2017 Symposium on RNA Biology XII: RNA Tool and...

October 19 & October 20

Genome Sciences BuildingUniversity of North Carolina at Chapel Hill

2017 Symposium on RNA Biology XII:

RNA Tool and Target

View an interactive campus map at: https://maps.unc.edu/

Event Spaces Parking Decks Route to Carolina Inn

2017 Symposium on RNA Biology XII:

RNA Tool and Target

Thank you to our generous sponsors

October 19 & October 20 Genome Sciences BuildingUniversity of North Carolina at Chapel Hill

Organizers Alain Laederach, UNC Chapel HillBill Marzluff, UNC Chapel HillGreg Matera, UNC Chapel HillRobin Stanley, NIEHSJane Richardson, Duke University Qi Zhang, UNC Chapel Hill

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2017 Symposium on RNA Biology XII: RNA Tool and Target

CONTENTSAGENDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

SPEAKERS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

KEYNOTE SPEAKERS: AbstractsDr. Douglas Black, UCLA........................................................................................10Dr. Eric Westhof, University of Strasbourg............................................................11Dr. Sandra Wolin, National Cancer Institute.........................................................12

INVITED SPEAKERS: AbstractsDr. Victoria D’Souza, Harvard University.................................................................13Dr. Kristen Lynch, University of Pennsylvania.......................................................14Dr. Jeffrey Kieft, University of Colorado, Denver....................................................15Dr. Robert Spitale, University of California, Irvine................................................16Dr. Gabriele Varani, University of Washington......................................................17

SELECTED SPEAKERS: AbstractsEmily Rogers, Georgia Institute of Technology......................................................18Paul Agris, University at Albany-SUNY...................................................................19Canan Kuscu, University of Virginia......................................................................20Shawn Lyons, Harvard Medical School.................................................................21Jim Giron, ThermoFisher Science.........................................................................22Monica Pillon, NIEHS............................................................................................23Kyle Mansfield, East Carolina University...............................................................24Jackson Trotman, Ohio State University.................................................................25Sujatha Jagannathan, FHCRC, Seattle...................................................................26Zhipeng Lu, Stanford University............................................................................27Reyad Elbarbary, Penn State University................................................................28Roger Alexander, PNDRI........................................................................................29Libby Snell, Oxford Nanopore Technologies...........................................................30

POSTERS1. Kripa Ahuja, University of North Carolina at Chapel Hill................................312. Laura Bisogno, Duke University.......................................................................323. Mark Boerneke, University of North Carolina at Chapel Hill...........................334. Alexander Borodavka, University of Leeds.....................................................345. Mauro Calabrese, University of North Carolina at Chapel Hill .......................356. Kausik Chakrabarti, University of North Carolina at Charlotte........................367. Becky Chen, University of North Carolina at Chapel Hill.................................378. Norman Chiu, University of North Carolina at Greensboro...........................38

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October 19 & October 20, 2017

9. Chia-Chieh Chu, Duke University....................................................................3910. Magdalena Cichewicz, University of Virginia..................................................4011. Mary Clay, Duke University ............................................................................4112. Richard Clayton, University of North Carolina at Chapel Hill ........................4213. Aaztli Coria, University of North Carolina at Chapel Hill ................................4314. Anita Donlic, Duke University............................................................................4415. Nichlas Engels, University of North Carolina at Chapel Hill............................4516. Shannon Farris, NIEHS/NIH............................................................................46 17. Nate Fry, East Carolina University...................................................................4718. Laura Ganser, Duke University .......................................................................4819. Nandan Gokhale, Duke University..................................................................4920. Kevin Goslen, National Institute of Environmental Health Sciences...............5021. Kelsey Gray, University of North Carolina at Chapel Hill................................5122. Torin Greenwood, Georgia Institute of Technology........................................5223. Hsiang-Ting Ho, Duke University.....................................................................5324. Isabel Hoerr, University of North Carolina at Chapel Hill...............................5425. Alyson Hoffman, Duke University...................................................................5526. Christopher Holmquist, University of North Carolina at Chapel Hill..............5627. Maryam Hosseini, Bowling Green State University..........................................5728. Megan Kelly, Duke University..........................................................................5829. Taejin Kim, National Cancer Institute/National Institutes of Health...............5930. Manjari Kiran, University of Virginia...............................................................6031. Jayashree Kumar, University of North Carolina at Chapel Hill........................6132. Lela Lackey, University of North Carolina at Chapel Hill................................6233. Brittany Law, Duke University.........................................................................6334. Noah Legall, University of North Carolina at Chapel Hill.................................6435. Yu-Hua Lo, NIEHS/NIH....................................................................................6536. Michael McFadden, Duke University..............................................................6637. Rita Meganck, University of North Carolina at Chapel Hill.............................6738. Dawn Merriman, Duke University...................................................................6839. Angelo Moreno, Duke University....................................................................6940. Brittany Morgan, Duke University...................................................................7041. Anthony Mustoe, University of North Carolina at Chapel Hill.........................7142. Chidinma Nnadi, Johns Hopkins University.....................................................7243. John Noto, University of North Carolina at Chapel Hill..................................7344. Lorena Parlea, National Cancer Institute/National Institutes of Health..........7445. Michelle Potter-Birriel, University of North Carolina at Chapel Hill................7546. Amanda Raimer, University of North Carolina at Chapel Hill.........................7647. Ramalakshmi Ramasamy, University of North Carolina at Chapel Hill............7748. Atul Kaushik Rangadurai, Duke University.......................................................7849. Thomas Ray, Duke University..........................................................................7950. Poorna Roy, University of Michigan................................................................8051. Casey Schmidt, University of North Carolina at Chapel Hill............................8152. Ashlyn Spring, University of North Carolina at Chapel Hill............................8253. Aline Umuhire Juru, Duke University..............................................................8354. Sarah Wicks, Duke University..........................................................................8455. Hannah Wiedne, University of North Carolina at Chapel Hill........................8556. Bo Zhao, University of North Carolina at Chapel Hill.....................................86

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12:00 & 12:30 pm Shuttles from Aloft Hotel to Genome Sciences Building

12:00 pm Registration

1:00 - 1:10 pm Opening Remarks: Alain Laederach, UNC-Chapel Hill

1:10 – 2:30 pm SESSION I: RNA StructureS Chair: Jane Richardson, Duke University

1:10 pm Ribometrix Sponsored Keynote Lecture: Eric Westhof, University of Strasbourg'The Third Genetic Code'

1:50 pm Selected Talk: Emily Rogers, Georgia Institute of Technology 'Profiling RNA secondary structures from a Boltzmann ensemble and its applications'

