely broadens the species coverage available.

By February 2009 ASTD and Integr8 will no longer have individual databases and web sites, but instead will be fully integrated into Ensembl. There will be a comprehensive suite of Ensembl type databases for all genomes including archaea, bacteria and metazoa. Ensembl will create the proteome sets from complete genomes. New developments within the Ensembl Genome Browser and EMBL-Bank will ensure capture of experimental data and variation data.

Release 1.1 notes (available mid Feb) :

a. Improved TSS predictions covering 80% of human genes and 68% of mouse genes. TSSs of a gene are grouped into clusters based on the TSS distribution. SIB-EPD (The Eukaryotic Promoter Database) evidences are mapped to the TSS positions.

b. Improved mapping of the ASTD transcript translations to Uniprot proteins. c. Rat transcripts are tagged with developmental stages and pathology states terms based on

cDNA libraries information extraction. Rat developmental stages evidences are mapped to the Witschi embryology classification. Anatomical system and disease states evidence are mapped to the MeSH ontology.

d. Normalized transcript digital expression values, introduced by the NCBI and known as TPM (transcript per million), are derived for each tissue/development/pathology related evidences from cDNA libraries. For instance, the tissue TPM values of transcripts are calculated as follow: for any transcript, divide the number of EST evidences found in a particular tissue by the total number of clones from the cDNA libraries related to this tissue. The resulting value is then normalized to one million.

e. Tissue/Development/Pathology fold-change is displayed in the transcript page.

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f. Digital differential expression significance of transcripts is calculated by t-tests with a p-value cutoff of 0.05. We applied a benjamini-Hochberg correction for false discovery rate of less than 5%. It provides a resulting set of statistically significant variant transcript between two conditions (normal vs. cancer for instance).

g. Experimental validations of mouse PolyA sites from the ATD consortium work described in Moucadel et al. (Beyond the 3' end: experimental validation of extended transcript isoforms. Moucadel V , Lopez F , Ara T , Benech P , Gautheret D - Nucleic Acids Res. [2007 (35) ] pp:1947-57)

h. miRNA target sites integration. See deliverable 8) below. i. Previous ASTD releases are archived. An archive search page enables users to trace the

history of genomic features. Work Package 7: Molecular Characterization of Splicing Substrates A huge number of proteins were identified upon analysis of RNP complexes formed on pre-mRNA regions containing splicing sites and their associated regulatory elements. We have now to investigate the biological significance of the presence of these numerous proteins, and how far they can influence splicing complex assembly. Further analyses of the interplay between nuclear factors at splicing regulatory regions in pre-mRNAs and the influence of pre-mRNA 2D structure have to be performed for a precise definition of the regulatory mechanisms of the studied cellular and viral pre-mRNAs. The influence that proteins have on the RNA structure will be considered taking into account the progress made in 3D structure determination of splicing regulatory proteins and their mode of interaction with RNAs. Studies on the effect of some point mutations found in genetic diseases on RNA 2D structure and association of regulatory proteins with pre-mRNA will be continued. The mechanisms of action of repeated RNA sequences in normal modulation of the splicing activity or in the generation of pathology linked to splicing defects will be investigated more deeply. Methods will be developed and used for more quantitative estimation of the relative affinities of regulatory proteins for pre-mRNAs in splicing conditions both in vitro and in cellulo. The possible dependence of the effect on splicing efficiency of the pre-mRNA 2D structure and protein binding on the identity of the transcriptional promoter, its efficiency and the identity of its activator has to be evaluated. Work Package 8: Genome-wide Analyses of Splicing Regulation After 24 months many of the initial objectives of this workpackage have been realised. A range of global approaches for AS analysis have been trialled in one or more groups. Some Approaches, including splice sensitive arrays and CLIP, have proven their worth and the expertise developed is being adopted by other groups and applied to other biological and medical problems. Other methods — for example genomic SELEX — appear to be of more restricted value. Successful application of genome wide techniques generates large datasets, which provide raw material for analysis by computational means, thereby generating a natural interface with WP6. For example, Ast’s group has been analyzing the set of identified PTB-regulated exons, identified by proteomic analysis, ExonArray and junction array, for distinctive motif enrichment, and has found an unexpected exonic enrichment of binding motifs. Some of the approaches, in particular the use of splice-sensitive arrays, are beginning to be applied in disease settings (see WP12, Splicing and Disease). In particular, the Affymetrix ExonArrays provide a global platform that has proven able to detect changes in AS in a number of experimental settings. Various data analysis procedures have been developed, compared and refined. At the same time, pilot experiments have been carried out using an Affymetrix in-house research array that features exon and junction probe-sets for all human (or mouse) Refseq and Ensembl isoforms. This platform was able to identify 5 times as many PTB-regulated events as the ExonArray. EURASNET is now in the process of negotiating access to the Affymetrix mouse and human junction arrays. As of January 18, 2008, we were given an informal indication that Affymetric were willing to make the human and mouse junction arrays available to use. At the time of submission of this report the legal agreement is in the process of being drawn up.

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A number of EURASNET groups would like access to either ExonArrays or the new junction arrays, but lack the expertise for this analysis. During the next period we intend to create a dedicated array bioinformatics postdoc position to support further development of data analysis procedures, to continue development of a user-friendly web interface, and to provide a service role for other EURASNET members who need access to array technology. The position will be located with the group of Didier Auboeuf and will be for a period of two years. A number EURASNET groups are interested in analyzing the global effects of knockdown of particular splicing regulators upon AS. The preliminary comparison of the Affymetrix ExonArray and junction arrays (with the PTB/nPTB knockdown experiment) suggests that the Affymetrix junction array will be an ideal platform. Analysis of several such knockdown experiments using a common array platform should generate an invaluable collection of data that will allow identification of AS events that are co-regulated by distinct combinations of splicing factors, indicate how different categories of splicing factor regulate AS and how different (combinations of) splicing regulators map onto different tissue-specific programmes of splicing regulation. Once we have formally secured access to the junction arrays we envisage that a future deliverable will involve a coordinated and more extended programme of analysis of splicing regulator knockdowns, that will build upon the starting point of individual labs’ interests in particular splicing regulators. Since the original EURASNET proposal, various deep-sequencing technology platforms have emerged. These have already been applied to genome sequencing, but they also have clear applications in alternative splicing research including discovery of AS events, quantitative profiling of AS events, and analysis of CLIP tags. In species for which there is limited EST and cDNA sequence data, deep sequencing approaches have an obvious and immediate application for AS discovery. However, they also have the potential to complement microarrays for the quantitative analysis of alternative splicing events. Indeed, since sequencing circumvents some of the pitfalls of arrays (e.g. non-responsive, or cross-hybridizing probes), there is even the possibility that microarrays may be displaced. Before the demise of arrays however, sequencing approaches will need to overcome serious challenges. Perhaps the most serious issue is the depth of sequence coverage that will be required to obtain statistically significant information about the quantities of alternatively spliced isoforms. The total amount of required sequence read will depend upon individual gene expression levels, and upon the magnitude of splicing changes between samples (a switch from 1% to 99% exon inclusion in a highly abundant mRNA will be readily quantifiable, but a switch from 40% to 60% exon inclusion in a poorly expressed gene will require a much higher level of sequence read). It may be that with current levels of sequence obtainable from individual runs it will prove necessary to focus the sequencing e.g. by using pools of reverse transcription primers that are targeted to AS regions of transcripts rather than using random priming. During the next period, various collaborative efforts will be made to test the applications of deep-sequencing for analysis of AS. These will involve both computational modelling exercises, as well as deep-sequencing of experimental samples that have previously been analyzed by splice-sensitive microarrays, and for which quantitative information is already available about changes in AS. Plant Alternative Splicing The three plant labs (Barta, Brown, Jarmolowski) continue to work as a highly integrated unit and have established a joint project to analyse aspects of alternative splicing in Arabidopsis. In the first 12 months, a pilot alternative splicing RT-PCR panel consisting of 96 events was tested using RNA from different plant organs, seedlings grown under different conditions (long and short days; dark/light; different temperatures) and seedlings of transgenic lines over-expressing particular SR proteins. The system was reproducible giving consistent results across biological reps and identifying statistically significant changes in alternative splicing (Simpson et al., 2008 Plant Journal, in press). On this basis, in months 12-24, all three groups participated in the selection of genes/AS events to expand the AS RT-PCR panel to contain 384 events. The AS events were selected from transcript information in the Alternative Splicing in Plants (ASIP) database and focused on genes encoding RNA-interacting proteins, transcription factors and genes involved in stress responses and signaling. Further refinement of the panel is now required because 1) transcripts from some genes are not detectable in any of the treatments (often reflecting low expression levels when expression databases such as MPSS are examined) and 2) some of the AS events are undetectable (reflecting rare ESTs or AS limited to a small number of cells or particular conditions). Results from the initial panel identified many novel transcripts. Cloning and sequencing of some of these transcripts demonstrated that they were novel, un-annotated AS events reflecting the more wide spread nature of AS in plants and the need for a systematic discovery programme for AS. Currently, our experiments with the expanded panel have identified >150 novel events which the three plant groups will attempt to

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characterize. In addition, we will use 454 high throughput sequencing to assess its utility in AS discovery in Arabidopsis. It will also be necessary to further develop the downstream statistical analysis to take into account multiple AS transcipts.

In months 12-24, the full 384 AS RT-PCR panel has been used to analyse mutants in the cap-binding proteins, cbp20, cbp80 and the double mutant, cbp20cbp80. Of the alternative splicing events which change significantly in the mutants, many are found in the first intron of the transcript consistent with a role of the CBC in splicing. In addition, the effects on AS were most apparent in the cbp80 and double mutant suggesting that CBP80 is more critical for the splicing phenotype. To complete the analysis of SR proteins and PTB-like proteins (PTBLs), transgenic over-expression lines and mutant lines are being prepared and checked genetically before being analysed (see WP11). An initial experiment with 96 AS events using two NMD mutants indicates that around one third of plant AS transcripts may be regulated by NMD. In addition, an initial experiment with a mutant in the microRNA processing pathway, hyl1, which affects the processing of around 25% of pri-miRNAs has also identified changes in AS in known miRNA targets and in other genes. Thorough analysis of SR and PTB proteins, NMD mutants and hyl1 (including testing levels of miRNAs together with checking accumulation of miRNA precursors using the Real-Time PCR) will be carried out between months 24 and 42. The above research has involved regular contact between the PIs, post-docs and PhD students from all three labs, exchanges for training in the use of the AS RT-PCR and a very valuable intranet workshop in November 2007.

The plant groups have presented their alternative splicing work at a range of plant meetings and are in the process of setting up interactions with other plant RNA/expression/development groups for the AS RT-PCR panel to be used to analyse particular genes or processes. For example, the human orthologue of Arabidopsis PRL1 is a spliceosomal protein and the prl1 mutant shows many pleiotropic defects showing AtPRL1 to be involved in signalling of hormone and sucrose responses. AtPRL1 also interacts with the SR protein, RSZ33, one of the SR proteins which we are studying. In collaboration with Prof. Csaba Concz, Cologne prl1 will be analysed on the AS RT-PCR panel. Similarly, collaborations with Prof. D. Staiger (Bielefeld), Dr. A. Maule (Norwich) and Dr. G. Simpson (Dundee) are being established to analyse AS effects of genes involved in circadian responses, heat shock and flowering time. Interaction with Alberto Kornblihtt on UV effects on alternative splicing in plants is being established and represents a further interaction within EURASNET. Finally, we intend to set up a collaboration with the Matzke laboratory (Gregor Mendel Institute, Vienna), to investigate the effect of mutants of the RNA-directed DNA methylation pathway on alternative splicing of plant genes. In some treatments or mutants, similar patterns of changes in AS among subsets of genes have been identified. These may indicate combinatorial control of these AS events. As more data from different over-expression and mutant lines is assembled, such patterns will be more easily determined. Work Package 9: Complexity of Spliceosomal Proteomes

After substantial insight has been obtained into the spliceosomal proteomes of canonical spliceosomes assembled in splicing extracts of cells from various organisms – including human, yeast and Drosophila – studies will be continued and intensified by various members of WP9 to (i) characterize the proteins associated with various viral regulated pre-mRNAs, (ii) to determine the proteomes of spliceosomes assembled on medically important pre-mRNA constructs that harbour mutations known to cause aberrant splicing and diseases, and to compare these with the spliceosomes assembled on normal pre-mRNA, and (iii) to investigate the RNP complexes assembled on artificial pre-mRNA constructs differing in respect of the presence of distinct enhancer and silencer elements.

The qualitative determination of the protein composition of the various isolated spliceosomal complexes represents an important first step in discovering the diverse roles played by the proteins in the regulation of the various splicing processes. However, the results obtained so far already show clearly that comparative proteomic studies of the spliceosome will have to be conducted on a quantitative basis. This is especially true of the influence of individual point mutations in pre-mRNAs upon the protein composition of the spliceosome. It is anticipated that in some cases only a few proteins will change in abundance, with consequent aberrant regulation of splicing. For this reason, future studies will have to be augmented by employing methods of relative quantification such as SILAC and iTRAQ. Feasibility studies with purified ‘B complex’ and ‘C complex’ spliceosomes have already been conducted successfully by both methods, and have shown the relative abundances of various sets of proteins in pre-catalytic and step I spliceosomes (Partner 1a + 1c)

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Ideally, one would like to know the absolute stoichiometry of the proteins in isolated spliceosomal complexes: this issue is at present not even understood in a rudimentary way. A possible approach to this question has been embarked upon by Partner 1a, and substantial progress has been made in this direction: a 2-dimensional gel system has been established that allows the separation of nearly all the proteins of the isolated human spliceosome (the B complex) without incurring loss of proteins in the transfer from the first to the second dimension of electrophoresis (for example, some proteins were inadequately soluble in the 2-D gel systems used earlier). Staining with sensitive dyes allows the absolute quantification of the individual proteins. When this technique is established as a routine method, it will raise substantially the amount of information obtainable in comparative studies of the spliceosome's proteome.

Finally, a further important goal of this workpackage is the detection and identification of protein–protein interactions in spliceosomal complexes. For example, our present “model” picture assumes that one important function of the SR proteins involves their undergoing protein–protein interactions. However, such interactions have never been demonstrated in splicing complexes. Furthermore, the interactions between proteins within the spliceosome are subject to major changes caused by the particle's structural dynamics. The situation is similar for protein–RNA interactions. Therefore, the direct observation of interactions between macromolecules in the spliceosome will constitute an important activity in this workpackage and in workpackage 7. Partner 9 (J. Beggs) has started to investigate such interactions in yeast spliceosomal complexes by chemical cross-linking. Partners 1a and 12c will carry out similar studies in purified human spliceosomal complexes. Pursuing the same goal but following different experimental strategies, several members of this workpackage will continue to characterise spliceosomal protein complexes in yeast, using the TAP tag technology(partners Seraphin and Beggs), or in human cells, using tagged stable cell lines(partner1a, Lührmann)

Work Package 10: Post-translational Modification and Dynamic Regulation The main goal of this WP remains to establish a link between signal transduction pathways and alternative splicing regulation. To summarise briefly some of the future goals expressed in the new set of deliverables for month 25-42, we will continue use of FRET/FLIM microscopy to extend the analysis to additional splicing components, we will also identify RNA targets for different trans-acting factors involved in splicing regulation and investigate how signalling pathways affect splicing activity. The role of protein kinases involved in splicing regulation (SRPK2, hPrp4, DNA topoisomerase I and Akt) will be further investigated.

Work Package 11: Function of Splicing Factor Isoforms The overall goal of WP11 is to contribute to understand the functional diversity that alternative splicing can provide to higher eukaryotic genes, with a natural focus on the extended family of splicing factors (spliceosomal components and splicing regulators), which is at the core of the activities of EURASNET groups. Insights obtained in the previous funding period (months 1-24) from a variety of experimental systems indicate that splicing factors are indeed among the gene families for which alternative splicing has a stronger impact and potential functional consequences. The previous work of the consortium has also provided enabling technologies, including splicing-sensitive microarrays and multiplexed RT-PCR to document variations in the relative expression of splicing factors isoforms. This description is necessary to frame physiologically relevant questions about differential expression and function of splicing factor variants. Such developments also provide technologies of general interest for the consortium and beyond and can have a durable impact on the outstanding questions and experimental designs used in the field. We expect that these reagents and data analysis methods will become of general use in the next period (25-40 months). In particular, remodeling of the spliceosomal proteome will be investigated during cell cycle, under conditions of activation of signaling cascades, both in Drosophila and in mammalian cells, and as potential markers for tumor progression. Also of great significance are findings contributed by several EURASNET groups regarding gene networks involving autoregulation and crosstalk among splicing factors and other RNA binding proteins in the form of regulated alternative splicing events. We intend to expand these studies and frame regulation of splicing factor isoforms in terms of gene networks from a systems biology perspective. Of particular importance will be

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the focus on alternative splicing events which affect regulatory sequences in untranslated regions of mRNAs, which facilitate or prevent the regulatory action of proteins and miRNAs. Emphasis will be placed in the next period in combining high throughput methods (including both CLIP, splicing microarrays and deep sequencing) with technologies for isoform-specific knock down (including siRNAs, esiRNAs and tRNA splicing-based technologies), with the goal of building regulatory maps specific for different isoforms. Previous work has focused on isoform variants of regulatory factors affecting early events in splice site recognition. We plan to explore, in addition, basal components of the spliceosome, in particular protein or RNA variants of spliceosomal snRNPs, because recent data suggest that regulation of alternative splicing can indeed be modulated not only at early points in spliceosome assembly but also in particllay or even fully assembled spliceosomes.

Regarding the work in plants, expansion of the AS RT-PCR panel is described in the report for WP8. Currently plant material is being prepared to allow the direct comparison of over-expression lines and mutant knock-out lines of the same SR protein isoforms. In addition, RSZ32 and RSZ33 are paralogues of the same SR protein gene. Mutants in each have been obtained and characterised for the two gene family members but unfortunately are in different Arabidopsis ecotype backgrounds. A double mutant and the appropriate F1 background control is being made to examine the effects of removing this gene. In addition, mutant lines of atRSp31 and atRSZ33 will be used for transformation with the alternatively spliced transcripts to investigate their respective activity. Over-expression constructs of GFP-fusions of PTB-like protein genes, PTBL1 and PTBL3 have been prepared and are currently being transformed into Arabidopsis. In addition, putative knock-out mutants have been identified and are being characterized genetically. Once prepared RNA from the SR and PTBL lines will be analysed on the 384 AS RT-PCR panel.

Work Package 12: Mis-splicing and Disease The first interdisciplinary focus meeting (IFM) on mis-splicing and disease was organized by F. Baralle and F. Pagani (ICGEB, Trieste – Italy) on January 17, 2007 in Cortina d’Ampezzo (Italy). The meeting dealt with gene expression regulation at the post-transcriptional level. The fundamental steps of eukaryotic RNA processing have been characterized in great detail, but knowledge of how the disruption of these processes contributes to human disease has only recently begun to emerge. Furthermore a whole section was devoted to specific talks by clinicians on inherited and common clinical problems derived from splicing regulation derangements such as retinitis pigmentosa, neurofibromatosis, some aspects of pain perception and pulmonary fibrosis. The meeting was arranged immediately after the EMBO Conference Series meeting entitled “pre-mRNA processing and disease” in order to increase the possibility of interaction offered to scientists and clinicians to exchange and acquire knowledge on the various aspects of Alternative Splicing among NoE members and the meeting participants. Most of the WP12 partners have attended and the clinical scientists invited to attend gave additional contribution to the meeting by presenting 30 minutes talks on their area of expertise. Additionally, we are grateful to Affymetrix for presenting their technologies during the meeting. From the scientific side, the meeting in Cortina provided numerous examples of mutations in cis-acting sequences and trans-acting factors that affect pre-mRNA splicing of genes important for human disease. Interestingly, it became evident that mutations in genes encoding general splicing factors might cause specific human diseases, such as adRP and SMA, instead of producing pleiotropic defects. Another important concept emerging from RNA analysis is that mutations in coding regions should not necessarily be assumed to be cSNPs or the cause of single amino-acid changes but could instead drastically affect splicing and lead to human disease. This should be taken into account during genotype screenings. More importantly, there is now evidence that the accumulated basic knowledge is slowly but steadily translating into molecular therapies designed to correct defective pre-mRNA processing.

Commercialization of the achievement is still far from being an option, although some of the therapeutic approaches derived from the improved knowledge of the mechanisms involved in pathological aberrant splicing are in the early stages of clinical trials.