2:10 pm NC RNA Society Stewardship Recipient: Paul Agris, University at Albany-SUNY'Novel small molecule antibacterial agents targeting tRNA regulation of transcription in Gram-positive bacteria'

2:30 pm Break

Thursday, October 19, 2017 Genome Sc iences Bu i ld ing , Un ivers i ty of North Caro l ina250 Be l l Tower Road , Chape l H i l l , NC 27599

Agenda

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3:00 – 5:00 pm SESSION II: REGULATORY RNAs Chair: Kate Meyer, Duke University

3:00 pm Invited Talk: Jeffrey Kieft, University of Colorado, Denver'A ribosome-induced RNA conformational change regulates translation: an RNA structure-based rheostat'

3:30 pm Selected Talk: Canan Kuscu, University of Virginia'Global Gene Repression by Dicer-independent tRNA fragments'

3:50 pm Selected Talk: Shawn Lyons, Harvard Medical School'tiRNA-mediated translation repression depends upon tetrameric G-quadruplex formation'

4:10 pm Invited Talk: Robert Spitale, University of California, Irvine'Light-Activated Chemical Probing of Nucleobase Solvent Accessibility Inside Cells'

4:40 pm ThermoFisher Sponsored Talk: Jim Giron, ThermoFisher Science'The QuantiGene® Platform: Unrivaled solutions to get you closer to true Biology'

5:00 - 5:45 pm Poster Session/Cocktails, GSB Lobby: Even Numbers Presenting

5:45 - 6:30 pm Poster Session/Cocktails, GSB Lobby: Odd Numbers Presenting

6:30 -9:00 pm BanquetCarolina Inn, 211 Pittsboro St, Chapel Hill, NC 27516

9:00 pm Shuttle from Carolina Inn to Aloft Hotel

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7:30 am Shuttles from Aloft Hotel to Genome Sciences Building

7:45 am Registration

8:00 am Breakfast, Genome Sciences Cafe, 1st Floor

8:30 – 10:00 am SESSION III: RNA FUNCTION Chair: Stacy Horner, Duke University

8:30 am Invited Talk: Victoria D’Souza, Harvard University'Capturing host proteins: Design of viral internal ribosomal entry sites'

9:00 am Selected Talk: Monica Pillon, NIEHS'Grc3 Activates the Essential Endoribonuclease Las1 for Specific RNA Cleavage'

9:20 am Selected Talk: Kyle Mansfield, East Carolina University'Role of the mRNA modification N6-methyladenosine in Hypoxia and Transformation'

9:40 am Selected Talk: Jackson Trotman, Ohio State University'RNA guanine-7 methyltransferase catalyzes the methylation of cytoplasmically recapped RNAs'

10:00 – 10:30 am Break

10:30 am – noon SESSION IV: RNPs Chair: Robin Stanley, NIEHS

10:30 am Keynote Lecture: Sandra Wolin, National Cancer Institute'Taking out the Trash: Tales of RNA Surveillance'

11:10 am Selected Talk: Sujatha Jagannathan, FHCRC, Seattle'When to shoot the messenger: Identifying modulators of mRNA surveillance'

Friday, October 20, 2017 Genome Sc iences Bu i ld ing , Un ivers i ty of North Caro l ina250 Be l l Tower Road , Chape l H i l l , NC 27599

Agenda

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11:30 am Invited Talk: Kristen Lynch, University of Pennsylvania'Alternative Splicing and the Human Immune System'

12:00 - 1:00 pm Lunch Genome Sciences Building Lawn

1:00 – 2:10 pm SESSION V: REGULATORY RNAs (II)Chair: Christopher Holley, Duke University

1:00 pm Invited Talk: Gabriele Varani, University of Washington'lncRNA, Structure, Function and Targeting'

1:30 pm Selected Talk: Zhipeng Lu, Stanford University'RNA Duplex Map in Living Cells Reveals Higher Order Transcriptome Structure'

1:50 pm Selected Talk: Reyad Elbarbary, Penn State University'Tudor-SN–mediated endonucleolytic decay of human cell microRNAs promotes G1/S phase transition'

2:10 – 2:40 pm Break

2:40 – 4:00 pm SESSION VI: ALTERNATIVE Splicing Chair: Jimena Giudice, UNC-Chapel Hill

2:40 pm Keynote Lecture: Douglas Black, UCLA'Mechanisms and programs of neuron gene regulation by Rbfox proteins'

3:20 pm Selected Talk: Roger Alexander, PNDRI'Extracellular RNA Communication Consortium Extracellular RNA Communication: Mechanisms, Biomarkers, and Therapies'

3:40 pm Selected Talk: Libby Snell, Oxford Nanopore Technologies'Highly parallel direct RNA sequencing on an array of nanopores'

4:00 pm Meeting Adjourns

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Mechanisms and programs of neuron gene regulation by Rbfox proteins

Douglas Black, PhDUniversity of California, Los Angeles

The third genetic code

Eric Westhof, PhDARN, IBMC-CNRSUniversity of Strasbourg, France

Keynote Speakers

Taking out the Trash: Tales of RNA Surveillance

Sandra Wolin, MD, PhDNational Cancer Institute

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invited speakers

Capturing host proteins: Design of viral internal ribosomal entry sites

Victoria D’Souza, PhDHarvard University

Alternative Splicing and the Human Immune System

Kristen Lynch, PhDUniversity of Pennsylvania

A ribosome-induced RNA conformational change regulates translation: an RNA structure-based rheostat

Jeffery Kieft, PhDUniversity of Colorado, Denver

Light-Activated Chemical Probing of Nucleobase Solvent Accessibility Inside Cells

Robert Spitale, PhDUniversity of California, Irvine

lncRNA, Structure, Function and Targeting

Gabriele Varani, PhDUniversity of Washington

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Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators, LASR. We find that Rbfox interacts with LASR via its C-terminal domain (CTD) and this domain is essential for its splicing activity. In addition to LASR recruitment, a low complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.

Mechanisms and programs of neuron gene regulation byRbfox proteins

Douglas Black, PhDUniversity of California, Los Angeles

Keynote Speaker AbstractDouglas B lack , PhD

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An integrative view of all the complex interaction networks between mRNA, tRNA, and rRNA is described: the stability of codon-anticodon trimers, the conformation of the anticodon stem-loop of tRNA, the modified nucleotides, and the interactions with bases of rRNA at the decoding site. An information-rich, alternative representation of the codon table is derived. The new organization of the 64 codons is circular with an asymmetric distribution of codons that leads to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. The advantage of integrating data in this circular decoding system is that all tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism and anticodon. Within this new representation, the multiplicity and complexity of nucleotide modifications, especially at positions 34 and 37 of the anticodon loop, segregate meaningfully and correlate well with the necessity to stabilize AU-rich codon-anticodon pairs and to avoid miscoding in split codon boxes. Modifications of U34 are critical to decode purine-ending codons in split codon boxes. This allows for diversity in codon usage depending on genomic GC content as well as on the number and types of isoacceptor tRNAs. Although universal, the genetic code is not translated identically and several differences exist between organisms in the three kingdoms of life. To decipher diversely but efficiently the genetic code, cells developed sophisticated arrays between tRNA pools and tRNA modifications, anchored in the cellular metabolic enzymatic pathways and guaranteeing protein homeostasis. We suggest that, beyond the table of the first genetic code and the second genetic code made of the recognition rules between aminoacyl-tRNA synthetases and their tRNA substrates, these integrating metabolic networks constitute a third genetic code.