A similar IFM will take place in Rome on May 31-June 5, 2008 to continue the discussions on RNA and disease with a more clinical approach. The organizers (Claudia Bagni, Francisco Baralle, Juan Valcarcel and Joel Richter) will follow the same pattern used in Cortina and preliminary details are already available at http://cwp.embo.org/cfs08-02/.

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We intend to consider multiple aspects of RNA formation and function such as RNA processing, mRNA translation, stability, biogenesis and the activities of small noncoding RNA, and how these processes impinge on human disease. The meeting will catalyze "translational science", bridging the gap between lab bench with the panoply of approaches revolving around RNA science and the clinic, and will form the basis of an exciting conference on RNA and the etiology of disease. We are confident that this second meeting will boost and strengthen the collaborations established so far between scientists and clinicians as well create the basis for new fruitful interactions. Work Package 13: Co-transcriptional Mechanism of Alternative Splicing Over the last two years, it has become clear that two themes are emerging as a major point of interaction among the work package members: 1) the role of transcription elongation rate in splicing outcome and 2) the mechanisms of co-transcriptional targeting of splicing regulators and spliceosomal components to actively transcribed genes in vivo. Our new deliverables clearly amplify on these themes and deepen the interactions among the groups. Two of our deliverables (100 & 142) received considerable input from the Kornblihtt, Ast, Bertrand and Aubeouf labs. A summary of the findings belonging to this deliverable can be found in the attached table, contributed to by all three groups. It compiles the effects of transcription elongation inhibitors on alternative splicing outcomes. In a related study, Neugebauer’s lab found that the level of co-transcriptional constitutive splicing (c-fos) increases when elongation is stalled by camptothecin (Listerman et al., 2006). These deliverables, aiming to investigate the relationship between elongation and splicing, should now be considered “done”; it is obvious that this theme now continues in more detail on the one hand, and broadens to include novel technologies on the other. Along these lines, Aubeouf’s lab demonstrated that mutation in Ewing Sarcoma of a transcriptional regulator (EWS-Fli) having an impact on transcription elongation, alter RNA splicing of cyclin D1 gene products. These data show that alterations of transcriptional regulators in cancer affect not only transcription, but also the processing of their target gene products, thereby increasing the expression of more oncogenic isoforms. This is the first demonstration of the physio/pathological impact of the coupling between transcription and mRNA maturation (manuscript in revision at PNAS). Moreover, Ast has generated a model system showing transition from exon inclusion to exon skipping as a function of time after transfection of the IKBKAP minigene into human cells lines. In collaboration with Kornblihtt, Ast’s lab could demonstrate that this transition is due to the elongation rate of RNA pol II – a slow RNA pol II restore the inclusion level whereas the fast one favor exon skipping. In addition, an especially fruitful collaboration between the labs of Kornblihtt and Bertrand, measuring the elongation rate of RNA polymerase II in vivo, has introduced new tools to the workpackage. The use of the FRAP system to measure Pol II elongation is a novel and key approach only possible thanks to the collaboration with Edouard Bertrand. Kornblihtt and Bertrand already published a paper (Boireau et al. 2007. J. Cell. Biol. 179(2):291-304.) using HIV-1 reporters, with which the system of studying mRNA biogenesis in real-time, by photobleaching specific transcription sites was characterized. They found that elongation proceeded at 1,9 kb/min, and polymerases remained at transcription sites 2.5 minute longer than nascent RNAs. On a total residency time of 333 seconds, 114 were assigned to elongation, 63 to 3'-end processing, and initiation likely took a large fraction of the remaining 156s. Remarkably, mRNAs were released seconds after the onset of polyadenylation, and analysis of polymerase density by ChIP indicated that they paused or lost processivity after passing the polyA site. Most importantly, this paper formally proved that the "slow" mutant of RNA pol II, used in previous studies to demonstrate the kinetic coupling between transcriptional elongation rates and alternative splicing is indeed slow in vivo. Complementary to this, Neugebauer and Bertrand will collaborate in future to determine whether splicing of HIV nascent transcripts is co-transcriptional. Bertrand’s model gene comprising a split MS2 reporter, in which the stem-loop binding site from MS2 BP can only form after the two halves are spliced together, will be used for ChIP as well as in vivo imaging. BAC-tagging in Bertrand’s cell line will also be attempted to enable splicing factor ChIP for comparison with the imaging work. This experiment could lead to a higher resolution dataset (in base pairs), informing us about the kinetics of co-transcriptional splicing -- i.e. how soon after the synthesis of the 3’splice site is the MS2 stem loop detected?

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The collaboration between Kornblihtt and Bertrand has continued and has led to another joint manuscript currently under review. The investigation of the mechanism by which UV radiation affects alternative splicing is relevant for the clinical importance of UV light as a cancer risk factor in combination with recent evidence on the relevance of alternative splicing in cancer. It was found that ultraviolet (UV) radiation affects alternative splicing of transfected and endogenous genes, including the upregulation of the pro-apoptotic splicing isoform of Bcl-x, an expected physiological response to DNA damage. Despite the fundamental roles played by the transcriptional activator p53 in response to DNA damage, the UV effect does not require p53 because it is still observed in p53 -/- cells. The UV effect is observed in transcripts of DNA templates transfected either before or after irradiation, which indicates that it is not caused by the actual damage of the DNA template in cis. The UV effect occurs only when splicing is co-transcriptional because in vitro synthesized pre-mRNAs transfected into cells allow for normal alternative splicing but this is not affected by UV light. This points at a systemic mechanism involving the kinetic coupling between transcription and splicing. UV light causes first the hyperphosphorylation of the carboxy terminal domain (CTD) of pol II, followed by its degradation. While the latter plays no role in the UV effect, CTD hyperphosphorylation at serines 2 and 5 is responsible for the alteration in alternative splicing through the inhibition of pol II elongation. Pol II mutants engineered to mimic the hyperphosphorylated state duplicate the effects of UV light on alternative splicing. Consistently, using fluorescence recovery after photobleaching (FRAP) to measure transcription elongation rates in vivo and in real time, we show that UV irradiation inhibits pol II elongation, similarly to a slow pol II mutant, which in turn affects splice site selection in the nascent pre-mRNA. Studies on elongation now extend also to the yeast system, with two groups (Neugebauer and Beggs) implementing in vivo elongation assays based on induction of gene transcription, followed by transcriptional shut-off and Pol II ChIP. Begg’s work specifically addresses the role of Prp45. In that work, the prp45-113 mutant has been shown to be sensitive to the transcription elongation inhibitors, 6-azauracil and mycophenolic acid. Furthermore, using antibodies that are specific for the different phosphorylated forms of RNA pol II CTD in ChIP assays, we found that the prp45-113 mutation caused a defect in the recruitment of serine 2-phosphorylated pol II to the yeast ASC1 gene. Together, these results are compatible with a defect in elongation rather than initiation of transcription. In a genome-wide screen we found synthetic genetic interactions between the prp45-113 mutation and genes encoding several chromatin remodeling factors. In the remaining months, Beggs will measure the effects of the prp45-113 mutation on transcription elongation directly by pol II transcription run-on assay (Birse et al, 1998) and/or by measuring the kinetics of induction of a PGAL-regulated gene in vivo (Mason & Struhl, 2005). In parallel, Neugebauer’s group has detected changes in cotranscriptional spliceosome assembly upon overexpression of the transcription elongation factor, TFIIS. Deliverable 102 examines how the polymerase controls the release of the nascent transcript. The group of Carmo-Fonseca had previously shown that transcripts derived from human β-globin genes containing splice site mutations are retained at the site of transcription. A key connection between transcription and mRNP biogenesis is provided by the carboxyl-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNA Pol II LS), which binds several proteins essential for pre-mRNA processing. In order to investigate the role of the CTD in β-globin transcript release, we generated murine erythroleukemia (MEL) cell lines that express α-amanitin resistant RNA Pol II LS with either full-length or truncated forms of the CTD. MEL cells expressing wild-type RNA Pol II LS maintained transcriptional activity in the presence of α-amanitin, indicating that the exogenous protein was functional. The mammalian CTD, which is essential for normal co-transcriptional maturation of mRNA precursors, comprises 52 heptad repeats. We show that a truncated CTD containing 31 repeats (heptads 1-23, 36-38 and 48-52) is sufficient to support transcription, splicing, cleavage and polyadenylation. Yet, the resulting mRNAs are mostly retained in the vicinity of the gene after transcriptional shutoff. The retained mRNAs maintain the ability to recruit components of the exon junction complex and the nuclear exosome subunit Rrp6p, suggesting that binding of these proteins is not sufficient for RNA release. We propose that the missing heptads in the truncated CTD mutant are required for binding of proteins implicated in a final co-transcriptional maturation of spliced and 3’ end cleaved and polyadenylated mRNAs into export-competent ribonucleoprotein particles. EURASNET support is acknowledged in the resulting paper: Custódio, N., Vivo, M., Antoniou, M., Carmo-Fonseca, M. (2007) Splicing and cleavage independent requirement of RNA polymerase II CTD for mRNA release from the transcription site. J Cell Biol 179(2):199-207. Mechanisms of splicing factor recruitment to sites of transcription (Deliverables 140 & 103) has received a major boost from the strategy of tagging splicing factors with GFP on bacterial artificial chromosomes (BACs) and then stably integrating the BACs into cell lines of choice. This system has the following advantages:

• the tagged splicing factor is expressed under the control of its own promoter; in extensive preliminary studies by Neugebauer’s lab, it was shown that protein expression levels in the

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stable cell lines are almost uniformly 1.5-fold of endogenous, as if the cells simply have an additional allele.

• the same tag can be used alternatively for imaging, for standard pull-downs or for ChIP. This is a significant advantage for ChIP, where background levels vary from antibody to antibody – here, the same antibody is used for every factor of interest.

• Moreover, splicing factors that simply do not “ChIP” with antibodies against endogenous epitopes (presumably a problem of epitope access) are detectable using antibodies against the tag.

• The BACs are portable, in that they can be transfected into any cell line, regardless of species or cell type. This is a significant saving in time and effort when developing new co-transcriptional alternative splicing assays.

Neugebauer has completed our survey of model genes, such as c-fos and, with Biamonti, SAT III. A manuscript is in preparation. Moreover, a number of the new deliverables are collaborative applications of this advance to other questions regarding co-transcriptional spliceosome assembly and/or recruitment of splicing regulators to endogenous gene targets. Currently we have tagged: CBP80, SmB, U1 70K, U2AF65, Prp8, PTB, SRp20, SF2, SC35, 9G8, SRp40, SRp55, SRp75.

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TABLE: Relationship of transcriptional elongation to alternative splicing: compilation of treatments that alter elongation rate and affect alternative splicing outcome (Deliverable 142) FACTOR OR TREATMENT

CHEMICAL NATURE

BASIC MECHANISM OF ACTION

CONSEQUENCES ON TRANSCRIPTION

EXAMPLES OF GENES WHOSE ALTERNATIVE SPLICING IS AFFECTED

5,6-dichloro-furanosyl-benzimidazole riboside (DRB)

Small organic molecule. Nucleotide analogue.

Inhibition of pTEFb (Cdk9 subunit), the protein kinase that phosphorylates the carboxy terminal domain (CTD) of RNA polymerase II

Inhibits transcriptional elongation

-Fibronectin - Bcl-X

Flavopiridol

Small organic molecule.

Inhibition of pTEFb (Cdk9 subunit)

Inhibits transcriptional elongation

-Fibronectin - NCAM - Bcl-X

Camptothecin

Small organic molecule.

Inhibition of DNA topoisomerase I

Creates raodblocks to transcriptional elongation

- Cyclin D1

TSA (Trichostatin A)

Small organic molecule.

Inhibitor of protein acetyl transferases

Favors histone hyperacetylation, subsequent chromatin opening and transcriptional elongation

- Fibronectin - NCAM

5 aza cytidine

Small organic molecule. Nucleotide analogue.

Inhibitor of DNA methyl transferases

Inhibits DNA methylation and favors chromatin opening and transcriptional elongation

- Fibronectin - NCAM

EWS

Protein

Human cell transcription factor

Stimulates transcriptional elongation

- Cyclin D1

VP16

Protein

Herpes virus transcription factor

Stimulates transcriptional elongation

- Fibronectin

SWI/SNF

Protein

Human cell transcription factor. Chromatin remodelling factor.

Interacts with Sam68 and creates raodblocks to transcriptional elongation.

- CD44

UV irradiation

-

-

Provokes RNA polymerase II hyperphosphorylation and inhibition of transcriptional elongation

- Fibronectin - Bcl-X

Slow mutant (C4) of RNA polymerase II

Protein

-

Displays lower transcriptional elongation rate

- Fibronectin - NCAM

Other small molecules that inhibit transcriptional regulators and impact splicing: H-7 Small molecule/ Inhibition of Cdk7/ Inhibits Pol II Ser5 phosphorylation/ Cyclin D1 Isoquinoline-5-sulfonic 2-methyl-1-piperazide H-8 Small molecule/ Inhibition of Cdk7/ Inhibits Pol II Ser5 phosphorylation/ Cyclin D1 N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride

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At the 2007 annual meeting in Ile de Bendor and an extended meeting at the Marseille airport, WP13 members met and unanimously agreed that what the field of co-transcriptional splicing needs is the ability to apply the many new in vivo techniques for examining the relationship between transcription and splicing (e.g. in vivo elongation assays, ChIP, chromatin modifications, etc) to novel and physiologically relevant systems. Therefore, we propose we would benefit as a group by committing ourselves to the deliverables described in the new JPA for WP13 month 25-42.

Work Package 14: Chemical Biology and Therapeutics After 24 months many of the initial objectives of this workpackage have been achieved. EURASNET members have collectively obtained hundred’s of small chemical molecules that interfere with constitutive or alternative pre-mRNA splicing and they have optimized strategies for using antisens oligonucleotide to modulate alternative splicing. Several groups have obtained active molecules to be tested in animal models. Constitutive splicing inhibitors: Plan Months 25-42 Fifteen compounds have been shown to inhibit splicing in vitro at different stages of spliceosome assembly (Partner 1a and 22) and therefore were used to establish the composition of stalled spliceosome. The aim for the future 18 month will be to identify direct molecular targets of these substances. First, we will test derivatives of these substances regarding their effect on pre-mRNA splicing in vitro. Synthesis of these derivatives will be performed by the research unit “Conception, Synthesis and Vectorisation of Biomolecules” at the Institut Curie (David Grierson, Paris/France) and/or by the “Scottish Hit Discovery Facility” at the University of Dundee (Julie Frearson, Dundee, Scotland). EURASNET acquired synthesis and support for these (and similar) purposes at both facilities. These analyses will identify the functional group(s) and overall scaffold required for inhibition by the substances. Comparison of these results within these chemicals, with other recently identified inhibitors of pre-mRNA splicing (partner 12a), and with well-characterized inhibitors of enzymatic activities as they are present in the spliceosome (e.g. kinases or phosphatases) will give indications how these substances inhibit pre-mRNA splicing in vitro. Second, we will try to identify the molecular targets of these inhibitors. For this, appropriately modified versions of the inhibitors will be attached to a column, and proteins that bind to these chemicals will be identified by affinity-chromatography followed by mass-spectrometry. Alternatively, an activatable crosslinker can be covalently attached to the inhibitors. After incubation with partially purified spliceosomes or total nuclear extract, crosslinking of the chemicals will be initiated and the modified proteins identified by mass-spectrometry. This will identify candidate factors, which will be analysed further. Both approaches require derivatives of the original inhibitors. The design of these will be helped by the analysis of inhibitor-derivatives as described above. Again, synthesis will be performed at the Institut Curie and/or the University of Dundee. The mass-spectrometry will be performed in collaboration with Henning Urlaub (partner 1c), who has longstanding experience in protein-protein and protein-RNA crosslinking analysis by mass-spectrometry. Specific inhibitors of ESE-dependent splicing (indole derivatives): Plan Months 25-42 Identification of 100 indole derivatives that selectively inhibit the activity of individual SR proteins by Partner 12a offered the exciting possibility to develop an alternative pharmacological approach for AIDS, aging, DMD and cancer treatment, based entirely on the use of “small molecules inhibitors “ to modulate splicing efficiencies of target pre-mRNAs. The broad objectives of this proposal are to design, synthesize and screen analogues of the initial molecule for maximum activity and minimum cytotoxic activity, to elucidate the interaction between selected analogues and SR proteins. The strategy has focused on developing drug like molecules disrupting the fused polyaromatic ring system of IDC16 (an SF2/ASF inhibitor) and generating a series of 200 ring opened analogues. We are currently testing whether newly synthesized molecules will maintain some of the putative essential structural features. It is anticipated that screening of libraries of analogues will result in identification of active compounds for lead optimization and for use in studies to elucidate the mechanism of SF2/ASF protein inhibition.

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HIV-1 and other retroviruses inhibition: collaboration Partner 12a and 12c. The main objective of this research proposal is to generate new and selective SF2/ASF inhibitors as lead compounds for HIV/AIDS drug development. A humanized mouse model for HIV-1 infection is developed by Partner 12a to test in parallel the efficacy and toxicity of the compounds. We will also use another mouse model in which a defect in splicing is associated with T-cell leukemia. This model is based on the infection of newborn mice from the so-called “ susceptible strains ” (Swiss, Balb, CBA, etc. ) with a mutant of the Moloney strain of MLV (Mo-MLV). In this model, inoculation of the parental Mo-MLV strain results exclusively in a T-cell leukemia, invariably characterized by a thymic and splenic involvement. These leukemia can be readily detected in all inoculated animals between 2 and 4 months of age. Leukemogenesis triggered by such a replication-competent MLV, which does not contain any oncogene, is a relatively slow process. According to the current model, integration, which follows a random pattern, in a “ sensitive ” region of the host genome leads to deregulation of (a) neighboring gene(s) normally involved in cellular proliferation and/or differentiation. This event would then participate to the positive selection of proliferating and transformed clones. Because alteration of the leukemia cell type in this case is entirely dependent on the alteration of an alternative splice site, we propose to use this model to test the compounds that target SR proteins and thus link splicing factors and splicing events to leukemogenic processes, specific or not of a leukemia cell-type.

Pre-clinical trials for metastatic breast cancer: collaboration Partner 12a and Partner 10. Partner 12a has recently installed a mouse mammary tumour model that consists of two metastatic tumour cell lines, MDA-MB-435 and MDA-MB-231. These cells were chosen because several indole derivatives were able to abrogate their motility in culture. In addition, when these cell lines are implanted into the mammary fat pads of syngeneic BALB/c mice, they form mammary carcinomas within a month. These cell lines disseminate from mammary fat pads and can be detected in lymph nodes and in lung. We are currently testing the efficacy of selected molecules to block spreading of these cell lines to lung. Selection of active molecules and pre-clinical trials for DMD: In collaboration with Genethon Partner 12a is currently screening the different libraries to identify new molecules capable of improving the efficacy of the exon skipping event using a luciferase reporter. The efficacy of selected compounds will then be assessed ex vivo using patient myoblasts, then in vivo using a humanized mouse model for this disease. In parallel NMD inhibitors will be tested for their suitability to correct cases of DMD in which the dystrophin gene harbours stop codons in the last exons which trigger mRNA degradation by NMD. The objective will be to produce truncated but functional Dystrophin Developement of a mouse model of HGPS and selection of inhibitors to correct aberrant LMNA splicing: Collaboration Partner 12a, Partner 12c, Partner 25 and Partner 1a and N. Levy clinician in Marsseille. The main objective will be to screen the different libraries to identify new molecules capable of improving the efficacy of authentic 5’ splice site downstream of exon 11 of LMNA using a luciferase reporter. Both in vitro and in vivo studies will be conducted with active molecules to determine which step of the aberrant splicing is affected. All the components (cell lines, reporter constructs for in vitro and in vivo studies, affinity purification, RNA structures of wild type and mutated LMNA substrates) have been obtained by the different partners. In collaboration with N. Levy Partner 12a has developed a mouse model in which mouse LMNA gene was replaced by a mutated version of the gene harbouring the GGC>GGT G608G single-base substitution. This model will be useful not only to test the efficacy of active molecules but also to determine tissue specific expression of LMNA aberrant splicing in correlation with the abundance of different SR proteins. Given that similar alteration of lamin A/C splicing was observed in aged individuals, Partner 12a is testing the hypothesis that the usage of the LMNA cryptic 5’ splice site with age can be dependent on the genetic background. To test this hypothesis, fibroblasts (50 lines) from individuals at different ages have been collected and the aberrant splicing of LMNA gene is quantified by RT-pPCR. Antisens oligonucleotide strategies to modulate alternative splicing

Correction of SMN expression in severe mouse model for SMA by U7-mediated transfer of antisens sequences:

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Partner 13 has extensively studied the U7 snRNP involved in histone RNA 3' end processing and its assembly, which is mediated by the SMN complex. For several years this partner has engineered modified U7 version (called U7 SmOPT) which no longer function in maturation of histone pre-mRNA but can carry antisens sequences. U7 SmOPT-derived RNA equipped with antisense sequences targeting specific splice sites should be ideally suited to manipulate the splicing patterns of individual target genes. Moreover, being embedded into snRNP particles, intracellularly expressed antisense sequences should be highly stable and resistant to nucleases. These vectors prove to be useful tools to induce exon skipping in several pathological models including β-globin gene in β-thalassimia, dystrophin gene in DMD, the cyclophilin A gene and the multiply spliced HIV-1 transcripts. However, this approach has never been tested in the context of exon inclusion. Since it has been hypothesised that the SMN2 C>T transition in exon 7 abolishes an ESE, Partner 19 designed an innovative strategy aimed at reintroducing an exogenous SF2/ASF binding site at the SMN2 exon 7 3'-ss. They rely on a so-called bifunctional oligonucleotide carrying an ESE tail linked to an antisense sequence necessary for specific targeting (TOE). Partner 13 adapted this principle by combining an antisense U7 snRNA with an additional ESE sequence capable of binding SF2/ASF. This strategy proved very efficient resulting in an exon 7 inclusion level superior to 30% in transient transfection. When integrated into stable Hela-SMN-LucF cell lines through a lentiviral vector, this construct even allows for a long term correction reaching 50% inclusion. To test the efficacy of this novel modified U7, Partner 13 has introduced in his lab a very severe mouse model for SMA where symptoms are clearly reduced and survival of the animals is prolonged. Moreover Partner 13 has made significant progress towards engineering inducible U7 snRNA cassettes. The plan for the next 18 months will be to introduce modified U7 in the model mice and test the efficacy of SMN2 correction. Work Package 15: Development of Enabling Technologies Several new technologies were investigated to enable a more complete and detailed understanding of alternative splicing. Some feasibility studies revealed limits of technologies (e.g., D2 bombardment, improvement of protein-RNA cross-linking, partner 1c). Alternative strategies will be explored (see future plans below). Important progress has been achieved in mass spectrometry analysis of RNA-protein cross-links (partner 1a). This strategy should be useful for many partners in the NOE. Similarly, constructs for stable expression of tagged mRNA species, for studies on alternative splicing and targeted, post-transcriptional RNA modification are now available (partners 1b, 3 and 10b). Development of enabling technology was also pursued in many unanticipated directions, due to new need in specific areas, this included:

- establishing splicing-sensitive microarray designs and data analysis (partner 2) - improving TAP tag purification and developing new protein interaction assays (partner 12b) - developing FLIM-FRET techniques for analyzing the interactions of protein splicing factors in vivo. - implementing new proteomics approaches for analyzing protein interaction partners using quantitative

mass spectrometry. Future plans/new JPA 1) Highly accurate ESI FT MS of cross-linked species will be performed. The exact masses of the cross-linked species, determined in this way, will serve as input information for the identification of the cross-linked peptide species: The exact mass and marker ions derived from the cross-linked peptide and RNA moiety will allow the identification of cross-linked species by database search using an appropriate search engine. This information will help all EURASNET collaborator and groups outside the network to map protein-RNA cross-links. 2) Enrichment strategies for cross-linked RNA-peptides will be used to identify cross-linked proteins/peptides in core 12S U2 snRNP, tri-snRNPs and spliceosomal B and C complexes. A similar enrichment strategy will be used to systematically investigate proteins with RRM domains complexes with various RNAs (collaboration between H. Urlaub (partner 1c) and F. Allain (partner 29). Such a systematic study will help (in the absence of highly resolved 3D protein–RNA structures) to elucidate the “code” of RNA interaction mediated by RRM domains. 3) Mass spectrometry-based identification and characterization of isoforms of spliceosomal proteins involved in (alternative) splicing: we plan to identify subsets of isoforms of proteins by multiple reaction monitoring

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experiments. We will also attempt to use quantitative mass spectrometry analysis to monitor changes in intracellular protein location, including factors involved in alternative splice choice. 4) The use of massive sequencing for the analysis of alternative splicing event will be investigated by several partners. 5) The use of efficient mutagenesis to identify protein interaction interfaces, a possible low resolution substitute for D2 bombardment, will be investigated. We will also investigate the use of FLIM FRET to monitor protein interaction in vivo. 6) A system for high throughput analysis of alternative splicing exon through cloning in minigenes will be set-up. 7) The feasibility of using ultracentrifucation to monitor assembly of RNA binding protein on RNA will be tested. Work Package 19: Public Understanding of RNA Biology The scientific officer (PSO) is central to the proposed activities in this work package. In principle, the PSO will oversee and help to coordinate all actions related to this area of activities. The PSO is in the process of generating an extended brochure describing the members and aims of the EURASNET as well as topics connected to alternative splicing. In addition, a new version of the EURASNET leaflet, including all YIPs will be generated in parallel. Another task of the public scientific officer is to directly communicate to the webpage master in Dundee for consistent report on PUS activities like press releases, accessible for EURASNET members and the public. The PSO will also continue to enrich and update the PUS part of the EURASNET webpage as well as take care of the teaching materials. The extended EURASNET brochure is supposed to inform the public about the impact of alternative splicing on life in general,. Target groups should be informed about the important role of RNA processing in mechanisms of human disease as well as in the developmental programme of higher organisms. Furthermore, a detailed insight into research topics and fields of all the network participants should be given. Presented articles will be richly illustrated with pictures, schemes and drawings for an easy understanding of the topic. Target groups to be reached were selected to act as “amplifiers”. These are medical doctors, teachers (especially biology teachers, intending to further instruct their pupils about the topic of alternative splicing) and higher educated pupils by themselves. First the brochure will be in English and German but later should be translated into several languages. The brochure is also intended to act as a more elaborate introduction to the network (including all 40 research groups). The extent of the brochure will be about 25 pages. To inform about current activities and remarkable findings within the network, a biannually appearing newsletter will be disseminated among researchers in the RNA field and selected target groups. This newsletter will comprise articles describing the impact of alternative splicing on one specific issue. A short description of the work of one research group as well as background information about their specific research topic will be given. To educate the scientists of the EURASNET in communicating science to the public, a media contest and a media workshop will be organized in the framework of the 1st International EURASNET Conference in Krakow (Poland) in May 2008. Work Package 20: SMEs and Technology Transfer We will use the in vitro splicing assay format developed to carry out a large scale small molecule inhibitor screen using the chemical compound library and robotic screening platform established at the University of Dundee by Professor Julie Frearson (www.drugdiscovery.dundee.ac.uk/). Any resulting chemical inhibitors of splicing will be characterised and made available to the EURASNET groups. Stable cell lines will be established in which the expression of differently coloured fluorescent proteins provide a reporter system for pre-mRNA splicing activity in vivo. These cells will be used as a secondary, in vivo screen for any small molecules that inhibit splicing in vitro and may also be used in a primary screen for in vivo inhibitors/modulators of RNA splicing if time and resources allow. We will continue develop our commercial links and an important part of our SME interactions will be to transfer more reagents from the member EURASNET laboratories for commercialisation by Dundee Cell Products. RNA splicing reagents, including an in vitro splicing kit, RNAase inhibitor and HeLa cell nuclear extracts, will be developed as commercial products

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by Dundee Cell Products and evaluated in collaboration with EURASNET groups. We will patent and commercialise any chemical inhibitors of splicing identified through the screening initiative. These intended activities of WP 20 are described within the new set of deliverables for the JPA (month 25-42): • In collaboration with EURASNET Groups, principally Group 22 (Lamond) Dundee Cell Products the components for carrying out standard in vitro splicing assays will be developed as a kit and commercialized. • Group 22 (Lamond) has negotiated with Professor Julie Frearson (University of Dundee) access to the Dundee based Drug Screening Facility and a major high throughput screen to identify splicing inhibitors is scheduled for summer 2008, involving a library of over 10,000 compounds selected for their chemical suitability for development as high affinity inhibitors. The timing of the screen will depend upon successful completion of optimizing the assay system developed in Deliverable 23 for use in this high throughput screening initiative. The resulting compounds will be made available for use by other NOE groups. Work was carried out during months 12-18 in conjunction with Dundee Cell Products to produce sufficient quantities of high quality HeLa cell nuclear extract and RNAase inhibitor at discounted price to support the high throughput screen and assay development and this activity in ongoing. • A stable cell line will be generated that expresses a transcript that will either encode a red or green fluorescent protein dependent upon whether splicing is active or inhibited. The cells will be used in conjunction with fluorescence microscopy to assess whether chemical compounds that inhibit splicing in vitro can also inhibit splicing in vivo. • Any chemical compounds that inhibit splicing either in vitro and/or in vivo, as determined by readout assays desribed above, will be characterized to determine their mechanism of action and their protein or RNA targets in the spliceosome. Work Package 21: Reachout to the Broader RNA Community The many and varied activities of the EURASNET members in promoting alternative splicing research and the Network will continue throughout the new JPA. The work programme for months 25-42 for WP21 will focus on efficient information gathering and quantification of these activities. In addition, the need for greater interaction with clinicians and medical scientists involved with splicing- and alternative splicing-related diseases will be addressed by identifying these individuals in the member countries and inviting them to attend meetings and workshops and sending information about EURASNET.

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18 MONTH JPA: WORKPACKAGE TIMECOURSE WORKPACKAGE 0 – 6 months 6 – 12 months 12 – 18 months DESCRIPTIONS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1. THE EURASNET WEB SITE (Stamm) 2. SHARING RESOURCES, TECHNOLOGY AND RELIABLE PROTOCOLS (Eperon)

4. THE ALTERNATIVE SPLICING DATABASE (Apweiler) 5. ENSURING DURABILITY (Carmo-Fonseca) 6. IN SILICO APPROACHES TO ALTERNATIVE SPLICING (Ast)

7. MOLECULAR CHARACTERIZATION OF SPLICING SUBSTRATES (Eperon)

8. GENOME-WIDE ANALYSES OF SPLICING REGULATION (Smith)

9. COMPLEXITY OF SPLICEOSOMAL PROTEOMES (Lührmann)

10. POST-TRANSLATIONAL MODIFICATION AND INTRACELLULAR DYNAMICS OF SPLICING FACTORS (Caceres)

11. FUNCTION OF SPLICING FACTOR ISOFORMS (Valcárcel)

12. MIS-SPLICING AND DISEASE (Baralle) 13. CO-TRANSCRIPTIONAL MECHANISMS OF ALTERNATIVE SPLICING (Neugebauer)

14. CHEMICAL BIOLOGY AND THERAPEUTICS (Tazi) 15. DEVELOPMENT OF ENABLING TECHNOLOGIES (Séraphin)

16. CONFERENCES AND MEETINGS (Beggs) 1st International meeting 2008 three workshops on areas of strategic relevance/4 IFMs

17. STAFF EXCHANGE AND TRAINING (Krämer)

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18 month JPA - Gantt chart (month 25-42) 15.2 PLANNING AND TIMETABLE

18. CAREER DEVELOPMENT (Kjems) career development workshop web-based seminars

19. PUBLIC UNDERSTANDING OF RNA BIOLOGY (Barta) Science and Society website training workshop

20. SMEs AND TECHNOLOGY TRANSFER (Lamond) 21. REACHOUT TO THE BROADER RNA COMMUNITY (Brown)

22. MANAGEMENT (Lührmann) Annual Meeting Report 2007 Steering Committee/Quarterly/Network-e-mail Annual Report 2007

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15.3 GRAPHICAL PRESENTATION OF WORK PACKAGES The figure shows the work packages of the NoE grouped by closeness of association, rather than by administrative hierarchy. Workpackages similar in nature are grouped together on a common background rectangle, and those with somewhat less close kinship are grouped close to one another but on different rectangles. Spheres of overlap are indicated by an overlap of individual workpackages over different rectangles, or by overlap of entire rectangles. The figure also shows a gradation from the more “individual” activities – career development, training – on the left, through research and central activities in the middle, to the “outwardly directed” activities on the right: external dissemination, collaboration and ultimately commercialisation. The “Management” activity (work package 22) is represented as underpinning the entire collection of network activities.

Pert-Diagram - Workpackage Interactions A more revealing picture of the high level of interactivity achieved within the network provides the following diagram of reported member interactions.

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Interactions among EURASNET participants.

EURASNET interactions 2007

ABa ABi AJ AKo AKr AL AS BS CB CS DA DB DG DS EB EE FA FB GAk GAs GB GTV HS HU IP JBe JBr JBuj JC JK JS JT JV KN MCF MZ PB RA RL SSAndrea Barta ABaAlbrecht Bindereif ABiArtur Jarmolowski AJAlberto R. Kornblihtt AKoAngela Krämer AKrAngus Lamond ALAnabella Srebrow ASBertrand Séraphin BSChristiane Branlant CBChristopher Smith CSDidier Auboeuf DADiana Baralle DBDavide Gabellini DGDaniel Schümperli DSEduard Bertrand EBEduardo Eyras EEFrédéric Allain FAFrancisco Baralle FBGöran Akusjärvi GAkGil Ast GAsGiuseppe Biamonti GBGlauco Tocchini-Valentini GTVHermona Soreq HSHenning Urlaub HUIan Eperon IPJean Beggs JBeJohn Brown JBrJanusz Bujnicki JBuJavier Caceres JCJørgen Kjems JKJames Stévenin JSJamal Tazi JTJuan Valcárcel JVKarla Neugebauer KNMaria Carmo-Fonseca MCFMihaela Zavolan MZPeer Bork PBRolf Apweiler RAReinhard Lührmann RLStefan Stamm SS

2007

YIPInformation received

245

15.4 TABLE OF MILESTONES (MONTH 25-42) milestone month 18 Annual NoE Meeting 28 19 Four interdisciplinary focus

meetings 36

20 EURASNET Website fully operational in all its modules

12

21 Third annual workshop programme completed

36

22 Second European Conference on Alternative Splicing

40

M18 Third annual NoE meeting. [This will have the character of an international meeting.] M19 Again, four interdisciplinary focus meetings (with one meeting on Splicing and Disease) have been held. M20 The EURASNET Website is fully operational with all its internally accessible modules as well as with modules directed to the medical community and the general public. M21 The third round of workshops has been completed by month 36. M22 The Second European Conference on Alternative Splicing in conjunction with the fourth annual NoE meeting. [This will instead be a normal reporting meeting.]

246

15.5 DELIVERABLES LIST (MONTHS 25-42)

Joint programme of activities (18 months period, month 25 - 42)

Del. no.

Deliverable name WP no. Lead participant

Nature Dissemination level

Delivery date

(proj. month)

127 Prepare Annual Report for 2007 period (month 25).

22 Lührmann R CO 25

234 Interdisciplinary focus meetings planned for 2008.

16 Beggs O PU 26-29

171 Array bioinformatics postdoc to be appointed in the Auboeuf group with the following remit: 1. develop methods for data analysis of ExonArrays (and junction arrays, when available – see next deliverable) 2. develop user-friendly web-interface (FAST-db) 3.provide service role for other EURASNET members (all month 27)

8 Smith O PU 27

259 Steering Committee Meetings at month 27,30, 33, 36, 39, 42

22 Lührmann O CO 27-42

260 Quarterly network e-mail at months 27,30, 33, 36, 39, 42

22 Lührmann O CO 27-42

233 Interdisciplinary focus meetings planned for 2008.

16 Beggs O PU 28-36

172 We will aim to reach an agreement with Affymetrix for access to their human and mouse research junction arrays (month 28)

8 Smith O CO 28

244 media contest (scientist write press releases) (month 29)

19 Barta O PU 29

19 a training workshop in public understanding of science for NoE scientists (month 29)

19 Barta O PU 29

239 Organization of a mentor lunch (month 29)

18 Kjems O PU 29

240 Organization of job interviews at the Krakow meeting (month 29)

18 Kjems O PU 29

247

114 First International EURASNET Meeting, Krakow The First International EURASNET Conference on Alternative Splicing, organised by participants J. Beggs, A. Jarmolowski, A. Krämer and S. Stamm, will be held May 21-23, 2008, followed by the Third Annual Meeting of the EURASNET NoE, May 24-25. (month 29)

16 Beggs O PU 29

119 Organization of a workshop on career development at the Krakow meeting (month 29)

18 Kjems O PU 29

118 Career development related round table discussions with representatives of both academia and industry (month 30).

18 Kjems O PU 30

175 Exon Array (and junction array, if available) microarray analysis of effects of hSlu7 knockdown upon alternative splicing (G Ast, month 30)

8 Smith O CO 30

116 Continue and further develop the use of travel bursaries for student and postdoc exchanges (also for necessary intra-workpackage meetings)(40 PMT/18 month) (month 30).

17 Krämer O PU 30

209 Use of minigene splicing assay to study the influence of mutations in neurofibromatosis type 1 exon/intron 29 splicing (month 30)

12 Baralle O CO 30

210 Analysis of the role of PTB in pseudoexon splicing (month 30)

12 Baralle O CO 30

257 Report on decisions made during the Annual Meeting 2008 (month 30).

22 Lührmann R CO 30

132 Improved tools for combined searches and user-friendly access to the ASD database; Integration of additional species to ASTD (continuation of del.7, month 30).

4 Apweiler O CO 30

N6 Computational structural model of splicing regulators hnRNP-L and hnRNP-L-like in complex with RNA (month 30)

6 Ast O CO 30

N7 Computational modeling and experimental testing of novel RNA binding domains (month 30)

6 Ast O CO 30

248

N8 Use of minigene splicing assay to study the influence of mutations in neurofibromatosis type 1 exon/intron 29 splicing (month 30)

12 Baralle O CO 30

N9 Analysis of the role of PTB in pseudoexon splicing (month 30)

12 Baralle O CO 30

72 Identification of more in vivo targets of the functional activity of splicing regulatory proteins (to be subsumed in WP8). (month 30)

7 Branlant O CO 30

243 an extended brochure for the general public describing the NoE, the concepts, participants and projects, as well as alternative splicing in development and disease (month 34)

19 Barta O PU 34

245 a new version of the EURASNET leaflet (month 34)

19 Barta O PU 34

248 Develop and market commercially a kit for in vitro splicing assays (month 36).

20 Lamond O CO 36

241 Evaluate available career development training courses within the EURASNET network for appropriateness for our pre- and postdocs (month 36)

18 Kjems O CO 36

254 Prepare circulation list of clinicians and scientists involved in therapeutic approaches to splicing diseases. The first year report suggested greater interaction with medical scientists and clinicians to raise awareness of alternative splicing. Some members of EURASNET interact with clinicians and medical scientists and consult or act as experts to groups involved in development of therapies for splicing-related diseases. We will, via these EURASNET contacts, establish a list of contacts for such scientists. Information on upcoming meetings and workshops organised by EURASNET will circulated to this group. (month 36).

21 Brown O PU 36

249

255 Modify proforma for capturing information. The proforma used to capture information on various activities in terms of broader outreach to the RNA community will be modified on the basis of feedback from members (month 36).

21 Brown O CO 36

146 Reorganization and re-siting of website (month 36)

1 Brown O PU 36

183 Replace low information content Arabidopsis AS events with other selected AS events and characterise novel events (month 36)

8 Smith O CO 36

147 Design of new website with new functionality and transfer of information (month 36)

1 Brown O CO 36

226 Proof of principle for identification of protein interaction surfaces by efficient mutagenesis (month 36)

15 Séraphin O CO 36

227 Set up a high-throughput cloning system for minigenes that allows rapid analysis of alternative exons (month 36)

15 Séraphin O CO 36

228 Development of FLIM-FRET for mapping protein-protein interactions at specific subcellular (and subnuclear) locations in vivo (month 36)

15 Séraphin O CO 36

148 Database of substances that change alternative splicing (month 36)

1 Brown O CO 36

236 Three workshops on areas of strategic relevance in 2009:

16 Beggs O PU 37

258 Prepare Annual Report for 2008 period (month 38).

22 Lührmann R CO 38

250 Develop a stable cell line for evaluating the effect of chemical inhibitors on pre-mRNA splicing in vivo (month 40).

20 Lamond O CO 40

235 2009 Annual Meeting, Rome, April, 2009 To be organized by participant G. Biamonti (month 40)

16 Beggs O PU 40

242 Pursue the possibility of selecting one program for a 1-2 day career development workshop in which participants get hands-on training in the chosen topic (eg writing the cv, giving a talk or an interview, making and presenting a poster) (month 41)

18 Kjems O PU 41

149 Liaise with targeted audiences and prepare appropriate content (month 42)

1 Brown O PU 42

250

150 Tools and resources repository updated (month 42).

1 Brown O CO 42

151 Implementation of an improved web entry form for protocols that takes into account feedback of EURASNET members and participants of the practical courses.