The third genetic code

Eric Westhof, PhDARN, IBMC-CNRS

University of Strasbourg, France

Keynote Speaker AbstractEr ic Westhoff, PhD

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Taking out the Trash: Tales of RNA Surveillance

Sandra Wolin, MD, PhDNational Cancer Institute

Keynote SpeakerSandra Wol in , PhD

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Capturing host proteins: Design of viral internal

ribosomal entry sites

Victoria D’Souza, PhDHarvard University

Invited SpeakerVictor ia D 'Souza , PhD

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Alternative Splicingand the Human Immune System

Kristen Lynch, PhDUniversity of Pennsylvania

Invited SpeakerKr isten Lynch , PhD

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Invited Speaker AbstractJeffery K ieft , PhD

Structured RNA elements within eukaryotic messenger RNAs regulate many processes through diverse mechanisms. These mechanisms include the ability to respond to diverse signals, change conformation, and communicate between different RNA elements to ultimately impact function and biology. To explore fundamental mechanisms of RNA structure, conformational change, and multi-domain communication, we are using the 3’ untranslated region (UTR) of the turnip yellow mosaic virus RNA. This UTR comprises two domains: the extreme 3’ end is the “tRNA Like Structure (TLS)” that drives aminoacylation of the viral genome, which in turn enhances translation of an upstream open reading frame. The second domain is the “Upstream Pseudoknot Domain (UPD)” which appears to stabilize the fold of the TLS and promote its function in unknown ways. Using functional assays, multi-dimensional chemical mapping, and biophysical approaches, we discovered that depending on the context of the UTR, ribosome-induced changes in the fold of the UPD affect the TLS, adjusting translation efficiency. Using x-ray crystallography to solve the structure of the entire two-domain viral 3’UTR, we revealed that the two structurally distinct domains use a spine of continuously stacked bases and an unusual strut-like linker to create a conduit for communication of conformational changes within the higher-order architecture. This reveals a mechanism by which structured RNA elements can sense the presence of RNA-interacting machinery through programmed conformational changes, then create a functional response by communicating that information to other RNA elements without direct structural interaction. We speculate that in the context of a viral RNA, the system may serve as an RNA-structure based rheostat to tune the rate of protein production in a ribosome-sensing feedback loop.

A ribosome-induced RNA conformational change

regulates translation: an RNA structure-based

rheostat

Jeffery Kieft, PhDUniversity of Colorado, Denver

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Invited Speaker AbstractRobert Sp ita le, PhD

The discovery of functional RNAs critical for normal and disease physiology continues to expand at a break-neck pace. Many RNA functions are controlled by the formation of specific structures; an understanding of each structural component is necessary to elucidate its function. Measuring solvent accessibility intracellularly with experimental ease is an unmet need in the field. Here, I will present a novel method for probing nucleobase solvent accessibility, Light Activated Structural Examination of RNA (LASER). LASER depends on light activation of a small molecule, nicotinoyl azide (NAz), to measure solvent accessibility of purine nucleobases. In vitro, this technique accurately monitors solvent accessibility and identifies rapid structural changes due to ligand binding in a metabolite-responsive RNA. LASER probing can further identify cellular RNA-protein interactions and unique intracellular RNA structures. Our photo-activation technique provides an adaptable framework to structurally characterize solvent accessibility of RNA in a myriad of environments.

Light-Activated Chemical Probing of Nucleobase Solvent AccessibilityInside Cells

Robert Spitale, PhDUniversity of California, Irvine

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Invited Speaker AbstractGabr ie le Varan i , PhD

The molecular basis for activity of long non coding RNAs and their structure-function relationship remain to be established. A commonly stated but unproven hypothesis is that their secondary structures are conserved and functional, despite low levels of primary sequence conservation. We have discovered a complex secondary structure in the functional core of Cyrano, a rare lincRNAs conserved over significant evolutionary distances, at the center of which is a strikingly conserved cloverleaf structure maintained over >420 million years of evolution. This structure provides protein interaction sites and is recognized by miR-7 in a non-canonical fashion. Structures within ncRNAs are functional, as we demonstrated for the promoter associated transcript controlling expression of the tumor suppressor E-cadherin, where a single SNP that alters RNA structure affects differential recruitment of epigenetic enzymes to the promoter and leads to different outcome in cancer patients. These RNA structures provide unexploited targets for intervention using peptidic and small molecule chemistry, as we illustrate with targeting of the microRNA precursor coding for the oncogenic miR-21.

Structure, evolution and targeting of long non

coding RNAs

Gabriele Varani, PhDDepartment of Chemistry University

of Washington

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Selected Talk AbstractEmi ly Rogers

Because of their simplicity, minimum free energy (MFE) predictions of RNA secondary structures are broadly used by many experimentalists. However, these methods are known by the computational community to be outperformed by methods that sample structures from the Boltzmann distribution. In particular, data mining a Boltzmann sample of a thousand structures yields substantial gains in accuracy, as well as a significantly more robust paradigm for analysis. We present our method of RNA profiling, which mines the structural information of a Boltzmann sample at the helical level. We demonstrate its superiority over MFE methods by discussing four major areas of application: 1) structure prediction accuracy; 2) structural pattern identification in regions; 3) quantifying of the conditioning of the underlying thermodynamic model; and 4) consensus structure prediction (and relatedly, multiple sequence alignment). Results from each of these four areas conclusively demonstrate the benefits of RNA profiling as both a prediction and analytical tool.