2 Stamm O CO 42

152 The current filemaker based system is slow and cumbersome to use. The pages will be reprogrammed using HTML with an easy to use interface (Month 36). As suggested by many users, there will be a stronger emphasis on background (why is this procedure done) as well as a searchable index.

2 Stamm O CO

153 Publication of the protocols as a pdf booklet on the EURASNET web site and publishing of a protocol book (Month 42)

2 Stamm O PU

154 Fast DB already contains an algorithm that allows analysis of Affymetrix splicearray datafiles. We will create an interface that allows direct visualization of splicearray data in FAST DB (month 42).

4 Apweiler O PU 42

155 Integration of an algorithm that computes the likelihood of sequences to be single stranded (PU) values into FAST DB (month 42).

4 Apweiler O PU 42

156 The EURASNET member Stamm published a method to calculate a statistical value (PU value) that expresses whether a given nucleotide in an RNA is single or double stranded. Experimental data show that exonic regulatory elements are preferably in single stranded conformation. We will implement a graphic representation of this algorithm in the FAST DB database (Hiller, M., Zhang, Z., Backofen, R., and Stamm, S. (2007). pre-mRNA secondary structure and splice site selection. PLOS Genetics 3, 2147-2155) (month 42).

4 Apweiler O PU 42

157 Maintain and further expand the information package/forum within the EURASNET website and meetings with updated announcements on funding possibilities.

5 Carmo-Fonseca

O PU 42

251

158 Maintain and further expand the liaison with relevant bodies at both national and EU level. (month 42)

5 Carmo-Fonseca

O CO 42

159 The Stamm lab will experimentally validated snoRNA target predictions generated by the Zavolan lab. This will be performed to identify the cis-elements that control HBII-52-dependent alternative splicing. The algorithm to determine snoRNA tartgets will be used with two other snoRNAs in the Prader-Willi critical region, HBII-438 and HBII-85. In addition, the algorithm to predict single-strandedness will be implemented in a suitable database and combined with binding motifs to analyse DNA array results for tra2-beta1 (month 42).

6 Ast O CO 42

160 The Zavolan lab implemented a snoRNA-mRNA binding model, and applied this method to the prediction of snoRNA targets in mouse (in collaboration with Stamm). They will apply this method to other systems such as plants. These latter predictions will be tested by the Brown lab in the plant Arabidopsis thaliana. In addition they will improve the quality of the miRNA target predictions, and will develop automated methods to update the predictions as more genomes and transcripts become available. Finally, they will develop methods to relate changes in mRNA expression to changes in the expression of miRNAs (month 42).

6 Ast O CO 42

161 The Bork lab will finalize a visualization tool that can display and integrate various datasets (EST, cDNA genomic DNA etc) and computes splice paths through the corresponding proteins. They will also examine a network of genomic sequence motifs involved in alternative splicing which includes prediction and validation of new alternative splicing variants (month 42).

6 Ast O CO 42

252

162 The Ast lab will compare splice sites and splicing factors that bind these sites among the entire eukaryotic kingdom (in collaboration with Eyras). They will compare the evolution of genes, exon-intron structure, intron gain and loss, and the effect of transposed elements in vertebrates and invertebrates (month 42).

6 Ast O CO 42

163 The Eyras group in collaboration with the Smith group will study the splicing regulation of introns in which the branch site sequence located more than 100 nucleotides from the 3’ss. They will use comparative methods to extract possible regulatory sequences and secondary structres that may be involved in the regulation. They will also identify the cases that are related to disease. In colaboration with the Ast group they will compare the splicing mechanism in eukaryotes. Using multiple eukaryotic species, they will compare the properties of protein factors and snRNAs involved in splicing. They will investigate the role of the SR and hnRNPs proteins in the origin of regulated splicing (month 42).

6 Ast O CO 42

164 The Bujnicki lab will develop a prototype version of template-based method for RNA 3D structure prediction by fragment matching and a prototype version of template-free method for RNA 3D structure prediction by coarse-grained folding simulation. They will also carry out modeling of hnRNPL&L-like protein-RNA complexes (in collaboration with the Bindereif group) and protein structures of novel domains involved in RNA-binding (in collaboration with the Stamm group (month 42)

6 Ast O CO 42

253

165 Identification of local pre mRNA secondary structures, the perturbations generated by mutations found in genetic diseases and their effects on splicing (continuation of deliverable 69) (month 42)

7 Branlant O CO 42

166 Complete identification of components in splicing regulatory complexes and assessment of their roles in the functions of splicing regulatory elements and splicing sites (continuation of deliverable 70) (month 42)

7 Branlant O CO 42

167 Expansion of work on detailed analysis of RNA-protein complexes formed with recombinant proteins and repeat sequences or pre-mRNA regulatory sequences, including structural analysis (continuation of deliverable 71) (month 42)

7 Branlant O CO 42

168 Development of methods for determination of the stoechiometry of binding of splicing factors on pre-mRNAs and to estimate their relative affinities in vitro and in cellulo. (month 42)

7 Branlant O CO 42

169 Development of cell imaging methods and single molecule methods to follow the kinetics of splicing factor association with pre-mRNAs (continuation of deliverable 73) (month 42)

7 Branlant O CO 42

170 Obtain 3D Structure information on splicing regulatory proteins by NMR approaches (Etr3 and Tra2β) by X-ray crystallography and 3D structure computer modelling (MBNL1 and CUG-BP) (continuation of deliverable N10) (month 42)

7 Branlant O CO 42

173 Further development of data analysis for ExonArrays (Bindereif, Auboeuf) (month 42)

8 Smith O CO 42

254

174 Application of splice-sensitive array analysis to biomedically relevant samples: 1. in a tuberculosis model system, Bindereif in collaboration with Stefan Kaufmann, Max-Planck-Institute for Infection Biology, Berlin (month 42). 2. ExonArray analysis of the transcriptome of a mouse model of breast cancer (Auboeuf, month 36)

8 Smith O CO 42

176 Exon Array (and junction array, if available) microarray analysis of effects of SF1 knockdown upon alternative splicing (Krämer, month 42)

8 Smith O CO 42

177 Validation of array predictions of AS changes in epithelial-mesenchymal transition, focusing mainly on alternative splicing of SR-proteins transcripts (Biamonti, month 42).

8 Smith O CO 42

178 We will develop simulations of alternative splice form sampling to study the theoretical limits of deep sequencing approaches to profiling of alternatively spliced isoforms (Zavolan).

8 Smith O CO 42

179 Application of Roche/454 and/or Solexa/Illumina sequencing for quantitative comparison of HeLa cell control and splicing regulator knockdown samples (PTB/nPTB, Smith, Valcarcel, Zavolan; hnRNP-L, Bindereif). (Month 36)

8 Smith O CO 42

180 Application of deep sequencing to CLIP tags of SF1 and U2AF65 (Krämer, month 42)

8 Smith O CO 42

181 Use of CLIP to identify RNA targets for the shuttling SR protein SF2/ASF. Use of CLIP will be complemented with subcellular fractionation to identify nuclear, cytoplasmic and polysomal targets (Caceres, month 42).

8 Smith O CO 42

255

182 Use of CLIP to identify RNA targets for hnRNP A1. Use of CLIP will be complemented with subcellular fractionation to identify nuclear, cytoplasmic and polysomal targets. Furthermore, we will identify RNA targets in cells subjected to stress, due to the reported role of hnRNP A1 in the stress response (Caceres, month 42).

8 Smith O CO 42

184 Continue to complete analyses of Arabidopsis NMD mutants and hyl1; SR protein overexpressor and mutant lines (month 42)

8 Smith O CO 42

185 The plant groups (will establish collaborations with other groups to analyse proteins which affect alternative splicing (month 42)

8 Smith O CO 42

186 Relativ quantitative mass-spectrometric analysis of spliceosomal complexes. (month 42)

9 Lührmann O CO 42

187 Establishing a map for spliceosomal proteins in a novel 2D electrophorese system without IEF. (month 42)

9 Lührmann O CO 42

188 Feasibilty studies for chemical cross-linking of proteins within purified spliceosomes and/or subspliceosomal protein complexes. (month 42)

9 Lührmann O CO 42

189 Role of Cwc21p in splicing specificity and fidelity (month 42)

9 Lührmann O CO 42

190 MS based proteomics of Brr2 contain complexes (month 42)

9 Lührmann O CO 42

191 Establishing of stable cell line L4-33K and studying cellular and viral protein–protein complexes containing L4-33K including subcellular localization. (month 42)

9 Lührmann O CO 42

192 Characterization of protein heteromeric complexes associated with regulated HIV RNA splice sites. (month 42)

9 Lührmann O CO 42

193 Relative quantitative mass spectrometry of proteins aossociated with regulated HIV RNA splice sites, RSV RNA and wildtype/mutant exon 5 of LMNA pre-mRNA. (month 42)

9 Lührmann O CO 42

256

90 Use the tap-tagged SRp30 protein to select and MS-analyze ribonucleoprotein complexes in nSBs (month 42).continued

10 Cáceres O CO 42

194 Bertrand will analyze in detail the binding of U1-specific proteins to the transcription site of the HIV-1 reporter RNA by FRAP, including (i) interpretation of the FRAP curves with a diffusion-reaction model, (ii) analysis of the binding of U1 proteins on reporter RNA containing mutation in their splice donor sequence (either stabilizing or destabilizing U1 binding), (iii) analysing the binding of mutant proteins, unable to bind U1 snRNP or other partners. He will determine the recruitment of splicing factors characteristic of a given spliceosomal complex (e.g. A, B, or C) on the transcription site of wild-type and mutant MINX reporter RNAs, and initiating the analysis of their binding dynamic by FRAP.

10 Cáceres O CO 42

195 Akusjarvi will extend the mutational analysis of L4-33K to other conserved regions of the protein with the aim of establishing the relationship between function, protein stability, subcellular localization and the phosphorylated status of the protein. He will also develop experimental reagents for overexpression of catalytically active and inactive C subunits of PKA (stable cell lines and/or recombinant adenoviruses) and will establish an in vitro system responding to PKA C subunit expression. (month 42)

10 Cáceres O CO 42

196 Lamond will perform FLIM/FRET analysis of additional spliceosomal proteins in mammalian cells. He will focus on the interactions between hnRNP M, CDC5 and PLRG1.

10 Cáceres O CO 42

257

197 Caceres will use the CLIP protocol (see WP8) to identify RNA targets of hnRNP A1 in control cells and cells subjected to stress and also to identify RNA targets for the shuttling SR protein, SF2/ASF. In both cases, use of CLIP will be complemented with subcellular fractionation to identify nuclear, cytoplasmic and polysomal targets of hnRNP A1 and SF2/ASF. This will result in the identification of endogenous targets for these antagonistic splicing factors as well as to identify a role for hnRNP A1 in the stress response (Month 42)

10 Cáceres O CO 42

198 Carmo-Fonseca will perform FRET analysis of splicing factor interactions in the spliceosome. She will use stable cell lines expressing a tandem array of transgenes tagged with the MS2-binding sequence. In these cells, co-transcriptional spliceosome assembly is visualized as a single fluorescent focus. (month 42)

10 Cáceres O CO 42

199 Lührmann will develop a more detailed picture of the mode of action of hPrp4 in the splicing machinery with particular respect to possible interaction partners. Lührmann and Urlaub will establish the phosphoproteom of human and yeast splicesomes. Lührmann will use phosphor-peptide specific antibodies for in situ localization of active spliceosomes (month 42)

10 Cáceres O CO 42

200 Stamm will determine how PP1 regulates the interaction of RNA with the splicing factos SF2/ASF, tra2-beta1 and SRp30c. (month 42)

10 Cáceres O CO 42

201 Tazi will investigate the relationship between B52 and the other candidate genes identified in the genetic screen. These factors could be regulators or antagonists of B52, but could also correspond to target genes whose expression is altered by B52 overexpression. (month 42)

10 Cáceres O CO 42

258

202 Srebrow will investigate the role of Akt as a novel SR protein kinase. The subcellular localization of SR proteins will be evaluated by observation of SR-GFP fusions in the presence of siRNA against AKT, and other established SR protein kinases, such as SRPK or CLK. (month 42)

10 Cáceres O CO 42

203 Description of splicing factor isoform changes upon activation of signaling pathways (month 42)

11 Valcárcel O CO 42

204 Description of isoform diversity and changes in core spliceosomal components in different tissues (month 42)

11 Valcárcel O CO 42

205 Analysis of effects of SF1 isoform overexpression on alternative splicing events identified by CLIP (month 42)

11 Valcárcel O CO 42

206 Investigate the role of U2AF35 isoforms and U2AF35-related proteins on splicing regulation / cell cycle progression (month 42)

11 Valcárcel O CO 42

207 Analysis of genetic circuits involving alternative splicing at 3’ UTRs and the interplay with cytoplasmic regulatory processes (month 42)

11 Valcárcel O CO 42

208 Analyze SR protein and PTBL over-expression and mutant lines using the 384 AS RT-PCR panel (month 42)

11 Valcárcel O CO 42

211 In depth characterization of trans-acting factors of particular interest with regards to their functionality, biochemical properties, and general importance for the cellular metabolism particularly in the development of tumors (Biamonti) (month 42)

12 Baralle O CO 42

212 Development of oligonucleotide-based or snRNP based approaches to correct splicing defects with special emphasis on investigating the molecular mechanisms affected by these novel reagents. (month 42)

12 Baralle O CO 42

213 Intensification of the interactions among medically interested people of EURASNET creating special interest groups centered on common clinical problems and diseases. (month 42)

12 Baralle O PU 42

259

214 Further development of techniques for use in diagnostic tests including the standard quality assurance required for diagnostic molecular genetic laboratories. (month 42)

12 Baralle O CO 42

215 To determine the molecular basis for the effect of Prp45p on transcription, Beggs will analyse the effects of prp45 mutant alleles on the recruitment of CTD kinases and chromatin remodeling factors to the ASC1 gene by in vivo ChIP assay, and the interaction of these proteins with Prp45p will be tested in pull-down assays in vitro. (month 42)

13 Neugebauer O CO 42

216 Aubeouf and Neugebauer will collaborate to establish splicing factor ChIP in MCF7 cell lines. These breast cancer cells are responsive to estradiol, which stimulates transcription of cyclin D1, PS2 and other genes. This cellular model will allow us to test the splicing process on gene products made in response to hormones. We will establish stable cell lines with integrated copies of BACs expressing GFP-tagged versions of U1 70K, U2AF65, Prp8, CBP80 and members of the SR protein family, in order to investigate the interplay between transcription, co-transcriptional spliceosome assembly and AS outcome. (month 42)

13 Neugebauer O CO 42

217 Kornblihtt will determine whether intronic siRNAs affect alternative splicing, using the EDI minigene reporter. If so, experiments will be initiated to determine whether the effect is via a gene silencing mechanism involving histone modifications. (month 42)

13 Neugebauer O CO 42

260

218 Smith and Neugebauer will collaborate to determine whether PTB is recruited co-transcriptionally to target genes identified in Smith lab by microarray analysis (see WP8). PTB-GFP BACs will be established (if possible) and stably transfected into cell lines, including mouse C2C12 myoblasts. Alternatively, PTB antibodies will employed in splicing factor ChIP. (month 42)

13 Neugebauer O CO 42

219 Bertrand and Neugebauer will collaborate to determine whether splicing of HIV nascent transcripts is co-transcriptional, using a model gene comprising a split MS2 reporter. ChIP of the MS2 binding protein will indicate when and where along the gene splicing catalysis occurs. (month 42)

13 Neugebauer O CO 42

220 Identification of novel molecules based on luciferase model substrates for DMD exon 51 skipping and LMNA aberrant splicing. (month 42)

14 Tazi O CO 42

221 Validation of the efficacy of novel synthesized compounds in model mice for: Mo-MLV, HIV-1, DMD, Breast metastic cancer and HGPS (month 42)

14 Tazi O CO 42

222 Validation of modified U7 cassetes to correct SMN expression in a mouse model for SMA (month 42)

14 Tazi O CO 42

223 Development of clinically useful drugs that direct the splicing pathway: both Antisense olinucleotides and small chemical molecules (month 42)

14 Tazi O CO 42

261

224 Schümperli's group will further exploit the mouse SMA model mentioned above to: (i) determine the time point when SMN production is critical and requires therapeutic boosting; (ii) study the ability of mutant SMN to counteract the SMA phenotype; (iii) study the role of SMN in motoneurons; (iv) develop ways to deliver the therapeutic U7 transgene to motoneurons. Additionally they will further improve their U7-based exon skipping strategy. Concerning the analysis of the role of PTB in pseudoexon splicing, the group has just started a study to look at whether PTB-nPTB or other neuron-specific splicing regulators affect the splicing of agrin, a protein important for synapse establishment and/or maintenance. (month 42)

14 Tazi O CO 42

225 Eperon's group will investigate the possibility of using TOES to stimulate splicing of exons damaged by mutation in cystic fibrosis (collaboration with Baralle lab) and of undamaged skipped exons in genes with roles in the development of leukaemia. (month 42)

14 Tazi O CO 42

229 Feasibility study to use mass spectrometrey based analysis for "Spatial Proteomics" where quantification of the relative distribution of cellular proteins between different cellular compartments will be analyzed in parallel (month 42)

15 Séraphin O CO 42

230 Identification and/or analysis of alternative splicing by high-throughput sequencing (month 42)

15 Séraphin O CO 42

231 Feasibility study to determine whether analytical ultracentrifugations can be used to determine the assembly of RNA binding proteins on model RNAs (month 42)

15 Séraphin O CO 42

232 Experimental setup to elucidate the “code” of RNA interaction as mediated by RRM domains (month 42)

15 Séraphin O CO 42

262

237 Continue and further develop the use of travel bursaries for student and postdoc exchanges for training and participation at intra-workpackage meetings (40 PMT/18 month) (month 42) (continuation of deliverable 116)

17 Krämer O CO 42

238 Establishing joint PhD committees for PhD students in the EURASNET. (month 42) (continuation of deliverable 16)

18 Kjems O CO 42

246 continuous enrichment and update of the PUS part of the webpage; as well as continuous collection of teaching material for RNA biology

19 Barta O PU 42

247 a newsletter describing contemporary findings within the EURASNET; the newsletter will appear twice a year, starting from month 34

19 Barta O PU 42

249 Screen at least 10,000 compounds in vitro for inhibitory effects on splicing (month 42).

20 Lamond O CO 42

251 Evaluate the mechanism of splicing inhibition by small molecule effectors identified in screening initiative (month 42)

20 Lamond O CO 42

252 Invite non-EURASNET RNA researchers to workshops/meetings to support interactions and collaborations. (month 42) (continuation of deliverable 123)

21 Brown O PU 42

253 Maintain updated contact list of RNA researchers and groups in Europe. (month 42). (continuation of deliverable 124)

21 Brown O PU 42

256 Regular requests for information from members. Requests for information will be sent out quarterly and collated. (month 42)

21 Brown O CO 42

Nature of a deliverable: R=Report O=Other Dissemination level for deliverables: PU=Public CO=Confidential (consortium members & Commission) Nn deliverables added after the second YIP integration (starting date is Jan 2008 = month 25)

263

15.6 JOINT PROGRAM OF ACTIVITIES (MONTH 25-42; January 2008-June 2009)

1. The EURASNET Web site Workpackage description (month 25-42)

Workpackage number 1 Starting day or starting event 0 month Participant id 14 Person months 8.5 Objectives • To establish, curate and maintain a EURASNET website as a tool to promote European research on alternative splicing by concentrating all available information, facilitating the exchange of information between NoE members and by disseminating the activities of the NoE to the wider scientific community and public interest groups Description of work The site will contain information that is open to the public and a password-protected area that is only available to the members of the NoE. The area restricted to NoE members will feature: (i) a collection of reliable protocols and the names of the curators, network wide-message postings; (ii) a list of and contact information for all members; (iii) EURASNET meeting abstracts, meeting reports and notes written by NoE members; (iv) training opportunities in NoE labs; (v) the advertisement of key technologies offered by different labs. The public area will contain: (i) links to alternative splicing databases (e.g. ASD, MASE, RNA workbench); (ii) links to all NoE member web pages; (iii) meeting announcements and virtual abstract books for the meetings; (iv) a collection of websites that are helpful for those working on alternative splicing; (v) advertisements for the young investigator program and job openings in NoE member labs, including e-mail addresses of contact persons. The site will also publicly disseminate the results of research carried out by the EURASNET by listing relevant publications and patents, including links to their hosting sites. In addition, these results will be explained in lay terms for the benefit of public interest groups, industry, a larger scientific audience and the general public. The need for the network to increase interactions with clinicians was highlighted in the first year report. Similarly, the first year report pointed out the need to establish a structure for ensuring the regular gathering of information on activities by the members of EURASNET. To achieve these goals requires time and effort to prepare content specifically for target audiences and to make and follow up on regular requests for information. This is being addressed by changing the management of the website and establishing a team of individuals to co-ordinate the activities of WP1, WP19 and WP21. Deliverables 146 Reorganization and re-siting of website (month 36) 147 Design of new website with new functionality and transfer of information (month 36) 148 Database of substances that change alternative splicing (month 36) 149 Liaise with targeted audiences and prepare appropriate content (month 42) 150 Tools and resources repository updated (month 42).