Profiling RNA secondary structures from a Boltzmann ensemble and its applications

Emily Rogers School of Computational Science and Engineering Georgia Institute of Technology

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NC RNA Stewardship Award Winner Talk AbstractPaul Agr is

Multidrug-resistant strains of numerous bacterial pathogens, including Streptococcus spp. and Staphylococcus aureus, necessitate the identification of new antibacterial agents exhibiting novel modes of action that preclude drug resistance. tRNA-dependent transcriptional regulation of the requisite genes for tRNA aminoacylation and amino acid biosynthesis in Gram-positive bacteria is an attractive target not found in the human host. A conserved, highly structured 5’-leader of mRNA, the ‘T-box’, binds unacylated tRNA to facilitate RNA polymerase read-through. Thus, inhibition of T-box function should result in bacterial cell death or growth arrest. In silico docking strategies yielded 200 small molecules as potential binders to the ‘Specifier Loop’ of the Bacillus subtilis’ tyrS T-box. A family of chemically related compounds selectively inhibited growth in a variety of Gram-positive bacteria and clinical isolates including methicillin-resistant S. aureus (minimum inhibitory concentrations, MIC, 15-64 µg/mL), but not Gram-negative bacteria, suggesting target specificity. An in vitro dissociation constant for compound binding to a truncated T-box was ~24 µM. Liquid and solid media assays indicated the development of resistance at an extremely low mutational frequency of 1.21 X 10-10. These compounds are as good or better than vancomycin in quenching Staphylococcus biofilms. In a mammalian membrane model system, compound transport occurred at a very slow rate reflecting this compound’s minimal cytotoxicity to mammalian cell culture, lack of toxicity when applied topically to an animal model, and demonstration of some wound healing. Structure activity relationship assays have revealed the core chemistry necessary for antibacterial activity. These data demonstrate the development of a new class of antibiotics against a novel target in Gram-positive pathogens that is refractory to the emergence of resistance.

Novel small molecule antibacterial

agents targeting tRNA regulation of

transcription in Gram-positive bacteria

Paul AgrisThe RNA Institute

University at Albany-SUNY

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Selected Talk AbstractCanan Kuscu

tRNA related RNA fragments (tRFs) are small RNAs as abundant as miRNAs and reported to be associated with Argonaute proteins, yet their function is unclear. We show that endogenous tRFs (18-22 bases) derived from 3’ end of tRNAs (tRF-3) repress genes. tRF-3 levels increase upon parental tRNA over-expression or tRNA induction by c-Myc. This represses target genes with a sequence complementary to the tRF-3 in the 3’ UTR. The tRF-3-mediated repression is Dicer-independent, Argonaute-dependent and the targets are recognized by 5’ seed sequence rules similar to miRNAs. Furthermore, tRF-3:target mRNA pairs in RISC associate with GW182 proteins, known to repress translation and promote the degradation of targets. RNA-seq demonstrates that endogenous target genes are specifically decreased upon tRF-3 induction. Therefore, Dicer-independent tRF-3s, generated upon tRNA upregulation such as c-Myc overexpression, regulate gene expression globally through Argonaute-GW182 containing RISC via seed sequence matches with target mRNAs.

Global Gene Repression by Dicer-independent tRNA fragments

Canan Kuscu Department of Biochemistryand Molecular Genetics University of Virginia

Kuscu C1, Kumar P1, Kiran M1, Su Z1, Malik A1, and Dutta A1

1 Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine

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Selected Talk AbstractShawn Lyons

As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they initiate several stress response pathways in order to protect the cells until transient stresses have passed. This is primarily accomplished by global translational arrest facilitated by phosphorylation of the translation initiation factor eIF2α, consequently, leading to the formation of non-membranous cytoplasmic bodies called stress granules (SGs). Functionally, SGs play diverse roles in cell adaptation to stress and promote cell survival and perturbations in SG dynamics are implicated in human diseases. However, translational attenuation is also moderated by non-eIF2α dependent mechanisms. Our lab has discovered one such mechanism that utilizes novel small non-coding RNAs that we have termed tRNA-derived stress-induced RNAs (tiRNAs). These are members of an ever-growing family of small (14 – 32 nucleotide) RNAs known as tRNA derived fragments (tRFs) that are produced through various different mechanisms, most of which have unknown function. We have identified two 5’-tiRNAs (5’-tiRNAAla and 5’-tiRNACys) that are potent inhibitors of protein synthesis. This ability requires a 5’ terminal oligoguanine (5’-TOG) motif. Here we show that this motif folds into parallel RNA G-quadruplex (G4) and that this structure, rather than solely 5’-TOG sequence, is required for bioactivity. By preventing the formation of G4 through ionic equilibration or through use of modified nucleic acids, we can prevent the activity of 5’TOG tiRNAs. Further, we demonstrate that the G4 structure is required for binding to YB-1, a multifunctional RNA binding protein with documented roles in translation regulation. Using CRISPR/Cas9 mediated deletions of YB-1 in tissue culture cells, we demonstrate that YB-1 is required for 5’-TOG containing tiRNA mediated SG formation, but is dispensable for translation repression activities. In vitro crosslinking studies and unbiased proteomic investigation of mRNP complexes in complex with 5’-tiRNAAla identified CNBP as a direct interacting partner of 5’tiRNAAla. Preliminary data suggests that interaction of CNBP with 5’-tiRNAAla is required for translation repression activity of 5’-tiRNAAla.

tiRNA-mediated translation repression

depends upon tetrameric G-quadruplex formation

Shawn Lyons Department of Medicine

Harvard Medical School / Brigham and Women’s Hospital

Shawn M. Lyons 1,2, Dorota Gudanis3, Zofia Gdaniec4, Pavel Ivanov 1,2,5,

Paul J. Anderson1,2

1 Department of Medicine, Harvard Medical School

2 Division of Rheumatology, Immunology and Allergy, Brigham and Women’s

Hospital

3 Institute of Bioorganic Chemistry, Polish Academy of Sciences

4 NanoBioMedical Centre, Adam Mickiewicz University

5 The Broad Institute of Harvard University and Massachusetts Institute of Technology

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Thermofisher Sponsored Speaker AbstractJim Giron

The branched DNA (bDNA) technology is a well-established and well–published signal amplification technology. Join us in a discussion to learn about Thermo Fisher Scientific’s portfolio of bDNA technology-based solutions for target gene expression studies in heterogeneous populations and at the single-cell level.The discussion will center around the QuantiGene Plex assay, which is designed to quantitatively measure up to 80 RNA transcripts directly from cell or tissue homogenates without RNA purification, or enzyme-dependent sequence amplification. The PrimeFlow RNA and ViewRNA assays will also be briefly introduced. PrimeFlow provides a unique tool to simultaneously investigate gene and protein expression at the single cell level using a flow cytometer while the View RNA assay allows RNA detection and localization at single-copy resolution, in cells and tissue sections

The QuantiGene® Platform:Unrivaled solutions to get you closer to true Biology

Jim Giron, MSApplication ScientistThermoFisher Science

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Selected Talk AbstractMonica P i l lon

Las1 is a recently discovered endoribonuclease that collaborates with Grc3-Rat1-Rai1 to process precursor ribosomal RNA (pre-rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here we report that the essential Las1 endoribonuclease displays weak RNase activity, however in the presence of the polynucleotide kinase Grc3, Las1 is reprogrammed for efficiently and specifically cleavage RNA. Our results establish that Grc3 specifically directs Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively towards single-stranded RNA. Together Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 crosstalk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/Las1 as a novel member of the RNaseL/Ire1 RNA Splicing Family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a new example of a protein guided programmable endoribonuclease.

Grc3 Activates the Essential

Endoribonuclease Las1 for Specific RNA

Cleavage

Monica Pillon Signal Transduction Laboratory

National Institute of Environmental Health Sciences

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Selected Talk AbstractKyle Mansfie ld

Proper response to nutrient deprivation is critical for cellular and organismal survival in both physiological and pathological situations. Our lab has had a long-standing interest in the cellular responses to these deprivations, particularly as it relates to posttranscriptional regulation of gene expression. Oxygen deprivation, or hypoxia, is a hallmark feature of many diseases such as heart attack, stroke, and also contributes to tumorigenesis. As such, understanding the cellular effects of hypoxia is critical to developing proper treatments and therapies for these diseases. While investigating the cellular posttranscriptional changes in response to hypoxia, we have discovered that upon exposure to 1% oxygen for 24 hours, HEK293T cells exhibit dramatic changes in the N6-Methyladenosine (m6A) content of their RNA. Isolation of cellular RNA fractions indicates that hypoxic exposure results in an increase in global messenger RNA (mRNA) m6A content. We have identified a number of hypoxia-related mRNAs, including GLUT1, MYC, and others, which exhibit specific increases in their m6A content upon exposure to hypoxic conditions. Interestingly, many of these same mRNAs also exhibit increased mRNA stability as well as continued association with heavy polysomes and faster recovery of translational efficiency following reoxygenation. We have also extended these studies to an in vitro model of breast cancer and show that the hypoxic increase in m6A is only seen in transformed, but not primary human mammary epithelial cells. Furthermore, increasing m6A levels by overexpression of the methyltransferases Mettl3 and 14 or hypoxic exposure increases the proliferation, migration, and invasion of transformed cells suggesting that m6A may play a key role in the acquisition and maintenance of a tumorigenic phenotype. We believe these changes in the cellular RNA epitranscriptome may represent an unexplored therapeutic target for the myriad of diseases that involve cellular hypoxia and are currently exploring the mechanisms underlying this phenomenon.

Role of the mRNA modification N6-methyladenosine in Hypoxia and Transformation

Kyle Mansfield Department of Biochemistry, Brody School of Medicine East Carolina University

Nate J. Fry, Kyle D. Mansfield*

Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University

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Selected Talk AbstractJackson Trotman

Cap homeostasis is a cyclical process of decapping and recapping that impacts a portion of the mRNA transcriptome. The metastable uncapped forms of recapping targets redistribute from polysomes to non-translating mRNPs, and recapping is all that is needed for their return to the translating pool. Previous work identified a cytoplasmic capping metabolon consisting of capping enzyme (CE) and a 5’-monophosphate kinase bound to adjacent domains of Nck1. The current study identifies the canonical cap methyltransferase (RNMT) as the enzyme responsible for guanine-N7 methylation of recapped mRNAs. RNMT binds directly to CE, and its presence in the cytoplasmic capping complex was demonstrated by pulldown assays, gel filtration, and proximity-dependent biotinylation. The latter also identified the RNMT cofactor RAM, whose presence is required for cytoplasmic cap methyltransferase activity. These findings guided developmentof an inhibitor of cytoplasmic cap methylation whose action resulted in a selective decrease in levels of recapped mRNAs.

RNA guanine-7 methyltransferase

catalyzes the methylation of cytoplasmically recapped RNAs

Jackson Trotman Center for RNA Biology

Ohio State University

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Selected Talk AbstractSujatha Jagannathan

The functional impact of protein-truncating genetic variants is often unpredictable because of the variable escape from RNA surveillance pathway, nonsense-mediated decay (NMD). The purpose of NMD is to identify and degrade messages that appear to encode truncated proteins. While efficient degradation of the protein-truncating variant causes loss of gene function, escape from NMD often leads to the production of a dominant, gain-of- function protein product. However, NMD efficiency is highly variable across genes, cell types and even individuals. Understanding the source of this variation is essential for accurate clinical interpretation of genetic variants, as well as understanding the tissue-tropism of certain cell type-specific diseases that involve NMD, such as facioscapulohumeral muscular dystrophy (FSHD). To understand how RNA surveillance is modulated by genetic and cellular determinants and how in turn, variable RNA surveillance influences cellular phenotypes, I have investigated mechanisms that modulate NMD in two experimental systems: A) Nonsense genetic variants that escape NMD and have risen to high frequency in the population. B) Loss of NMD efficiency in human muscle cells with FSHD.

By combining analysis of published genomic datasets, with reporter-based molecular validation, I identified mechanisms including alternative splicing, alternative translation initiation, stop codon read through, and C-terminal truncation that enable protein production from genes carrying high-frequency nonsense genetic variants. In the FSHD system, I have shown that NMD efficiency is modulated via the ubiquitin proteasome system, uncovering a novel mechanism by which NMD efficiency is tuned. These findings are important for the functional interpretation of genetic data, including non-pathogenic as well as disease-associated variation.

When to shoot the messenger: Identifying modulators of mRNA surveillance

Sujatha Jagannathan Computational BiologyPublic Health Sciences Division - Fred Hutchinson Cancer Research Center, Seattle

Sujatha Jagannathan1, 2, 3, Stephen J. Tapscott2 and Robert K. Bradley3

1 Computational Biology, PHS

2 Human Biology Division

3 Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA

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Selected Talk AbstractZhipeng Lu

RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Base pairing dominates the energetics of both RNA folding and RNA-RNA interactions. However, there are no generally applicable methods for the determination of base pairing interactions in living cells. Here we develop PARIS (Psoralen Analysis of RNA Interactions by Sequencing), a method based on reversible psoralen-crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher order architectures, and RNA:RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on a single molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures, and discovered conserved long-range and alternative structures. These conserved long-range interactions and alternative structures are validated by SHAPE (Selective 2’Hydroxyl Acylation Profiling Experiments) -based flexibility measurement of RNAs in living cells and evolutionary conservation. Using PARIS, we also identified many novel RNA-RNA interactions with near base pair resolution. XIST, a lncRNA essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. Using a combination of PARIS and RNA-protein crosslinking methods, we found that the RNA structure domains are formed together with distinct sets of associated proteins into functional modules. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher order assembly of the key epigenetic silencing protein SPEN.PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome.