264

2. Sharing Resources, Technology and Reliable Protocols

Workpackage description (month 25-42)

Workpackage number 2 Start date or starting event: Month 0 Participant id All Person months 52.5

Objectives

• To create a seamless, integrated network that allows each lab to draw upon the knowledge and resources available in other labs. • To promote and encourage shared use of already existing facilities belonging to individual institutes whenever this is compatible with existing on site work loads and work flows. • To actively ensure transfer of a technology fully developed in one laboratory to another participating laboratory still lacking this technology.

Description of work 1. Expertise, methods and facilities are available for 2D DiGE (Cy2/3/5) (CS), MALDI-TOF and MS-MS protein mass spectrometry (CS, KN, RL, AL), monoclonal antibody production (KN), transgenic mice via direct injection or specific targeting in ES cells (HS, IE), high throughput screening (KN), DNA sequencing (HS, AL), SNP analysis (HS), electron microscopy (HS, AL, RL), recombinant protein purification (HS), biostatistic services (HS) and microarray analysis (AL, JV). Expertise is also available in confocal microscopy and photobleaching, RNA-FISH, FRET and related techniques (MCF, GAs, KN, GB, HS), minigene analysis (SS, GAs, GB), microarray analysis of mRNA isoforms (JV) and mRNA from yeast with splicing defects (JBe), and analysis of splicing in plants (JBr, ABa, AJ). This collection of technologies offered by the network will be the basis for direct one on one contacts between participating labs to set up a highly connected network of various kinds of cooperation on single research projects or shared uses of facilities and equipment. 2. Establishment of an online knowledge database (controlled access) containing uniformly formatted protocols and useful information such as a list of sequences used for successful down-regulation of target genes by RNAi (GAk, SS, RL) and proteomics data (RL, AL). This database will be curated to ensure that reviewed information entering the database is always kept up to date. User feedback and a list of contact persons for each deposited protocol will be incorporated into the database structure. 3. Sharing of facilities set up by participants, such as datasets and analysis programs (GAs). 4. Identification and network dissemination of the availability of mouse monoclonal antibodies and rabbit antisera as they become available (RL, SS). These arrangements will be followed during the remainder of the funding period by more widespread sharing of expertise, which will usually involve movement of young trainee scientists from one laboratory to another (usually in different countries). In addition, some laboratories run intensive workshops, and these can be opened up to post-docs and students from the network (e.g., the FISH and immunocytochemistry workshop run annually in Jerusalem: HS).

Deliverables 151 Implementation of an improved web entry form for protocols that takes into account feedback of EURASNET

members and participants of the practical courses. (month 42) 152 The current filemaker based system is slow and cumbersome to use. The pages will be reprogrammed using

HTML with an easy to use interface (Month 36). As suggested by many users, there will be a stronger emphasis on background (why is this procedure done) as well as a searchable index.

153 Publication of the protocols as a pdf booklet on the EURASNET web site and publishing of a protocol book (Month 42)

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4. The Alternative Splicing and Transcript Diversity (ASTD) Database

Workpackage description (month 25-42) Workpackage number 1 Starting day or starting event 0 month Participant id 2 3 5a 5b 6 7 8 23 28 Person months 1.5 1.5 2 8 1.5 1.5 1.5 1.5 1.5

Objectives – To maintain, update and expand the ASTD hosted at the EBI.

Description of work a. Maintenance of the operation of the ASTD database at the EBI. b. Collection of feedback from users to improve the presentation and tools offered by the ASTD (in particular

combined search tools and links to other databases). c. Collection of curated information about alternative splicing available through Network members and other

“private” databases, and incorporate that information into the ASTD. d. Development of download modules that will allow the use of the ASTD data by bioinformaticians running

various formats of computational pipelines. e. Exploring the possibilities for combined presentation and cross-reference of other major efforts in classification of alternative splicing events and genomic information.

Deliverables 132 Improved tools for combined searches and user-friendly access to the ASD database; Integration of additional species to ASTD (continuation of del.7, month 30). 154 Fast DB already contains an algorithm that allows analysis of Affymetrix splicearray datafiles. We will create an interface that allows direct visualization of splicearray data in FAST DB (month 42). 155 Integration of an algorithm that computes the likelihood of sequences to be single stranded (PU) values into FAST DB (month 42). 156 The EURASNET member Stamm published a method to calculate a statistical value (PU value) that expresses whether a given nucleotide in an RNA is single or double stranded. Experimental data show that exonic regulatory elements are preferably in single stranded conformation. We will implement a graphic representation of this algorithm in the FAST DB database (Hiller, M., Zhang, Z., Backofen, R., and Stamm, S. (2007). pre-mRNA secondary structure and splice site selection. PLOS Genetics 3, 2147-2155) (month 42).

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5. Ensuring Durability

Workpackage description (month 25-42) Workpackage number 5 Start date or starting event: 13 month Participant id 1a 2 3 9 16 Person months 1.5 1.5 1.5 1.5 8

Objectives

• To ensure a durable, functional alternative splicing network that will serve as a model for cooperative research and innovation in the field of RNA splicing and RNA biology in Europe long beyond the NoE funding period.

Description of work

The NoE will work towards maintaining its research, training and dissemination activities beyond its five year funding period.To ensure that this is more than simply the sum of individual fragmented efforts of the NoE participants, the NoE will seek to both promote such efforts and to complement them by integrating a dedicated workpackage into the NoE's structure. It will focus upon perceived needs for supporting durable collaboration, communication and coordination by the following means:

1. The EURASNET website (see WP1) will be maintained – and, if considered appropriate at the time, further expanded, beyond the current support period. This will be financed by present NoE members, with possible additional external funding according to need and resources.

2. The website will contain an information package (web page) to inform participants of available funding possibilities – e.g., short- and long-term fellowships, national programmes, and intergovernmental and international programmes and other initiatives.

3. Liaison will be maintained by the programmes organisers with relevant scientific, technological, educational and cultural bodies in the EU. In this way close contact will be maintained between the Network's leading participants (and the Network as a whole) and the mainstream of European science and its public perception. This will be done with the goal of maintaining a sensitive response to perceptions of future need, and influencing this perception where necessary.

4. Towards the end of the five-year period of the NoE, a strategic meeting will be held to review needs, prospects and opportunities for future collaboration within the field. This will allow the development of new strategies to meet new needs in a coherent and integrated manner.

Although the activities in this workpackage will be forward-looking, they will commence at the time of inception of the NoE. However, they will increase during the second half of the NoE's five-year term. Thus, most measures to ensure durability of the Network – perpetuation of the website and end-of-term strategic review – will be implemented in the latter part of the NoE's five-year period.

Deliverables

157 Maintain and further expand the information package/forum within the EURASNET website and meetings with updated announcements on funding possibilities. (month 42)

158 Maintain and further expand the liaison with relevant bodies at both national and EU level. (month 42)

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6. In silico approaches to alternative splicing Workpackage description (month 25-42)

Workpackage number 6 Start date or starting event: 0 month Participant id 3 5a 6 28 30 31 Person months 8 8 42 6 8 8

Objectives • Improved identification of known regulatory sequence motifs • Computational prediction of novel regulatory motifs • Computational prediction of regulatory proteins and RNAs • Investigation of correlations between promoter structure and alternative splicing • Investigations on the evolution of alternative splicing • Study evolution of gene structure.

Description of work 1.Generation of computational tools to assemble the information currently available on regulatory factor binding sites, as well as visualization tools to screen genomic/pre-mRNA sequences for binding sites, with particular emphasis on clusters or regulatory elements. 2. Develop algorithms for identification of novel regulatory motifs focusing on three aspects: Enrichment of / bias against sequence elements located in the proximity of weak or regulated splice sites. a.Enrichment of / bias against sequence elements in exons alternatively spliced in a tissue-specific fashion …b. Phylogenetic conservation of regulatory elements 3. Identification of proteins with domains characteristic of splicing regulators and description of potential patterns of expression based on EST information. 4. Exploration in silico of possible binding sites for micro-RNAs in alternatively spliced regions of pre-mRNAs, with particular emphasis on genes of interest to the experimental groups of the Network. 5. Exploration of possibilities for developing computational tools that correlate promoter structure with alternative splicing events. 6. Comparative analyses of the regulatory sequences and factors identified in points 2–4 for various species.

Deliverables N6 Computational structural model of splicing regulators hnRNP-L and hnRNP-L-like in complex with RNA (month 30) N7 Computational modeling and experimental testing of novel RNA binding domains (month 30) 159 The Stamm lab will experimentally validated snoRNA target predictions generated by the Zavolan lab. This will be performed to identify the cis-elements that control HBII-52-dependent alternative splicing. The algorithm to determine snoRNA tartgets will be used with two other snoRNAs in the Prader-Willi critical region, HBII-438 and HBII-85. In addition, the algorithm to predict single-strandedness will be implemented in a suitable database and combined with binding motifs to analyse DNA array results for tra2-beta1 (month 42). 160 The Zavolan lab implemented a snoRNA-mRNA binding model, and applied this method to the prediction of snoRNA targets in mouse (in collaboration with Stamm). They will apply this method to other systems such as plants. These latter predictions will be tested by the Brown lab in the plant Arabidopsis thaliana. In addition they will improve the quality of the miRNA target predictions, and will develop automated methods to update the predictions as more genomes and transcripts become available. Finally, they will develop methods to relate changes in mRNA expression to changes in the expression of miRNAs (month 42). 161 The Bork lab will finalize a visualization tool that can display and integrate various datasets (EST, cDNA genomic DNA etc) and computes splice paths through the corresponding proteins. They will also examine a network of genomic sequence motifs involved in alternative splicing which includes prediction and validation of new alternative splicing

268

variants (month 42). 162 The Ast lab will compare splice sites and splicing factors that bind these sites among the entire eukaryotic kingdom (in collaboration with Eyras). They will compare the evolution of genes, exon-intron structure, intron gain and loss, and the effect of transposed elements in vertebrates and invertebrates (month 42). 163 The Eyras group in collaboration with the Smith group will study the splicing regulation of introns in which the branch site sequence located more than 100 nucleotides from the 3’ss. They will use comparative methods to extract possible regulatory sequences and secondary structres that may be involved in the regulation. They will also identify the cases that are related to disease. In colaboration with the Ast group they will compare the splicing mechanism in eukaryotes. Using multiple eukaryotic species, they will compare the properties of protein factors and snRNAs involved in splicing. They will investigate the role of the SR and hnRNPs proteins in the origin of regulated splicing (month 42). 164 The Bujnicki lab will develop a prototype version of template-based method for RNA 3D structure prediction by fragment matching and a prototype version of template-free method for RNA 3D structure prediction by coarse-grained folding simulation. They will also carry out modeling of hnRNPL&L-like protein-RNA complexes (in collaboration with the Bindereif group) and protein structures of novel domains involved in RNA-binding (in collaboration with the Stamm group (month 42)

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7. Molecular Characterization of Splicing Substrates

Workpackage description (month 13-30) Workpackage number 7 Start date or starting event: 0 month Participant id 4 7 10a 12c 17 19 23 29 Person months 8 10 8 8 21 8 8 8

Objectives • To develop a strategy with selected pre-mRNA substrates that would provide a complete description of what the spliceosomal components encounter when they come into proximity with the pre-mRNA and how they are directed to splice sites.

Description of work 1. Agreement on common model substrates, so that the expertise of various groups can be applied to a selected number of constitutive and alternative sites and exons. 2. Characterisation of pre-mRNA structure and accessibility of selected introns and exons, before and at early stages of spliceosome assembly; investigations of heterogeneity in complexes. Methods used will include chemical, enzymatic and microarray analysis of structure and accessibility, use of RNase H to measure stability of bound components, and Fe-BABE to investigate proximity of different parts of the substrate to each other. 3. Identification of any modifications of bases or ribose groups in selected pre-mRNA. 4. Mapping protein distribution and stoichiometry along pre-mRNA in extracts. Protein components will be identified in WP9. Mapping will be done by both site-specific cross-linking along the RNA and FRET with fluorescent proteins and oligoribonucleotide analogues. 5. Reconstitution of events involved in splice site selection with pure components on selected pre-mRNAs (analysis of RNA-protein interactions, dissociation rates, cooperative and competitive binding, spreading vs long-range interactions, boundary formation, etc.). After this initial work on these examples, future investigations will use the expertise and knowledge gained to address substrates in which mutations affecting splicing via exonic or intronic sequences cause human disease, viral substrates that provide appropriate examples of the diverse ways in which viral sequences exploit cellular proteins regardless of the normal functions of such proteins, and plant substrates. Satellite III transcripts would be included because they act to sequester processing factors after heat shock. Additional substrates will be fed in via WP6.

Deliverables 72 Identification of more in vivo targets of the functional activity of splicing regulatory proteins (to be subsumed in WP8). (month 30) 165 Identification of local pre-mRNA secondary structures, the perturbations generated by mutations found in

genetic diseases and their effects on splicing (continuation of deliverable 69) (month 42) 166 Complete identification of components in splicing regulatory complexes and assessment of their roles in the

functions of splicing regulatory elements and splicing sites (continuation of deliverable 70) (month 42) 167 Expansion of work on detailed analysis of RNA-protein complexes formed with recombinant proteins and

repeat sequences or pre-mRNA regulatory sequences, including structural analysis (continuation of deliverable 71) (month 42)

168 Development of methods for determination of the stoechiometry of binding of splicing factors on pre-mRNAs and to estimate their relative affinities in vitro and in cellulo. (month 42)

169 Development of cell imaging methods and single molecule methods to follow the kinetics of splicing factor association with pre-mRNAs (continuation of deliverable 73) (month 42)

170 Obtain 3D Structure information on splicing regulatory proteins by NMR approaches (Etr3 and Tra2β) by X-ray crystallography and 3D structure computer modelling (MBNL1 and CUG-BP) (continuation of deliverable N10) (month 42)

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8. Genome-wide Analyses of Splicing Regulation

Workpackage description (month 25-42)

Workpackage number

8 Start date or starting event: 0 month

Participant id 1b 3 5b 6 9 10a

10b 11 14 15 16 18 19 21 23 24 26 28

Person months 10 8 8 10 9 8 8 8 8 8 8 8 8 8 42 8 6 7

Objectives

• To develop and share relevant expertise, techniques and reagents.

• To test the feasibility of and to optimize key techniques e.g. splicing factor ChIP, CLIP, iTRAQ analysis of alternative splicing. Where possible, to coordinate such tests within common model systems.

• To develop a co-ordinated strategy for genome-wide investigation of splicing factor activity, aiming to maximize complementarity and minimize overlap between participants, particularly in the area of mammalian splicing factor activity, where the majority of participants are active. To identify and eliminate major “gaps” (e.g. important splicing factors not currently being analyzed).

Description of work Experimental model systems for analysis of splicing factors include human and mouse cells, Drosophila, C. elegans and Arabidopsis. Approaches for analyzing pre-mRNA/mRNA binding targets will include chromatin immunoprecipitation (ChIP) followed by microarray analysis (KN), conventional immunoprecipitation and microarray analysis (JC, JT), cross-link immunoprecipitation (CLIP) tag analysis (DA, JC, AKr) and Genomic SELEX (JK). Functional target analysis will involve perturbation of splicing factor levels via RNAi (ABi, JC, MCF, AKr, RL, CS) by genetic depletion (JBr), sequestration by SatIII RNA overexpression (GB) and by splicing factor overexpression (JBr, SS, CS, JT). The functional consequences of alterations in splicing factor levels will be analyzed by splice-sensitive microarrays (DA, ABi, JC, MCF/JV). In addition, the potential for direct quantitative proteomic analysis of alternative splicing by isotope tagging for relative and absolute quantitation (iTRAQ) will be tested (CS). Given the large number of participating investigators a major activity of the initial 18-month period will be coordination of subsequent activities and transfer of optimized techniques. However, some early attempts at coordination will be developed e.g. the differentiating C2C12 myogenic cell model is being used to validate alternative splice-sensitive microarrays (MCF/JV), and will also be used for proteomic analysis of alternative splicing by iTRAQ (CS), and for ChIP analysis of SR proteins (KN).

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Deliverables Arrays: 171 Array bioinformatics postdoc to be appointed in the Auboeuf group with the following remit: 1. develop methods for data analysis of ExonArrays (and junction arrays, when available – see next deliverable) 2. develop user-friendly web-interface (FAST-db) 3.provide service role for other EURASNET members (all month 27) 172 We will aim to reach an agreement with Affymetrix for access to their human and mouse research junction arrays (month 28) 173 Further development of data analysis for ExonArrays (Bindereif, Auboeuf) (month 42) 174 Application of splice-sensitive array analysis to biomedically relevant samples: 1. in a tuberculosis model system, Bindereif in collaboration with Stefan Kaufmann, Max-Planck-Institute for Infection Biology, Berlin (month 42). 2. ExonArray analysis of the transcriptome of a mouse model of breast cancer (Auboeuf, month 36) 175 Exon Array (and junction array, if available) microarray analysis of effects of hSlu7 knockdown upon alternative splicing (G Ast, month 30) 176 Exon Array (and junction array, if available) microarray analysis of effects of SF1 knockdown upon alternative splicing (Krämer, month 42) 177 Validation of array predictions of AS changes in epithelial-mesenchymal transition, focusing mainly on alternative splicing of SR-proteins transcripts (Biamonti, month 42). Deep sequencing approaches: 178 We will develop simulations of alternative splice form sampling to study the theoretical limits of deep sequencing approaches to profiling of alternatively spliced isoforms (Zavolan). (month 42) 179 Application of Roche/454 and/or Solexa/Illumina sequencing for quantitative comparison of HeLa cell control and splicing regulator knockdown samples (PTB/nPTB, Smith, Valcarcel, Zavolan; hnRNP-L, Bindereif). (Month 36) 180 Application of deep sequencing to CLIP tags of SF1 and U2AF65 (Krämer, month 42) CLIP: 181 Use of CLIP to identify RNA targets for the shuttling SR protein SF2/ASF. Use of CLIP will be complemented with subcellular fractionation to identify nuclear, cytoplasmic and polysomal targets (Caceres, month 42). 182 Use of CLIP to identify RNA targets for hnRNP A1. Use of CLIP will be complemented with subcellular fractionation to identify nuclear, cytoplasmic and polysomal targets. Furthermore, we will identify RNA targets in cells subjected to stress, due to the reported role of hnRNP A1 in the stress response (Caceres, month 42). Plant AS (Barta, Brown, Jarmolowski): 183 Replace low information content Arabidopsis AS events with other selected AS events and characterise novel events (month 36) 184 Continue to complete analyses of Arabidopsis NMD mutants and hyl1; SR protein overexpressor and mutant lines (month 42) 185 The plant groups (will establish collaborations with other groups to analyse proteins which affect alternative splicing (month 42)

272

9. Complexity of Spliceosomal Proteomes Workpackage description (month 25-42)

Workpackage number 9 Start date or starting event: 0 month Participant id 1a 4 8 9 12a 12b 12c 19 Person months 39 8 8 9 8 10 8 8

Objectives

• Determine the composition of enhanceosomes assembled on different pre-mRNA substrates containing defined cis-acting regulatory elements.

• Elucidate qualitative and quantitative changes in the composition of spliceosomes formed under different physiological conditions (i.e., after viral infection or heat shock), in different cell types, and at different stages of the cell cycle.