RNA Duplex Map in Living Cells Reveals Higher Order

Transcriptome Structure

Zhipeng Lu Department of Dermatology

Stanford University

Zhipeng Lu,1,9 Qiangfeng Cliff Zhang,1,2,9 Byron Lee,1 Ryan A. Flynn,1 Martin A. Smith,3,4

James T. Robinson,5 Chen Davidovich,6,7,8 Anne R. Gooding,6 Karen J. Goodrich,6 John S.

Mattick,3,4 Jill P. Mesirov,5 Thomas R. Cech,6 and Howard Y. Chang1,*

1 Center for Personal Dynamic Regulomes, Stanford University

2 Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua

University3 RNA Biology and Plasticity Group, Garvan

Institute of Medical Research4 St Vincent’s Clinical School, UNSW Medicine

5 Department of Medicine and Moores Cancer Center, University of California San

Diego6 HHMI and Department of Chemistry and

Biochemistry, University of Colorado7 Department of Biochemistry and Molecular

Biology, Biomedicine Discovery Institute8 EMBL Australia and the ARC Centre of

Excellence in Advanced Molecular Imaging9 Co-first author

28


Selected Talk AbstractReyad E lbarbary

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We found that functional miRNAs are degraded in human cells by the endonuclease Tudor-SN (TSN). In vitro, recombinant TSN initiated the decay of both protein-free and Argonaute 2–loaded miRNAs via endonucleolytic cleavage at CA and UA dinucleotides, preferentially at scissile bonds located more than five nucleotides away from miRNA ends. Cellular targets of TSN-mediated decay defined using microRNA sequencing followed this rule. Inhibiting TSN-mediated miRNA decay by CRISPR-Cas9 knockout of TSN inhibited cell cycle progression by up-regulating a cohort of miRNAs that down-regulates mRNAs that encode proteins critical for the G1-to-S phase transition. Our study indicates that targeting TSN nuclease activity could inhibit pathological cell proliferation.

Tudor-SN–mediated endonucleolytic decay of human cell microRNAs promotes G1/S phase transition

Reyad Elbarbary Orthopaedics and Rehabilitation Penn State University-College of Medicine

Elbarbary RA, Miyoshi K, Myers JR, Du P, Ashton JM, Tian B, Maquat LE, Tudor-SN- mediated endonucleolytic decay of human-cell microRNAs promotes G1-to-S phase transition, Science 2017; 356:859-62.

29


Selected Talk AbstractRoger A lexander

In 2013, the NIH Common Fund and National Center for Advancing Translational Sciences funded the Extracellular RNA Communication Consortium (ERCC), an in-depth examination of the newly recognized field of extracellular RNA (exRNA). It is now known that RNA are present outside of cells in a variety of forms, including bound by proteins and encapsulated in extracellular vesicles (EV). The main goals of the ERCC are to understand mechanisms of exRNA biogenesis and function and to find exRNAs that can be used as biomarkers of and therapies for disease. In this talk, after outlining what we know about the RNA profiles in different biofluids, I will briefly overview examples of consortium research in each of these areas. First, I will review work from Vanderbilt University on the role of the oncogene K-RAS in packaging Argonaute-2-bound microRNAs into secretory exosomes. Second, I will outline work at UCSD on the promise of exRNA biomarkers for glioblastoma, a devastating and difficult-to-biopsy brain cancer. Third, I will review work from the Mayo Clinic Florida showing that EVs from mesenchymal stem cells can rescue mice from otherwise lethal liver failure, and that Y-RNA-1 in the EVs plays a role. Finally, I will direct the audience to a poster on the exRNA Portal and exRNA Atlas, resources provided by the consortium for the research community to track developments in the field and to download, contribute, and analyze exRNA datasets.

Extracellular RNA Communication

Consortium Extracellular RNA Communication:

Mechanisms, Biomarkers, and Therapies

Roger Alexander Pacific Northwest

Diabetes Research Institute

30


Selected Talk AbstractLibby Sne l l

Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing – information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies’ sequencing technology is capable of electronically analysing a sample’s DNA directly, and in real-time. In this presentation, we demonstrate the ability of an array of nanopores to sequence RNA directly, and we show it applied to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analyzing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.

Highly parallel direct RNA sequencing on an array of nanopores

Libby Snell Applications Oxford Nanopore Technologies

31


Poster 1Kr ipa Ahu ja

The American Cancer Society reports that this year there will be an estimated 600, 920 deaths due to cancer in the United States. Current cancer research includes the use of biomarkers on the surface of cancer cells to distinguish the cancerous cells from normal body cells. Molecular cloning can enhance these biomarkers. Over the past thirty years, molecular cloning has progressed immensely. From digestion to plasmid insertion, the possibilities are endless. The AAV (Adeno Associated Virus) CXCL 12(C-X-C Motif Chemokine Ligand 12) is a Protein Coding gene that shows great promise with cloning and plasmid insertion. Our project aims to use this gene to bind tightly to biomarkers on the surface of cancer cells. However before this optimal binding can occur, it is essential to know more about the AAV CXCL 12 Gene itself. For this reason, our project includes multiple gel electrophoresis assays, plasmid insertion/digestion assays, and PCR purification. From the results of these assays, the efficacy of AAV CXCL 12 to bind to cancer biomarkers will become clear. In particular, the cloning assay for the AAV CXCL 12 gene holds great potential, as it is possible to clone extraneous DNA into a different host. If extraneous DNA can be cloned into a different host, then there is the possibility of that DNA binding to a biomarker on a cancer cell.

Molecular Cloning Involving the AAV

CXCL12 Gene

Kripa Ahuja Lineberger Comprehensive

Cancer Center University of North Carolina

at Chapel Hill

32


Poster 2Laura B isogno

Post-transcriptional events are crucial determinants of gene expression, the misregulation of which can result in disease. For example, cancer has traditionally been viewed as being driven by aberrant transcriptional regulation and signaling events, though, over the past several years, many RNA binding proteins and non-coding RNAs have emerged as critical players in tumor development. It is now recognized that regulation of post-transcriptional processes, such as mRNA stability and translation, robustly influence cancer-related gene expression patterns of proto-oncogenes, growth factors, cytokines, and cell cycle regulators. Here we present the application of Digestion-Optimized Ribonucleoprotein Immunoprecipitation coupled with RNA-sequencing (DO-RIP-seq) to identify and quantify transcriptome-wide binding sites for the RNA binding protein HuR during oncogenic Ras transformation of immortalized human mammary epithelial cells (HMECs). HuR quantitatively, but not qualitatively, changed association at individual mRNA binding sites during Ras transformation. Mechanistically, we identified a GU-rich secondary motif associated with a quantitative decrease in HuR binding during transformation, and our data suggest that HuR may cooperate with the CELF1 protein to positively regulate the translation of a subset of mRNAs and promote the oncogenic Ras induced EMT phenotype. Furthermore, HuR expression was necessary for the maintenance of cancer traits, including proliferation, anchorage independent growth, migration and invasion. RNA-sequencing of HuR CRISPR knockout cell lines demonstrated that our identified targets of HuR, as a group, decreased expression in response to HuR knockout. The size of this response correlated with both binding site strength and number of binding sites. Overall, HuR is necessary for the maintenance of tumorigenic phenotypes, DO-RIP-seq revealed that HuR targets are not gained or lost, but rather quantitatively changed during tumorigenesis, and these quantitative changes are reflected in the total transcriptome.