Description of work To learn more about the composition of enhanceosomes and how different environmental conditions affect the complexity of the spliceosome, spliceosomal complexes will first be assembled under splicing conditions in cellular extracts and, after affinity purification, mass spectrometry will be employed to reveal the nature, including posttranslational modification status, and quantity of the spliceosomal proteins present. In one set of experiments, enhanceosomes will be allowed to form on both naturally occurring and artificially-engineered human pre-mRNA substrates containing different exonic splicing enhancers that are specifically recognised by a particular SR protein (e.g. SF2/ASF, SC35, Tra2beta and SRp55). In a second set of experiments, spliceosomes will be isolated from different types of cells or after cells are either subjected to different environmental conditions, including heat shock or infection with adenovirus, or are stalled at a specific stage of the cell cycle. In a third set, spliceosomes will be assembled in vitro in the presence or absence of the HIV-1 Rev protein on a pre-mRNA substrate containing an RRE. The information obtained from these experiments will potentially complement studies described in work packages 10 and 11 (i.e., posttranslational modification and function of splicing factor isoforms). Finally, tagged pre-mRNA substrates or alternative methods suitable for the isolation of spliceosomal sub-complexes from plants (as well as other organisms) will be constructed and initially tested on an analytic scale.

Deliverables 186 Relativ quantitative mass-spectrometric analysis of spliceosomal complexes. (month 42) 187 Establishing a map for spliceosomal proteins in a novel 2D electrophorese system without IEF. (month 42) 188 Feasibilty studies for chemical cross-linking of proteins within purified spliceosomes and/or subspliceosomal protein complexes. (month 42) 189 Role of Cwc21p in splicing specificity and fidelity (month 42) 190 MS based proteomics of Brr2 contain complexes (month 42) 191 Establishing of stable cell line L4-33K and studying cellular and viral protein–protein complexes containing L4-33K including subcellular localization. (month 42) 192 Characterization of protein heteromeric complexes associated with regulated HIV RNA splice sites. (month 42) 193 Relative quantitative mass spectrometry of proteins aossociated with regulated HIV RNA splice sites, RSV RNA and wildtype/mutant exon 5 of LMNA pre-mRNA. (month 42)

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10. Post-translational Modification and Dynamic Regulation

Workpackage description (month 25-42) Workpackage number 10 Start date or starting event: 0 month Participant id 1a 3 4 10a 12a 12d 15 16 20b 22 26 Person months 10 8 8 8 8 6 42 8 8 8 6

Objectives

• The aim of this work package is to characterize molecular interactions that play a function role in linking signal transduction pathways and the regulation of alternative splicing.

• Study how post-translational modifications of protein splicing factors affect their activity and/or subcellular localization.

• Assess using mutational studies the functional role of specific modifications in regulating alternative splicing pathways. The ultimate goal is to understand how extracellular signals modulate gene expression through mechanisms involving changes in alternative pre-mRNA splicing.

Description of work 1. Biochemical characterization of post-translational modifications (phosphorylation, sumoylation, ubiquitination, etc.) on all types of trans-acting factors involved in constitutive and/or alternative pre-mRNA splicing.

2. Determine the significance of post-translational modifications for constitutive and alternative splicing. Characterization of protein kinases/phosphatases regulating alternative splicing (or the equivalent enzymes causing other modifications).

3. Regulation of alternative splicing through the common (and new) signal transduction pathways (ERK, MAPK, p38 etc pathways). Effect of extracellular cues on alternative splicing (UV, stress etc).

4. In vivo analysis of splicing factor localization, dynamics and protein-protein interactions using quantitative fluorescence microscopy techniques. 5. Remodelling of the host cell RNA splicing machinery during viral infections.

Deliverables 90 Use the tap-tagged SRp30 protein to select and MS-analyze ribonucleoprotein complexes in nSBs (month 42).continued 194 Bertrand will analyze in detail the binding of U1-specific proteins to the transcription site of the HIV-1 reporter RNA by FRAP, including (i) interpretation of the FRAP curves with a diffusion-reaction model, (ii) analysis of the binding of U1 proteins on reporter RNA containing mutation in their splice donor sequence (either stabilizing or destabilizing U1 binding), (iii) analysing the binding of mutant proteins, unable to bind U1 snRNP or other partners. He will determine the recruitment of splicing factors characteristic of a given spliceosomal complex (e.g. A, B, or C) on the transcription site of wild-type and mutant MINX reporter RNAs, and initiating the analysis of their binding dynamic by FRAP. (month 42) 195 Akusjarvi will extend the mutational analysis of L4-33K to other conserved regions of the protein with the aim of establishing the relationship between function, protein stability, subcellular localization and the phosphorylated status of the protein. He will also develop experimental reagents for overexpression of catalytically active and inactive C subunits of PKA (stable cell lines and/or recombinant adenoviruses) and will establish an in vitro system responding to PKA C subunit expression. (month 42) 196 Lamond will perform FLIM/FRET analysis of additional spliceosomal proteins in mammalian cells. He will focus on the interactions between hnRNP M, CDC5 and PLRG1. (month 42)

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197 Caceres will use the CLIP protocol (see WP8) to identify RNA targets of hnRNP A1 in control cells and cells subjected to stress and also to identify RNA targets for the shuttling SR protein, SF2/ASF. In both cases, use of CLIP will be complemented with subcellular fractionation to identify nuclear, cytoplasmic and polysomal targets of hnRNP A1 and SF2/ASF. This will result in the identification of endogenous targets for these antagonistic splicing factors as well as to identify a role for hnRNP A1 in the stress response (Month 42) 198 Carmo-Fonseca will perform FRET analysis of splicing factor interactions in the spliceosome. She will use stable cell lines expressing a tandem array of transgenes tagged with the MS2-binding sequence. In these cells, co-transcriptional spliceosome assembly is visualized as a single fluorescent focus. (month 42) 199 Lührmann will develop a more detailed picture of the mode of action of hPrp4 in the splicing machinery with particular respect to possible interaction partners. Lührmann and Urlaub will establish the phosphoproteom of human and yeast splicesomes. Lührmann will use phosphor-peptide specific antibodies for in situ localization of active spliceosomes (month 42) 200 Stamm will determine how PP1 regulates the interaction of RNA with the splicing factos SF2/ASF, tra2-beta1 and SRp30c. (month 42) 201 Tazi will investigate the relationship between B52 and the other candidate genes identified in the genetic screen. These factors could be regulators or antagonists of B52, but could also correspond to target genes whose expression is altered by B52 overexpression. (month 42) 202 Srebrow will investigate the role of Akt as a novel SR protein kinase. The subcellular localization of SR proteins will be evaluated by observation of SR-GFP fusions in the presence of siRNA against AKT, and other established SR protein kinases, such as SRPK or CLK. (month 42)

275

11. Function of Splicing Factor Isoforms Workpackage description (month 25-42)

Workpackage number 11 Start date or starting event: 0 month Participant id 1a 2 10b 12a 14 15 16 21 Person months 10 42 8 8 8 8 8 8

Objectives

• To analyse functional differences between alternatively spliced isoforms of splicing factors and regulators.

Description of work 1. Generation of databases of genes encoding spliceosomal components and splicing regulators, and

their patterns of alternative splicing, in human, Drosophila melanogaster, Caenorhabditis elegans and the plant Arabidopsis thaliana.

2. Selection of isoform-specific DNA probes for production of splicing-sensitive microarrays, and of dsRNA probes for RNA interference.

3. Production of microarrays and synthesis of siRNAs/dsRNAs. 4. Analysis of changes in alternative splicing patterns upon knockout of individual isoforms.

Deliverables 203 Description of splicing factor isoform changes upon activation of signaling pathways (month 42) 204 Description of isoform diversity and changes in core spliceosomal components in different tissues (month 42) 205 Analysis of effects of SF1 isoform overexpression on alternative splicing events identified by CLIP (month 42) 206 Investigate the role of U2AF35 isoforms and U2AF35-related proteins on splicing regulation / cell cycle progression (month 42) 207 Analysis of genetic circuits involving alternative splicing at 3’ UTRs and the interplay with cytoplasmic regulatory processes (month 42) 208 Analyze SR protein and PTBL over-expression and mutant lines using the 384 AS RT-PCR panel (month 42)

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12. Mis-splicing and Disease Workpackage description (month 25-42)

Workpackage number 12 Starting day or starting event 0 month Participant id 4 7 10

a 11 12

a 12c

16 19 25 26 27 32

Person months 8 42 8 8 8 8 8 8 8 8 6 8 Objectives • Identifying the connection between derangements in RNA splicing and disease • Dissemination of splicing technology and knowledge to patient care through the establishment of a task force to bring to the attention of practicing clinicians the role of RNA splicing derangements in disease. The Human Genetic Societies meetings and other relevant gatherings will be the target of this activity. WP 16 and 21 can assist with this. • Setting up best practice protocol for the interpretation of nucleotide changes seen in diagnostic sequencing. Description of work The study of mis-splicing in disease can be schematically divided into three work phases: 1. Identification of aberrant splicing. This will be done by direct evaluation of patient’s RNA samples. When RNA is not available, or when it has a tissue-specific expression, appropriate experimental models (minigene and in vitro splicing assays) will be produced. These will be useful also for diagnostic purposes and for subsequent elucidation of the basic mechanism(s) as described in point 2. Dedicated databases and in silico prediction tools will be created in parallel. 2. Elucidation of the basic mechanism(s) that cause mis-splicing. This phase will be done in close cooperation with WP7, WP9 and WP11. This includes several complementary activities:

a. The identification of common classes of regulatory elements disrupted by the mutations (enhancer, silencer, composite elements etc.),

b. The investigation of the role of secondary structure determinants and trans-acting splicing factors in mis-splicing

c. The identification of pathological splicing mechanisms. 3. Initial evaluation of the potential therapeutic effect of compounds developed by high throughput screening and the study of changes in the expression of trans-acting splicing factors by means of RNAi (see also WP14). We expect some time-frame overlapping among the work-phases due to differential progress of one activity and/or discovery of new mechanisms. Deliverables 209 Use of minigene splicing assay to study the influence of mutations in neurofibromatosis type 1 exon/intron 29 splicing (month 30) 210 Analysis of the role of PTB in pseudoexon splicing (month 30) 211 In depth characterization of trans-acting factors of particular interest with regards to their functionality, biochemical properties, and general importance for the cellular metabolism particularly in the development of tumors (Biamonti) (month 42) 212 Development of oligonucleotide-based or snRNP based approaches to correct splicing defects with special emphasis on investigating the molecular mechanisms affected by these novel reagents. (month 42) 213 Intensification of the interactions among medically interested people of EURASNET creating special interest groups centered on common clinical problems and diseases. (month 42) 214 Further development of techniques for use in diagnostic tests including the standard quality assurance required for diagnostic molecular genetic laboratories. (month 42) N8 Use of minigene splicing assay to study the influence of mutations in neurofibromatosis type 1 exon/intron 29 splicing (month 30) N9 Analysis of the role of PTB in pseudoexon splicing (month 30)

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13. Co-transcriptional Mechanisms of Alternative Splicing

Workpackage description (month 25-42)

Workpackage number 13 Start date or starting event: 0 month Participant id 1b 4 6 9 10a 12b 19 20a 23 24 26 Person months 42 8 10 9 8 10 8 8 8 8 6

Objectives

• Analysis of the coupling between transcriptional and splicing regulation in order to ultimately explain how alternative mRNAs are expressed in vivo. The overall aim is to optimize and extend key techniques. A particular focus of this period will be exchanging expertise and reagents, such as Pol II mutants, antibodies, and newly established cell lines, for application among the experimental systems.

Description of work The novel application of the ChIP technique to co-transcriptional splicing is a key aim. By describing co-transcriptional spliceosome assembly in yeast, we will provide a kinetic description of how spliceosomal components are added to nascent RNA on the time scale of transcription. This will be enhanced by the use of fluorescence microscopy to study the dynamics of splicing factor recruitment to transcription units and to measure the kinetics of splicing catalysis. We will examine how transcriptional elongation rates - studied with RNA polymerase mutants that proceed at different rates and the activities of elongation factors - influence the selection of alternative exons. Finally, we will investigate how particular alternative splicing factors participate in transcriptional regulation. These joint efforts will break significant new ground by providing a comprehensive picture of the kinetics of transcription and splicing and their interplay.

Deliverables 215 To determine the molecular basis for the effect of Prp45p on transcription, Beggs will analyse the effects of prp45 mutant alleles on the recruitment of CTD kinases and chromatin remodeling factors to the ASC1 gene by in vivo ChIP assay, and the interaction of these proteins with Prp45p will be tested in pull-down assays in vitro. (month 42) 216 Aubeouf and Neugebauer will collaborate to establish splicing factor ChIP in MCF7 cell lines. These breast cancer cells are responsive to estradiol, which stimulates transcription of cyclin D1, PS2 and other genes. This cellular model will allow us to test the splicing process on gene products made in response to hormones. We will establish stable cell lines with integrated copies of BACs expressing GFP-tagged versions of U1 70K, U2AF65, Prp8, CBP80 and members of the SR protein family, in order to investigate the interplay between transcription, co-transcriptional spliceosome assembly and AS outcome. (month 42) 217 Kornblihtt will determine whether intronic siRNAs affect alternative splicing, using the EDI minigene reporter. If so, experiments will be initiated to determine whether the effect is via a gene silencing mechanism involving histone modifications. (month 42) 218 Smith and Neugebauer will collaborate to determine whether PTB is recruited co-transcriptionally to target genes identified in Smith lab by microarray analysis (see WP8). PTB-GFP BACs will be established (if possible) and stably transfected into cell lines, including mouse C2C12 myoblasts. Alternatively, PTB antibodies will employed in splicing factor ChIP. (month 42) 219 Bertrand and Neugebauer will collaborate to determine whether splicing of HIV nascent transcripts is co-transcriptional, using a model gene comprising a split MS2 reporter. ChIP of the MS2 binding protein will indicate when and where along the gene splicing catalysis occurs. (month 42)

278

14. Chemical Biology and Therapeutics

Workpackage description (month 25-42)

Workpackage number 14 Start date or starting event: 0 month

Participant id 1a 3 10b 12a 12c 13 17 22 25 32

Person months 10 8 8 42 8 16 16 8 8 4

Objectives

• Characteriziation of the five novel inhibitors of pre-mRNA splicing regarding their mode of action and molecular target

• Final optimization and implementation of the high-throughput screening for inhibitors of in vitro pre-mRNA splicing using large libraries (up to 100,000 compounds)

• Development of indole based probes for the comprehension of their mode of interaction with SR proteins, and RNA

• Compound library synthesis using alternative scaffolds-pharmacological profiling

• Validation of the efficacy of novel synthesized compounds

• Development of clinically useful drugs that direct the splicing pathway.

Description of work

One focus of the future work will be on the further characterization of the novel inhibitors of pre-mRNA splicing identified by Partners 1a, 12a, and 22. First, to identify the functional group(s) and overall scaffold required for inhibition by the various compounds, we will test derivatives of these chemicals regarding their effect on pre-mRNA splicing in the biochemical and cell-biological assays developed by Partners 1a, 3, 10a, 12a, 12c, 22 and 25. This parallels a standard drug development approach, involving the optimization of active molecules (“hits”) to obtain highly specific compounds (“leads”) with drug-like properties (e.g. solubility, good adsorption, low cytotoxicity). Second, to identify the molecular targets of the various inhibitors, the compounds well be modified by: (i) incorporating photoaffinity agents like azide groups, which can then be crosslinked to the protein or RNA target. These crosslinks can be identified by mass-spectrometry with the help of Henning Urlaub (partner 1c), who has longstanding experience in protein-protein and protein-RNA crosslinking analysis by mass-spectrometry. And (ii) attaching a linker arm at an appropriate position of the novel inhibitors such that the resultant compounds can be used for affinity chromatography. Again, proteins that bind to these chemicals can be identified by mass-spectrometry. These approaches will be performed with individual components (e.g. SR proteins, which are the likely targets of the indole compounds identified by partner 12a), partially purified splicing compexes, or total nuclear extract. Comparison of these results within these chemicals, and with well-characterized inhibitors of enzymatic activities as they are present in the spliceosome (e.g. kinases or phosphatases) will give indications how these substances inhibit pre-mRNA splicing in vitro.

Furthermore, the in vitro screening procedure for the identification of inhibitors of splicing of the MINX pre-mRNA developed by partner 1a will be analyzed regarding its robustness, reproducibility, and sensitivity under actual screening conditions. This will allow a final optimization so that we can then start to screen larger collections of small molecules. Close to 100,000 chemicals that can be screened for inhibitors of pre-mRNA splicing in vitro are accesible to EURASNET via the Institut Curie, the University of Dundee, and the Max-Planck-Society. In parallel, we will adopt the fluorescent mRNA detection assay for alternatively spliced pre-mRNAs. Using pre-mRNA substrates whose incorrect splicing is implicated in a disease, this will pave the way to start screening for compounds that correct the disease-causing splicing mode and thus might be leads for the development of therapeutic agents.

Deliverables

220 Identification of novel molecules based on luciferase model substrates for DMD exon 51 skipping and LMNA aberrant splicing. (month 42) 221 Validation of the efficacy of novel synthesized compounds in model mice for: Mo-MLV, HIV-1, DMD, Breast metastic

cancer and HGPS (month 42)

222 Validation of modified U7 cassetes to correct SMN expression in a mouse model for SMA (month 42)

279

223 Development of clinically useful drugs that direct the splicing pathway: both Antisense olinucleotides and small chemical molecules (month 42) 224 Schümperli's group will further exploit the mouse SMA model mentioned above to: (i) determine the time point when SMN production is critical and requires therapeutic boosting; (ii) study the ability of mutant SMN to counteract the SMA phenotype; (iii) study the role of SMN in motoneurons; (iv) develop ways to deliver the therapeutic U7 transgene to motoneurons. Additionally they will further improve their U7-based exon skipping strategy. Concerning the analysis of the role of PTB in pseudoexon splicing, the group has just started a study to look at whether PTB-nPTB or other neuron-specific splicing regulators affect the splicing of agrin, a protein important for synapse establishment and/or maintenance. (month 42) 225 Eperon's group will investigate the possibility of using TOES to stimulate splicing of exons damaged by mutation in cystic fibrosis (collaboration with Baralle lab) and of undamaged skipped exons in genes with roles in the development of leukaemia. (month 42)

280

15. Development of Enabling Technologies

Workpackage description (month 25-42)

Workpackage number 15 Starting day or starting event 0 month Participant id 1a 1c 2 3 10b 12b 22 Person months 10 6 10 8 8 42 8 Objectives

• To identify and develop new technologies necessary to advance research on alternative splicing.

Description of work

Our ability to dissect the regulatory mechanisms of alternative splicing is, at present, greatly limited by currently available techniques. The main goal of the “Enabling techniques” workpackage is thus to develop new strategies for the analysis of alternative pre-mRNA splicing. All groups participating in the EURASNET, in the context of their research programs, will be involved in improving techniques that facilitate the analysis of alternative splicing. New technologies of particular interest to the network will be defined by the Enabling Technique Steering Committee (ETSC) at regular intervals. Two current priority areas are developing methods for (i) the identification and mapping of transient protein-protein or protein-RNA interactions within spliceosomes or enhanceosomes, and (ii) the generation in vivo of tagged pre-mRNA molecules of defined size, for the purpose of isolating and characterizing spliceosomes or enhanceosomes formed in vivo. Initial promising approaches for mapping protein-RNA and protein-protein interactions that will be tested/optimized include RNA-protein crosslinking coupled with mass spectrometry and deuterium exchange footprinting. To isolate specific splicing complexes formed in vivo that are suitable for the subsequent analysis of their composition by mass spectrometry, cell lines stably expressing a tagged version of a particular pre-mRNA will first be established. Methods to site-specifically cleave RNAs in the cell nucleus will also be developed to enable the isolation of splicing complexes formed on a pre-mRNA of defined size. New technologies (as well as standard, currently-used) protocols will be made available to the entire network through internet (protocols), through the organization of training sessions (theoretical and/or practical) and/or by organizing the visit of EURASNET members to the expert laboratory (see WP17 for details). Finally, to incorporate novel, state of the art technologies into this network, the ETSC will actively recruit young scientists with expertise in a particular enabling technology to participate in the network either via the Young Investigator Programme and or by becomming an NoE core member. Deliverables 226 Proof of principle for identification of protein interaction surfaces by efficient mutagenesis (month 36) 227 Set up a high-throughput cloning system for minigenes that allows rapid analysis of alternative exons (month 36) 228 Development of FLIM-FRET for mapping protein-protein interactions at specific subcellular (and subnuclear) locations in vivo (month 36) 229 Feasibility study to use mass spectrometrey based analysis for "Spatial Proteomics" where quantification of the relative distribution of cellular proteins between different cellular compartments will be analyzed in parallel (month 42) 230 Identification and/or analysis of alternative splicing by high-throughput sequencing (month 42) 231 Feasibility study to determine whether analytical ultracentrifugations can be used to determine the assembly of RNA binding proteins on model RNAs (month 42) 232 Experimental setup to elucidate the “code” of RNA interaction as mediated by RRM domains (month 42)

281

16. Conferences and meetings

Workpackage description (month 25-42) Workpackage number 16 Start date or starting event: 0 month

Participant id 1b 2 3 5a 6 7 9 16 17 21 23 32 Person months 1.5 1 0.5 0.5 0.5 0.5 4 0.5 0.5 0.5 0.5 0.5

Objectives To organise three types of conferences and meetings:

• Biennial European Conference on Alternative Splicing, participation open world-wide, for the exchange of information on the latest advances in the field.