DO-RIP-seq identifies quantitative changes in HuR binding at specific sites during Ras transformation

Laura Bisogno Molecular Genetics & Microbiology Duke University

Laura Bisogno, Matt Friedersdorf and Jack Keene

33


Poster 3Mark Boerneke

RNA virus pathogenicity is regulated in significant part by complex higher-order RNA structures across their genomes. Structured RNA elements clearly play important roles in viral replication, protein synthesis, packaging, evasion of host immune factors, and the hijacking of host-cell machinery, but the full extent to which viral RNA genomes contain true higher-order structures whose functions are critical to viral processes is poorly understood. Dengue virus (DENV) is a serious mosquito-borne pathogen that threatens more than one-third of the world’s population, and there are currently no broadly effective vaccines or therapeutics. Here we use single-molecule correlated chemical probing to define well-determined secondary and tertiary structures across the RNA genome of DENV serotype 2. These higher order RNA structures involve more than one-third of nucleotides in the DENV genomic RNA and promote a compact global RNA genome architecture. Disruption of these higher order RNA structures significantly impairs DENV replication and infectivity. These novel RNA regulatory motifs in DENV may be exploited in the development of critically needed and broadly effective anti-dengue virus therapeutics and vaccination strategies.

Higher Order RNA Structures in the Dengue

Virus RNA Genome Regulate Replication and

Infectivity

Mark Boerneke Chemistry Department

University of North Carolinaat Chapel Hill

34


Poster 4Alexander Borodavka

Segmented RNA viruses, including influenza viruses and rotaviruses, are ubiquitous human, animal and plant pathogens. A major challenge in understanding their assembly is the combinatorial problem of a non-random selection of a full genomic set of distinct single-stranded (ss)RNAs. This process involves multiple, complex RNA-RNA and protein-RNA interactions, which to date have been obscured by non-specific binding and aggregation at concentrations approaching in vivo assembly conditions. To interrogate specific inter-segment interactions in rotaviruses, we employ two-color fluorescence cross-correlation spectroscopy (FCCS) for detecting stable RNA-RNA interactions taking place in complex RNA and protein mixtures. We show that binding of the rotavirus non-structural protein NSP2 to ssRNAs results in RNA conformational rearrangements conducive to forming stable contacts between RNA segments. To identify the sites of inter-segment interactions, we developed an RNA-RNA SELEX approach for mapping the RNA sequences mediating stable inter-molecular base-pairing between the interacting ssRNAs. Our findings elucidate the molecular basis underlying inter-segment interactions in rotaviruses, paving the way for studying genome packaging of other segmented RNA viruses. The integrated approach expands the arsenal of techniques much needed for delineating dynamic RNA-RNA interactions involved in the assembly of large ribonucleoprotein complexes.

Protein-assisted RNA folding mediates specific RNA-RNA genome segment interactions in segmented RNA viruses

Alexander Borodavka Astbury Centre forStructural Molecular Biology University of Leeds

Alexander Borodavka1,3*, Eric C Dykeman2, Waldemar Schrimpf3, Don C Lamb3

1 Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds

2 York Centre for Complex Systems Analysis and Departments of Mathematics and Biology, University of York

3 Department of Chemistry and Center for Nano Science, Ludwig Maximilian University of Munich

35


Poster 5Mauro Ca labrese

Long noncoding RNAs (lncRNAs) direct Polycomb Repressive Complexes (PRCs) to specific genomic regions but the mechanisms are unclear. We profiled H3K27me3, a proxy for PRC-targeted regions, in cells that express Xist, Kcnq1ot1 and Airn, three lncRNAs that require PRCs for repressive activity. Unexpectedly, H3K27me3 enrichment extended for megabases beyond previously defined target regions of Kcnq1ot1 and Airn. Extent of H3K27me3 enrichment correlated with pre-existing genome architecture and intra-nuclear distance to the lncRNA locus. Catalytic components of PRCs showed broad enrichment on lncRNA targeted alleles, but punctate enrichment at active promoters on untargeted alleles. Increasing Airn expression from 9 to 30 copies per cell increased H3K27me3 over a 14-megabase span. Our data indicate that megabase-scale control of PRC deposition is an inherent property of a class of lncRNAs, not only Xist, and that the repressive potency of such lncRNAs can be regulated by altering their abundance on chromatin.

Megabase-scale control of Polycomb deposition

by long noncoding RNAs

Mauro Calabrese Department of Pharmacology

and Lineberger Comprehensive Cancer Center

University of North Carolina at Chapel Hill

36


Poster 6Kaus ik Chakrabart i

New technological advances are providing transcriptome-wide insights into mechanisms of RNA processing. We have used high throughput RNA sequencing approaches to study global RNA folding at various processing events of mRNAs and noncoding RNAs in malaria pathogen Plasmodium falciparum to identify RNA structural folds that could be ultimately important for maturation of disease related transcripts. Malaria caused by Plasmodium falciparum is still the no.1 killer in parasitic diseases in the world. According to the World Health Organization (WHO), about 3.2 billion people are at risk of malaria infection and there were more than 200 million new cases of malaria in 2015. Unfortunately, Plasmodium falciparum has developed drug resistance to all currently used anti-malarials worldwide. We hope that insights from these global transcriptomics data will allow us to employ new biochemical strategies to define and target unique structural folds and protein–RNA interactions that will lay the groundwork to selectively target malaria.