• Annual Interdisciplinary Focus Meetings, open for NoE members and invited participants, for strategic planning and the establishment of collaborations across research field boundaries. • Longer "hands-on" courses/workshops for graduate scientists and clinical researchers/clinicians • Annual NoE meeting, open to NoE members. Description of work 1. Organization of a large (300 participants) conference on Alternative Splicing: choosing a venue, financial planning, contacting keynote speakers, advertising, management, and documentation. 2. Organization of small (30 participants) focus meetings: choosing a venue, contacting external experts, financial planning, and documenting conclusions. Topics will include:

• Combining computational and experimental approaches to study alternative splicing • Biophysical methods and functional genomics tools to study alternative splicing • Cell biology, signaling and alternative splicing • Alternative splicing and clinical research

3. Organize longer "hands-on" courses/workshops for graduate scientists and clinical researchers/clinicians 3. Organization of an annual NoE meeting (~150 participants): choosing a venue, financial planning, management, and documentation

Deliverables Events planned for 2008: 114 First International EURASNET Meeting, Krakow The First International EURASNET Conference on Alternative Splicing, organised by participants J. Beggs, A. Jarmolowski, A. Krämer and S. Stamm, will be held May 21-23, 2008, followed by the Third Annual Meeting of the EURASNET NoE, May 24-25. (month 29) 233 Interdisciplinary focus meetings planned for 2008. 1. “Cell Biology”, April 2008, Montpellier, organised by participants E. Bertrand and M. Carmo-Fonseca. (month 28) 2. “Mis-splicing and Disease” will be held June 3, 2008 in Villa Mondragone, near Rome, Italy, organised by participants F. Baralle and J. Valcarcel, as an independent meeting alongside an EMBO meeting “RNA and Disease: RNA Metabolism and Associated Pathologies” (month 30) 3. “Biophysical Methods” will be held in Aarhus, end Oct., 2008, organised by participant J. Kjems (month 35) 4. “High Throughput Technologies for the Analysis of Alternative Splicing” organised by participants J. Valcarcel and C. Smith, will be held in Valencia, Spain, in November/December 2008 (month 36)

282

234 Workshops in 2008. 1. Alternative Splicing & Disease, Montpellier February 18-23, 2008. A one-week in-depth practical and theoretical introduction into alternative splicing related to human disease, organised by participants J. Tazi and S. Stamm. (month 26) 2. “RNA splicing and genetic diagnosis”, to be held March 3, 2008 London, organised by participant Diana Baralle. (month 27) 3. “Bioinformatics” in Krakow, May 22, 2008, organized by participant G. Ast (month 29) This represents a full programme of WP16 activities planned for 2008. Events planned for 2009: 235 2009 Annual Meeting, April, 2009 To be organized by participant G. Biamonti (month 40) 236 Three workshops on areas of strategic relevance in 2009: 1) UK RNA Splicing Workshop, Cumbria, UK, January 2009, organized by J. Beggs & J. Brown. In addition to the seven UK-based EURASNET groups (Baralle, Beggs, Brown, Caceres, Eperon, Lamond, and Smith) we will invite approximately 14 non-EURASNET groups. Therefore, this would be an outreach activity as well as promoting staff exchange for EURASNET groups. Students and postdocs will be encouraged to give talks, thereby promoting training and career development of young researchers. (month 37) 2) "Analysis of RNA-protein interactions techniques workshop", Barcelona, Spain. This will be a “hands-on” practical workshop including lectures and practicals aimed at graduate scientists, organised by participant J. Valcarcel.

283

17. Staff Exchange and Training

Workpackage description (month 25-42) Workpackage number 17 Start date or starting event: 4 month Participant id all Person months 28.5

Objectives

• To increase the technical competence of PhD students and postdocs in the EURASNET by giving them opportunities to learn state-of-the-art techniques in other member laboratories.

• To encourage staff exchange among EURASNET laboratories for the purpose of training specific techniques.

• To promote scientific collaborations, in particular those of a multidisciplinary nature, by supporting the exchange of personnel between EURASNET member laboratories.

Description of work Staff exchange and training in the groups of EURASNET participants will be continued and the travel bursaries will also be used for participation of students and postdocs at intra-workpackage meetings. EURASNET members will be reminded at regular intervals about these possibilities. As predicted, the number of training exchanges has doubled from 2006 to 2007; however most exchanges are relatively short (less than two weeks on average in 2007), resulting a low number of PMT. We expect a further increase in staff exchange after initiation of new collaborations between EURASNET groups. In addition, with workpackages evolving, we expect that travel bursaries will increasingly be used for attendance at intra-workpackage meetings. Several requests for 2008 have already been received.

Deliverables 116 Continue and further develop the use of travel bursaries for student and postdoc exchanges (also for necessary intra-workpackage meetings)(40 PMT/18 month) (month 30). 237 Continue and further develop the use of travel bursaries for student and postdoc exchanges for training and participation at intra-workpackage meetings (40 PMT/18 month) (month 42) (continuation of deliverable 116)

284

18. Career Development

Workpackage description (month 25-42) Workpackage number 18 Start date or starting event: 3 month Participant id all Person months 27

Objectives

• To prepare young scientists for careers in science, teaching and industry

• To facilitate the transition from a post-doctoral fellow to an independent group leader

• To give young scientists the opportunity to raise the profile of their research and their research groups, and to establish lasting contacts/collaborations among the young generation of European scientists

Description of work We will continue to provide opportunities for personal development that equip young scientists and postdocs for later stages of their scientific careers. The instruments will be to arrange 2 web-based seminars in 2007 about “Effective scientific communication - the art of writing scientific papers” and “Innovation, patents and entrepreneurship”. In addition, these seminars will be distributed as a DVD to all partners. This initiative will be followed by a satellite session on Career Development in conjunction with the biennial European Conference on Alternative Splicing in 2008. At this meeting many young scientists will attend which will give the initiative more impact. At this meeting we plan, with the consent of the younger participants, to video record their presentations and subject them to professional critics. We will also arrange round table discussions with representatives of both academia and industry where the individual career opportunities of the young scientists are discussed. We will constantly try to improve the contacts between PhD students and senior scientists in EURASNET, through the travel bursaries program and joint thesis committees. Finally, the EURASNET website will continuously be updated with information regarding job opportunities for network members looking for employment at the end of their PhD thesis or post-doctoral work.

Deliverables 118 Career development related round table discussions with representatives of both academia and industry (month 30). 119 Organization of a workshop on career development at the Krakow meeting (month29) 238 Establishing joint PhD committees for PhD students in the EURASNET. (month 42) (continuation of deliverable 16) 239 Organization of a mentor lunch (month 29) 240 Organization of job interviews at the Krakow meeting (month 29) 241 Evaluate available career development training courses within the EURASNET network for appropriateness for our pre- and postdocs (month 36) 242 Pursue the possibility of selecting one program for a 1-2 day career development workshop in which participants get hands-on training in the chosen topic (eg writing the cv, giving a talk or an interview, making and presenting a poster) (month 41)

285

19. Public Understanding of RNA Biology Workpackage description (month 25 – 42)

Workpackage number 19 Start date or starting event: Month 0 Participants: 7 8 11 14 18 Person month 0.5 4 0.5 0.5 0.1 Objectives

• To inform the public about the important role of RNA processing in mechanisms of human disease and for the developmental program of organisms, and to explain in general terms the major aims and results of the NoE consortium.

• To discuss with a general audience issues of ethical and social concern raised by progress in the RNA splicing field and to explain the impact of this research for future medical intervention and disease management

• To educate and support scientists in the NoE to enhance their skills in communication with the public, media and politicians

Description of work (from 24 - 42) The scientific officer (PSO) is central to the proposed activities in this work package. In principle, the PSO will oversee and help to coordinate all actions related to this area of activities. The PSO is in the process of generating an extended brochure describing the members and aims of the EURASNET as well as topics connected to alternative splicing. In addition, a new version of the EURASNET leaflet, including all YIPs will be generated in parallel. Another task of the public scientific officer is to directly communicate to the webpage master in Dundee for consistent report on PUS activities like press releases, accessible for EURASNET members and the public. The PSO will also continue to enrich and update the PUS part of the EURASNET webpage as well as take care of the teaching materials. The extended EURASNET brochure is supposed to inform the public about the impact of alternative splicing on life in general,. Target groups should be informed about the important role of RNA processing in mechanisms of human disease as well as in the developmental programme of higher organisms. Furthermore, a detailed insight into research topics and fields of all the network participants should be given. Presented articles will be richly illustrated with pictures, schemes and drawings for an easy understanding of the topic. Target groups to be reached were selected to act as “amplifiers”. These are medical doctors, teachers (especially biology teachers, intending to further instruct their pupils about the topic of alternative splicing) and higher educated pupils by themselves. First the brochure will be in English and German but later should be translated into several languages. The brochure is also intended to act as a more elaborate introduction to the network (including all 40 research groups). The extent of the brochure will be about 25 pages. To inform about current activities and remarkable findings within the network, a biannually appearing newsletter will be disseminated among researchers in the RNA field and selected target groups. This newsletter will comprise articles describing the impact of alternative splicing on one specific issue. A short description of the work of one research group as well as background information about their specific research topic will be given. To educate the scientists of the EURASNET in communicating science to the public, a media contest and a media workshop will be organized in the framework of the 1st International EURASNET Conference in Krakow (Poland) in May 2008.

Deliverables

286

19 a training workshop in public understanding of science for NoE scientists (month 29) 243 an extended brochure for the general public describing the NoE, the concepts, participants and projects, as well as alternative splicing in development and disease (month 34) 244 media contest (scientist write press releases) (month 29) 245 a new version of the EURASNET leaflet (month 34) 246 continuous enrichment and update of the PUS part of the webpage; as well as continuous collection of teaching material for RNA biology (month 42) 247 a newsletter describing contemporary findings within the EURASNET; the newsletter will appear twice a year, starting from month 34

287

20. SMEs and Technology Transfer

Workpackage description (month 25-42)

Workpackage number 20 Starting day or starting event 4 month Participant id 1a 2 10b 12a 12c 13 17 22 24 Person months 1.5 1 0.5 0.5 0.5 0.5 0.5 4 0.5

Objectives • To provide NOE members with advice, information and training that will alert them to commercial opportunities in their research field and enhance their ability to interact successfully with the private sector.

• To facilitate the commercial exploitation of reagents, technology and intellectual property in the field of RNA splicing and cognate areas; to forge commercial links between NOE members and existing European companies.

• To enhance the commercial viability of a spin-out drug discovery company using RNA splicing factors as drug targets.

Description of work Responsibility for coordinating and planning SME & Technology Transfer activities of the network will rest with the HTE-Fund Committee, to be chaired by RL. The committee will provide formal annual reports to the network members in addition to regular email contact and informal bilateral and multilateral discussions. The committee will initially comprise AL, RL, JT, CB, GTV and JV, who all have prior experience as consultants and/or members of commercial scientific advisory boards. When the NOE commences, membership of the committee will be expanded to incorporate other EURASNET members with relevant expertise and to ensure broad representation from across the EU member states. An indicative list showing prior commercial experience and experience of EURASNET members is provided elsewhere in this application (Section 6.3 d). The committee will review the prior and current commercial experience and commercial activities of all NOE members and will use this information to establish priorities and develop a commercialization strategy best suited to the NOE. Building on established links between EURASNET members and companies operating in Europe, the HTE-Fund Committee will coordinate negotiations to obtain preferential terms for commercialization of reagents, such as antibodies, expression vectors, probes, cell lines, proteins and cell extracts, that have been generated in the laboratories of EURASNET members. This activity will stimulate commercialization by improving access and communication between scientists and companies in different EU member states. The HTE-Fund Committee will monitor regularly the requirements of all research projects within the network and review the opportunities for recruiting commercial expertise and technology provision to aid the advancement of research within the network. Thus we will establish which commercial sources can supply services needed by Network members, such as DNA sequencing, antibody production, protein expression, microarray analysis, generation of transgenic mice etc., and negotiate favourable access terms. We will be proactive in seeking opportunities to offer European companies access to expertise and IP generated within the Network. This will help provide a competitive advantage to European companies by allowing them to expand their range of commercial products, services and technologies. The HTE-Fund Committee will provide a bridge between members of the NOE and industry. The resulting interactions will enhance the transfer of technology, IP and “know-how” from the splicing field into the commercial sector. To ensure that the NOE resources are managed as efficiently and productively as possible, we will employ the following strategy. An initial two-year phase, corresponding to a ‘proof of concept’ stage, will be established by the NoE; this will be essential to make the NoE a genuinely attractive partner for future links with the pharmaceutical industry. During the proof of concept stage several independent projects will be carried out by NOE participants and the results evaluated by the HTE-Fund Committee, in concert with the appointed external consultants, to assess which assay formats and screening targets best meet the criteria required for conducting an industrial strength high throughput small molecule screening programme to identify splicing inhibitors. This work will be coordinated with research activity in WP14 by the HTE-Fund Committee and will offer all interested NOE members the opportunity to participate in and contribute to the commercial development of splicing factor research.

Deliverables 248 Develop and market commercially a kit for in vitro splicing assays (month 36). 249 Screen at least 10,000 compounds in vitro for inhibitory effects on splicing (month 42). 250 Develop a stable cell line for evaluating the effect of chemical inhibitors on pre-mRNA splicing in vivo (month 40). 251 Evaluate the mechanism of splicing inhibition by small molecule effectors identified in screening initiative (month 42)

288

21. Reachout to the Broader RNA Community

Workpackage description (month 25-42) Workpackage number 21 Start date or starting event: 7 month Participant id all Person months 30.5

Objectives

• To identify and make contact with other EU-funded groups and other relevant organizations in Europe, to produce a database of e-mail contacts of their members. • To establish contact with these members and make them aware of our existence and our aims. • To promote exchange of expertise within this extended network. • To collect and disseminate information about conferences and workshops on relevant topics. • To identify sources of grant funding for applications to extend these activities • To identify commercial organisations to support and promote RNA research

Description of work: The many and varied activities of the EURASNET members in promoting alternative splicing research and the Network will continue throughout the new JPA. The work programme for months 25-42 for WP21 will focus on efficient information gathering and quantification of these activities. In addition, the need for greater interaction with clinicians and medical scientists involved with splicing- and alternative splicing-related diseases will be addressed by identifying these individuals in the member countries and inviting them to attend meetings and workshops and sending information about EURASNET.

Deliverables 252 Invite non-EURASNET RNA researchers to workshops/meetings to support interactions and collaborations. (month 42) (continuation of deliverable 123) 253 Maintain updated contact list of RNA researchers and groups in Europe. (month 42). (continuation of deliverable 124) 254 Prepare circulation list of clinicians and scientists involved in therapeutic approaches to splicing diseases. The first year report suggested greater interaction with medical scientists and clinicians to raise awareness of alternative splicing. Some members of EURASNET interact with clinicians and medical scientists and consult or act as experts to groups involved in development of therapies for splicing-related diseases. We will, via these EURASNET contacts, establish a list of contacts for such scientists. Information on upcoming meetings and workshops organised by EURASNET will circulated to this group. (month 36). 255 Modify proforma for capturing information. The proforma used to capture information on various activities in terms of broader outreach to the RNA community will be modified on the basis of feedback from members (month 36). 256 Regular requests for information from members. Requests for information will be sent out quarterly and collated. (month 42)

289

22. Management

Workpackage description (month 25-42) Workpackage number 22 Start date or starting event: 0 month Participant id 1a 2 3 9 15 16 21 22 Person months 45 13.5 13.5 0.5 0.5 0.5 0.5 0.5

Objectives • Manage the research, integration and dissemination components of the NoE by the management team

Description of work Organize and survey network operations, manage financial operations. Provide assistance for all organizational, legal and financial problems. Organize and survey integration and dissemination activities.

Deliverables 127 Prepare Annual Report for 2007 period (month 25). 257. Report on decisions made during the Annual Meeting 2008 (month 30). 258. Prepare Annual Report for 2008 period (month 38). 259. Steering Committee Meetings at month 27,30, 33, 36, 39, 42 260. Quarterly network e-mail at months 27,30, 33, 36, 39, 42

290

15.7 WORK PACKAGE LIST (MONTHS 25-42)

Work-package

No

Workpackage title Lead

Contractor No

Person

months

Start month

End month

Deliverable No

1 The EURASNET Web site 14 8.5 25 42 146-150

2 Sharing resources, technology and reliable protocols

3 60 25 42 151-153

4 The Alternative Splicing Database 5b 21 25 42 132, 154-156

5 Ensuring durability 16 14 25 42 157-158

6 In silico approaches to alternative splicing

6 80 25 42 N6, N7, 159-164

7 Molecular characterization of splicing substrates

12c 100 25 42 72, 165-170

8 Genome-wide analyses of splicing regulation

23 180 25 42 171-185

9 Complexity of spliceosomal proteomes 1a 98 25 42 186-193

10 Post-translational modification and intracellular dynamics of splicing factors

15 120 25 42 90, 194-202

11 Function of splicing factor isoforms 2 100 25 42 203-208

12 Mis-splicing and disease 7 126 25 42 209-214, N8, N9

25 Co-transcriptional mechanisms of alternative splicing

1b 125 25 42 215-219

14 Chemical biology and therapeutics 12a 128 25 42 220-225

15 Development of enabling technologies 12b 92 25 42 226-232

16 Conferences and meetings 9 11 25 42 114, 233-236

17 Staff exchange and training 21 28.5 25 42 116, 237

18 Career development 19 27 25 42 118-119, 238-242

19 Public understanding of RNA biology 8 5.5 25 42 19, 243-247

20 SMEs and technology transfer 22 9.5 25 42 248-251

21 Reachout to the broader RNA community

14 30.5 25 42 252-256

22 Management 1a 74.5 25 42 127, 257-260

15.8 PMs FOR THE 3rd 18 MONTH PERIOD (MONTH 25 to 42)

MPG

1a

MPG

1b

MPG

1c

CR

G

UER

LN

UU

EMB

L5a

EMB

L5b

TAU

ICG

EB

MU

W

UED

IN

CN

R 1

0a

CN

R 1

0b

LUG

CN

RS

12a

CN

RS

12b

CN

RS

12c

CN

RS

12d

UN

IBE

SCR

I

MR

C

IMM

Uni

leic

AM

U

UA

AR

FCEN

-U

BA

20a

FCEN

-U

BA

20b

UN

IGE

UN

IVD

UN

UC

AM

- D

BIO

C

HU

JI

CER

BM

-G

IE

IN

SER

M

FCSR

ISB

-SIB

Joint Programme of Activities Integrating activities1 WP1 2.5 6 WP2 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 WP4 1.5 1.5 2 8 1.5 1.5 1.5 1.5 1.5 WP5 1.5 1.5 1.5 1.5 8 Jointly executed research activities3 WP6 8 8 42 6 WP7 8 10 8 8 42 8 8 WP8 10 8 8 10 9 8 8 8 8 8 8 8 8 8 42 8 6 7 WP9 39 8 8 9 8 10 8 8 WP10 10 8 8 8 8 6 42 8 8 8 6 WP11 10 42 8 8 8 8 8 8 WP12 8 42 8 8 8 6 8 8 8 8 6 WP13 42 8 10 9 8 10 8 8 8 8 6 WP14 10 8 8 42 8 16 16 8 8 WP15 10 6 10 8 8 42 8 Spreading of Excellence activities3 WP16 1.5 1 0.5 0.5 0.5 0.5 4 0.5 0.5 0.5 0.5 WP17 1.5 1.5 0.5 1 1.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 WP18 1.5 1.5 1 1 1.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 1 0.5 WP19 0.5 4 0.5 0.5 WP20 1.5 1 0.5 0.5 0.5 0.5 0.5 4 0.5 WP21 1.5 1.5 1 1 1.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 0.5 4 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 1 0.5 TOTAL JPA

96 59.5 63 59.5 44.5 13.5 19 68.5 57.5 16.5 37 43 35.5 19.5 77.5 65 27.5 19.5 22.5 62.5 37 62 11.5 46.5 11 11 24.5 32.5 63 19.5 19

Consortium Management Activities WP22 Management

45 13.5 13.5 0.5 0.5 0.5 0.5 0.5

TOTAL per participant

141 59.5 10 76.5 67 44.5 13.5 19 68.5 57.5 16.5 37.5 43 35.5 19.5 77.5 65 27.5 10 19.5 28.5 63 37.5 62 11.5 46.5 11 11 25 33 63 19.5 19 22 10 17.5

Overall total efforts

(all numbers refer to person months) 291

1 except management of the consortium activities

ETH

Z

IIM

CB

UPF

SOTO

N-

HG

D

TOTA

L

Joint Programme of Activities Integrating activities2 WP1 8.5 WP2 1.5 1.5 1.5 1.5 60 WP4 0.5 21 WP5 14 Jointly executed research activities3 WP6 8 8 80 WP7 8 100 WP8 180 WP9 98 WP10 120 WP11 100 WP12 8 126 WP13 125 WP14 4 128 WP15 92 Spreading of Excellence activities3 WP16 0.5 11 WP17 0.5 0.5 1 0.5 28.5 WP18 0.5 0.5 0.5 0.5 27 WP19 5.5 WP20 9.5 WP21 0.5 0.5 1 1 30.5 TOTAL JPA

11 11 12 16.5

Consortium Management Activities WP22 Management

74.5

TOTAL per participant

11 11 12 16.5

Overall total efforts

1439

292

2 except management of the consortium activities

293

16 Project management level description of resources and grant grant [month 25-42; Jan 2008-June 2009] The EURASNET consortium has been granted a maximum of 10 million Euros from the EU. This is estimated to be not more than 30% of expected expenditure required to carry out the activities specified in the JPA of this proposal. We have included a headcount of 147 researchers, which is in excess of 100 researchers required for a requested grant of 10 million euros. We also have 80 doctoral students, all of which justifies a contribution from the EU of the amount granted. The content and scope of the EURASNET JPA represent a very substantial effort that will require considerable human, material and financial resources. The network consortium implicates 30 laboratories and almost 150 investigators. This equates to an equivalent of roughly 10,000 person months manpower capacity. At current rates, the estimated manpower cost for this effort represents over 25 M EUR in stipends alone. We estimate that the actual cost of the research itself in terms of reagents, etc. will be in the range of 25-40 M EUR for the five years period again, at current prices. The EU funds granted to support JPA research are therefore considerably lower than the actual costs of the planned research. Importantly, these estimates do not consider equipment acquisition or maintenance, a particularly relevant consideration taking into account the heavy component of development and use of new technologies outlined in the application. It is also important to point out that the JPA-related research is just part of the activities –in fact largely a newly conceived activity- of the research being carried out by the Network, whose overall budget would easily triplicate these figures. The EURASNET NoE uses 10 M€ to support the Joint Programmes of Activities of the consortium in research, integration and dissemination, as well as the overall network management (see Fig. 7). 6.05 M€ are assigned to research activities. This includes the annual allocation for each participant as well as the High-Throughput-Enabling Fund. The Integration Programme, supported with 2 M€, comprises the YIP Programme, the interdisciplinary meetings and the web-services. The Dissemination Programme includes the meetings and workshops and public science activities and receives 1.25 M€. Management is supported with 0.7 M€. Below, we give a detailed account of NoE-dedicated resources which the participants have available in order to execute the Joint Program of Activities. Joint Research 1. Annual Allocation It is partly because the requested budget represents only a fraction of the real research costs, that the Network members decided to have a flat distribution of the research budget among the individual laboratories, which corresponds to 40,000 EUR per laboratory per year during the first three years and 30,000 EUR/year during the fourth and fifth year. This amount will cover a larger fraction of the costs for smaller groups, whose workload will also be generally less extensive. This arrangement has been deemed fair by Network even though smaller research groups will benefit relatively more from the amount received. The cost to employ a postdoc vary considerably among European countries. If we assume an average annual cost of 50,000 € and if we assume that each network partner will have at least two persons dedicated full time to Network research efforts (24 person months), then it becomes very clear that the allocated research funds alone are insufficient to support the research goals of the Network. The estimated costs will be three to four times higher than the requested funds. It will be left to the team leaders discretion on what the annual research fund would best be spend in his or her group. No additional funds beyond the 40,000 € (30,000 € in fourth and fifth year) will be available for consumables and equipment.

294

Joint Programme of Activities RESEARCH

(60 month period)

Joint Programme of Activities RESEARCH

(3rd 18 month period)(month 25-42)

Estimated costs for Jointly Executed

Research

Requested Grant for Jointly

Executed Research

Estimated costs for Jointly Executed

Research

Requested Grant for Jointly Executed

Research MPG 1a 630,000 € 180,000 € 210,000 € 55,000 € MPG 1b 630,000 € 180,000 € 210,000 € 55,000 € CRG 2 630,000 € 180,000 € 210,000 € 55,000 € UERLN 3 630,000 € 180,000 € 210,000 € 55,000 € UU 4 630,000 € 180,000 € 210,000 € 55,000 € EMBL 5a 630,000 € 180,000 € 210,000 € 55,000 € EMBL 5b 630,000 € 180,000 € 210,000 € 55,000 € TAU 6 630,000 € 180,000 € 210,000 € 55,000 € ICGEB 7 630,000 € 180,000 € 210,000 € 55,000 € MUW 8 630,000 € 180,000 € 210,000 € 55,000 € UEDIN 9 630,000 € 180,000 € 210,000 € 55,000 € CNR 10a 630,000 € 180,000 € 210,000 € 55,000 € CNR 10b 630,000 € 180,000 € 210,000 € 55,000 € LUG 11 630,000 € 180,000 € 210,000 € 55,000 € CNRS 12a 630,000 € 180,000 € 210,000 € 55,000 € CNRS 12b 630,000 € 180,000 € 210,000 € 55,000 € CNRS 12c 630,000 € 180,000 € 210,000 € 55,000 € UNIBE 13 630,000 € 180,000 € 210,000 € 55,000 € SCRI 14 630,000 € 180,000 € 210,000 € 55,000 € MRC 15 630,000 € 180,000 € 210,000 € 55,000 € IMM 16 630,000 € 180,000 € 210,000 € 55,000 € Unileic 17 630,000 € 180,000 € 210,000 € 55,000 € AMU 18 630,000 € 180,000 € 210,000 € 55,000 € UAAR 19 630,000 € 180,000 € 210,000 € 55,000 € FCEN-UBA 20

630,000 € 180,000 € 210,000 € 55,000 € UNIGE 21 630,000 € 180,000 € 210,000 € 55,000 € UNIVDUN 22

630,000 € 180,000 € 210,000 € 55,000 €

295

UCAM-DBIOC 23

630,000 € 180,000 € 210,000 € 55,000 € HUJI 24 630,000 € 180,000 € 210,000 € 55,000 € CERBM-GIE 25

630,000 € 180,000 € 210,000 € 55,000 € TOTAL 18,900,000 € 5,400,000 € 6,300,000 € 1,650,000 € The new contractors incorporated after completion of the first and second YIP round are shown in the budget of the Joint Program of Integration! 2. The High-Throughput-Enabling Fund “High-throughput enabling” (HTE)-Fund for securing new technologies. This special fund, amounting to € 650,000, will be instituted. This will have two purposes: (i) to support the exploitation of high-throughput technologies in the study of alternative splicing, e.g., micro-array technology used for the genome-wide identification of alternatively spliced mRNAs, as explained in detail in Section 6.2 c of Annex_I, and (ii) to support essential feasibility studies required for the development and effective use of new assay formats by NoE member groups to support the high-throughput screening of chemical compound libraries to identify inhibitors and modulators of splicing (Section 6.2 i). Of this fund, our indicative proposal is that € 400,000 will be directed towards the former purpose and € 250,000 to the latter. However, the final allocation of resources from the fund will be closely linked to the success and development of these respective projects. To ensure best practice, the progress of all projects will be regularly reviewed by the HTE-Fund Committee, with advice from the appointed external consultants, and funding allocated accordingly to reinforce the most successful work. In the case of feasibility studies that involve the use of screening facilities at the University of Dundee, labour and material costs incurred may be paid by the NOE member for whom the study is performed; however, we note that the facility will participate on a non-profit basis. The NoE member in question can apply to the HTE-Fund Committee for reimbursement of these costs. either in whole or in part, from the HTE-Fund. The HTE-Fund of € 650,000 supporting array technology and high-throughput screening of chemical compound libraries will be under the administration of the Chairman of the HTE-Fund Committee. While the funds are primarily intended to support the efforts of the participants in the corresponding work packages 11 and 14, they remain also available to proposed projects by other participants. To obtain support from the HTE-Fund for either of the above activities, members of the NoE will in the first instance submit an informal written application for this support to the chairman of the HTE-Fund Committee. Applications will be considered, and allocation made, by the HTE-Fund Committee, usually not later than three months after receiving a request. To ensure objective and transparent criteria in the decision-making process, the Committee's decisions will be reviewed by two members of the external Scientific Advisory Board. Any member of the NOE whose application is turned down by the committee will have the opportunity to appeal the decision by writing to the chairman of the Scientific Advisory board, whose decision, after review, will be regarded as final. The High-Throughput-Enabling Fund can only cover part of the actual costs involved in developing and exploiting these key technologies. Substantial additional funding through additional grants of participants and support from their institutions will be required. We estimate that actual costs will be higher by a factor of 10.

296

60 month period 18 month period Estimated costs

for Jointly Executed Research

Requested Grant for

Jointly Executed Research

Estimated costs for Jointly

Executed Research

Requested Grant for

Jointly Executed Research

4,000,000 €

400,000 €

1,200,000 €

120,000 €

micro-array technologies

MPG 1a =Coordinator and Head of

HTE-Committee

2,500,000 €

250,000 €

750,000 €

75,000 €

small

chemical compound screening

TOTAL

6,280,000 €

650,000 €

1,807,000 €

195,000 €

297

Fig. 7 Budget

298

Integration 1. Young Investigator Programme (YIP) The YIP budget amounts to 1.2 M€. These funds will be distributed during two rounds of open calls for 5 young investigators each. Support will be 40,000 € annually for a 3-year period. Since all the winners of YIP awards remain to be determined, these funds can not be allocated to specific participants yet. Like the founding members of the network, YIP members are expected to contribute at least 24 person months per year towards EURASNET research goals. 2. Interdisciplinary Meetings Four interdisciplinary focus meetings will be organized annually around the following topics by the indicated NoE members:

• Combining computational and experimental approaches to study alternative splicing (G. Ast, P. Bork)

• Biophysical methods and functional genomics tools to study alternative splicing (R. Lührmann, C. Smith, I. Eperon)

• Cell biology, signaling and alternative splicing (J. Cáceres, A. Lamond) • Alternative splicing and clinical research (F. Baralle, S. Stamm)

Funds in the following table have been allocated accordingly. Each meeting is supported by 10,000 €, except for the meeting on splicing and disease which will receive 20,000 € instead (250,000 € total over the 5-year period). Funds will provide support for travel and accommodation of invited speakers. These meetings will have a limited number of participants (max. 30) and will include both NoE members whose research is directly connected to the topic, as well as outside experts from other fields that will be invited to participate and give a 30 minute talk about their technologies or areas of expertise. These conferences also provide the opportunity to get in touch with representatives of SMEs. To underscore the importance of the scientific exchange between alternative splicing researchers and clinicians, one meeting each year will be specially dedicated to this theme. This meeting will be assigned the increased budget of 20,000 € and 50 participants will be admitted. 3. Web-Services Fundamental for integration are platforms for communication and infrastructure, particularly those that are web-based. We plan to create web-based tools that will be both professionally useful and user-friendly, with the goal that they will become regular instruments of day-to-day research. Although the contents will be ultimately dictated by the investigators’ needs, we aim for high caliber, highly professional web pages that will be created with the goal of durability and potential for expansion and review. We plan to jump-start these resources with the help of Network funds with the perspective that, once established, they will maintain a durable structure that will be, at least in part, self-supported through small users’ fees. As pointed out in numerous places throughout this Annex, the EURASNET Website is one of the most critical points in the successful design of a European Network of Excellence as it is intended by the European Commission. Therefore an amount of 550,000 € over the five year period is earmarked for developing and maintaining the EURASNET Website. This site will be the hub for all Network- and Public Science-related information traffic. Activities within this budget are organized and overseen by John Brown, Dundee. The amount of 550,000 € includes the webmaster employment (80,000 €/y over 5 years) in Dundee, a part time position (20,000 €/y over 5 years) located at the Web-server site in Dundee, UK and additional 50,000 €/5y for necessary hardware and software.

299

Joint Programme of Activities

INTEGRATION (60 month period)

Joint Programme of Activities

INTEGRATION (18 month period,

month 25-42, Jan '08 – June "09)

Estimated costs

Requested Grant

Estimated costs

Requested Grant

1. YIP MPG 1c * 420,000 €/3y 120,000 €/3y 210,000 € 60,000 € CNRS 12d *

420,000 €/3y 120,000 €/3y 210,000 € 60,000 €

INSERM 26 *

420,000 €/3y 120,000 €/3y 210,000 € 60,000 €

FCSR 27 * 420,000 €/3y 120,000 €/3y 210,000 € 60,000 € ISB-SIB 28 *

420,000 €/3y 120,000 €/3y 210,000 € 60,000 €

* First round integrated by month 6 (July 2006).

FCEN-UBA20b**

420,000 €/3y 120,000 €/3y 70,000 € 60,000 €

ETHZ 29** 420,000 €/3y 120,000 €/3y 70,000 € 60,000 € IIMCB 30**

420,000 €/3y 120,000 €/3y 70,000 € 60,000 €

UPF 31 ** 420,000 €/3y 120,000 €/3y 70,000 € 60,000 € SOTON-HGD 32 **

420,000 €/3y 120,000 €/3y 70,000 € 60,000 €

2 rounds of 5 YIP awards

4,200,000 €

1,200,000 €

1,400,000 €

600,000 €

** Second round integrated by month 25 (Jan 2008).

Winners of the second round of YIP awards start their contract during the third 18 month period in month 25 (Jan 2008). 2. Interdisciplinary Meetings MPG 1a 50,000x1/3 € 50,000x1/3 € 10,000x1/3 € 10,000x1/3 € UERLN 3 100,000x1/2 € 100,000x1/2 € 20,000x1/2 € 20,000x1/2 € EMBL 5a 50,000x1/2 € 50,000x1/2 € 10,000x1/2 € 10,000x1/2 € TAU 6 50,000x1/2 € 50,000x1/2 € 10,000x1/2 € 10,000x1/2 € ICGEB 7 100,000x1/2 € 100,000x1/2 € 20,000x1/2 € 20,000x1/2 € MRC 15 50,000x1/2 € 50,000x1/2 € 10,000x1/2 € 10,000x1/2 € Unileic 17 50,000x1/3 € 50,000x1/3 € 10,000x1/3 € 10,000x1/3 € UNIVDUN 22

50,000x1/2 € 50,000x1/2 € 10,000x1/2 € 10,000x1/2 €

300

UCAM-DBIOC 23

50,000x1/3 € 50,000x1/3 € 10,000x1/3 € 10,000x1/3 € 3. Web-Services

400,000 € 400,000 € 120,000 € 120,000 € SCRI 14*

50,000 € 50,000 € 15.000 € 15.000 € EMBL 5b 100,000 € 100,000 € 30,000 € 30,000 € * took over from UERLN 3 effective July 2007 (month 19) TOTAL 5,000,000 € 2,000,000 € 1,615,000 € 815,000 € Dissemination 1. Training, Meetings and workshops Scientific exchange in the form of annual meetings and topical workshops is deemed essential for durable integration and flexible planning. Two of the annual meetings will be coincident with the Biennial International Conference on Alternative Splicing, and will highlight recent developments and technologies through the invitation of keynote speakers. Annual meetings will have a budget of 100,000 EUR, and 70,000 EUR in the years of Biennial International Conferences. The latter is expected to be lower as some funds will be recovered from non-network members registration fees. During international conferences all sessions will be open to participants worldwide, except those where internal Network issues are discussed. In order to provide a venue at which data can be exchanged at a maximal transparence, the presence of SME representatives might not be desirable, although this prevents suppport from sponsoring companies. The registration fees will be waived for Network members and – if funds permit – Third Parties, who become actively involved in the activities of the network without receiving EU funding. The annual meeting funds will include the travel and accommodation for two members of each lab, and for invitation of SAB members, keynote speakers and candidates for the YIP. The SAB will be invited and costs covered to attend both the International conference and the annual NoE meeting. 1.05 M€ will be dedicated to staff exchange and scientific exchange. As elaborated at various points in the proposal, staff mobility is critical for integration, for optimizing the use of available resources and for career development. We expect that a substantial number of exchanges will take place involving virtually every lab of the consortium (approx. 90 staff exchanges during the period of the grant). On average, travel and accommodation allowances for periods of three months or more will amount to 3,000 EUR bursaries. Angela Krämer will act as the coordinator of this program. Workshops on three areas of strategic relevance for the consortium, with a strong focus on developing technologies and a reduced number of participants (around 30), are planned annually, with a budget of 20,000 EUR per workshop. These funds should cover travel and accommodation costs of Network members and invited speakers. Workpackage 18 (Career development) will be supported with funds for two workshops during the 5-year period (20 k€ each). Lead contractor for this workpackage is J. Kjems.

301

The Meeting and conference programme over 60 months month 25-42 2 biennial international conferences on alternative splicing

140,000 €

-

3 annual conferences 300,000 € 100,000 € 90 training bursaries 270,000 € 81,000 € workshops 300,000 € 80,000 € 2 career development workshops

40,000 €

20.000 €

Total 1,050,000 € 281,000 €

For the next 18 month period (Jan 2008-June 2009) EURASNET requests funds for an annual meeting (100.000 €) and the second career development workshop. 2. Public Science Activities Aware as we are of the lack of tradition and structure in scientific communication with the public, the Network has decided to devote a significant amount of resources to reverse this trend and provide attractive platforms for exchange of information with society, including a dedicated web-page, round-table discussions, workshops, institutional open days, and other initiatives detailed in the JPA. Public Science activities are organized and overseen by Andrea Barta, Vienna. 60,000 € are reserved for public science activities over the 5-year period. For the employment of a Public Scientific Officer, located in Vienna and reporting directly to Andrea Barta, the amount of 100,000 € is planned for the 5-year period. The workload for the PSO will be significantly higher during the first year of the Network. This should be reflected in a full-time employment during this initial phase and part-time employment for the time thereafter. The PSO will organize various public science activities as outlined in WP19. In addition, public science efforts will be supported by two training workshops on public understanding of science, both associated with the annual meeting (20 k€ each, maximum of 30 participants).

(60 month period)

(month 25-42 period)

Participant Estimated costs Requested Grant Estimated costs Requested Grant

MUW 8

60,000 € 100,000 € 40,000 €

60,000 € 100,000 € 40,000 €

18,000 € 30,000 € 12,000 €

18,000 € 30,000 € 12,000 €

TOTAL 200,000 € 200,000 € 60,000 € 60,000 € Management 0.7 M€ are used to ensure a robust and efficient Management structure. As described and justified in the JPA description, 1 full-time and 2 half-time positions, which represents the minimum for such a large network, will integrate the Managing Team. The full amount of the management budget is assigned to the management team. Audit costs are not included in this budget, they will have to be carried instead by

302

each individual participating lab and are not eligible for refund. Although annual audits are mandatory, nevertheless every effort should be made to keep the enormous amount of estimated 300.000 € in audit costs over the five year period at a minimum. We therefore request the provisions of special clauses 32 and 39 being considered and applied to the participants of this network whenever appropriate.

MANAGEMENT (60 month period)

MANAGEMENT

(month 25-42 period)

Estimated costs Requested Grant Estimated costs Requested Grant MPG 1a 360,000 € 360,000 € 108,000 € 108,000 € CRG 2 150,000 € 150,000 € 45,000 € 45,000 € SCRI 14* 180,000 € 180,000 € 54,000 € 54,000 € Travel costs 10,000 € 10,000 € 3,000 € 3,000 € * takes over from UERLN 3 effective July 2007 (month 19)

TOTAL 700,000 € 700,000 € 210,000 € 210,000 €

Total over all activities:

32,130,000 € 10,000,000 € 10,273,000 € 3,211,000 €