Cracking the code of transcriptomics to study RNA processing in Malaria

Kausik Chakrabarti Biological Sciences University of North Carolinaat Charlotte

37


Poster 7Becky Chen

Background. FXR1 is one of two homologues of the X-linked FMR1 (fragile X mental retardation) gene that encodes for a RNA-binding protein. Mutations in FMR1 gene cause mental retardation in humans also known as the fragile X syndrome. Different FXR1 isoforms exist due to alternative splicing of four regions: an 87 nt insert within exon 12, the first 78 nt of exon 13 (alternative 3’ splice site), and the cassette exons 15 (81 nt) & 16 (92 nt) (Kirkpatrick et al Genomics 1999). Relative abundance of the splice variants depends on cell and tissue types. This balance is altered in myotonic dystrophy, facioscapulohumeral muscular dystrophy, and diabetes, suggesting that FXR1 splicing plays a functional role. In mice, FXR1 expression is essential for postnatal viability. FXR1 depletion disrupts internal muscle cell architecture, reduces muscle mass, and induces perinatal lethality likely due to cardiac or respiratory failure (Mientjes et al Hum Mol Genet 2004). No definitive explanations exist yet for the lethal phenotype. We hypothesize that FXR1 regulates striated muscle development through translational control of specialized cytoskeletal structures and that alternative splicing is one of the mechanisms involved.

Aims. This study has three main aims: a) investigate FXR1 role in translational control of cytoskeletal structures in muscle cell differentiation, b) describe how Fxr1 is regulated by alternative splicing during mouse development in a tissue specific manner and c) identify the functional consequences of Fxr1 splicing.

Results. By using reverse transcription PCR assays we show that Fxr1 exhibits significant alternative splicing changes during postnatal development and generates isoforms that vary from tissue to tissue. In particular, exons 15 &16 are gradually included between neonatal and adult stages in different skeletal muscle types and parts of the heart. We confirmed the expression of the diverse splice variants at the protein level in multiple tissues. Furthermore, FXR1 splicing is conserved between mouse and human striated muscle development. These mRNA transitions are reproduced during differentiation of murine C2C12 myoblast into myotubes and overall FXR1 depletion strongly impairs differentiation into myotubes. We are currently elucidating the mechanisms behind the strong phenotypes by measuring the expression levels of potential Fxr1 targets (Igf1, p21, Talin 2, etc). In addition, we are modulating splicing patterns using antisense oligonucleotides in C2C12 cells and we plan to deliver morpholinos into the flexor digitorum brevis muscles in adult mice to evaluate its impact on differentiation and cell architecture.

Conclusions. So far, our ongoing studies demonstrate: a) Fxr1 is required for C2C12 myoblast differentiation into myotubes, b) Fxr1 splicing is regulated in a developmental stage- and tissue-specific manner, c) Fxr1 depletion in undifferentiated stages upregulates the IGF1 and Sema7a transcripts which may implicate Fxr1 in the AKT and Map kinase pathways, and d) Fxr1 splicing regulation also occurs in humans, suggesting evolutionarily conserved functions that are muscle specific.

Understanding the contribution of the

Fragile X-related protein-1 (Fxr1)

to muscle cell differentiation and its

regulation by alternative splicing

Becky Chen Cell Biology and Physiology

University of North Carolinaat Chapel Hill Medical School

R. Eric Blue, Ennessa Curry*, Becky Chen*, Eunice Y Lee, Susan Ngo, Nichlas Engels,

Jimena Giudice

Department of Cell Biology & Physiology, University of North Carolina Chapel Hill

* denotes equal contribution and presenter authors

38


Poster 8Norman Ch iu

Mass spectrometry has proven to be an accurate tool for measuring biomolecules. For mass spectrometry of RNA, through the development of soft ionization techniques, the molecular ions of entire RNA molecule can be detected. However, with the high molecular mass of RNA molecules, the accuracy on using mass spectrometry to identify specific RNA biomarker is lowered, which is due to the presence of natural heavy carbon-13 isotopes in RNA. To address the issue on mass accuracy as well as the challenge on measuring isobaric RNA ions, our research group has developed a number of new tools for measuring structural isomers of microRNA (SimiR). As much as 55% of human mature microRNA are identified to be SimiR, which are defined as microRNA having the same nucleotide composition but different RNA sequences. We reported the biggest group of SimiR that could be found in the human body had as many as 13 different microRNA. Two bottom-up mass spectrometry methods for measuring microRNA were developed in our group. In the first case, an intrinsic mass signature of specific microRNA was created by using the enzymatic activity of RNase T1. In the second case, a simple yet compatible chemical method to partially hydrolyze microRNA was developed, which can be used to generate RNA sequencing ladders. In both cases, RNA was directly measured by using mass spectrometry without the use of any chromatography technique. To assist the analysis of mass spectrometric data collected from human microRNA samples, our group has recently developed a new software program called MicroRNA MultiTool, which can analyze both unmodified and modified microRNA.

Tools for Mass Spectrometric Measurements of Structural Isomers of MicroRNA

Norman Chiu Department of Chemistryand Biochemistry University of North Carolina at Greensboro

Norman H. Chiu, Joseph Mwangi, John Cui, and Dickson Wambua

Department of Chemistry and Biochemistry, University of North Carolina at Greensboro

39


Poster 9Chia-Ch ieh Chu

Antiretroviral combination therapies suppressing HIV-1 replication have transformed the fatal illness of AIDS into a long-term controllable disease. However, emergence of drug-resistant HIV-1 strains has raised the risks of transmission of the drug-resistant strains among individuals, and development of drugs targeting new replication steps is needed. Rev response element (RRE) is a highly conserved viral cis-acting RNA element which plays an essential role in promoting the nuclear export of unspliced or incompletely spliced intron-containing HIV-1 RNAs through cooperative Rev binding. Rev protein initially binds to a high affinity purine-rich site in RRE stem-loop IIB, and minor mutations in stem IIB have detrimental effects on viral RNA exportation and replication. In this study, we used NMR spectroscopy to characterize the various conformational states that are sampled by stem IIB in RREIIB and RREII three-way junction. Relaxation dispersion NMR data reveal the existence of low abundance and short-lived conformational excited states in RREIIB that reshuffle mispairs which may play critical roles in Rev recognition. RRE mutants stabilizing these excited states (ES traps) were designed to investigate their effects in the context of the Rev-RRE interaction, and these ES traps show significant inhibition to Rev binding in fluorescence and cell-based assay. These ESs will be the alternative targets for the future small molecule screening.

Uncovering Conformational Excited

States in HIV-1 Rev Response Element RNA as New Small Molecule

Targets

Chia-Chieh Chu Department of Biochemistry

Duke University

40


Poster 10Magdalena C ichewicz

We report a long non-coding RNA (lncRNA) MUNC that promote myogenesis, differentiation process of skeletal muscle myoblasts to myotubes. MUNC (MyoD Upstream NonCoding Regulatory Element) is encoded 5 kb upstream of the Transcription Start Site of MyoD, with its 5’ end overlapping with the cis-acting Distal Regulatory Region (DRR) of MyoD. MUNC is specifically expressed in skeletal muscle tissue. It exists as an unspliced and spliced isoform. In vitro knockdown of MUNC reduces myoblast differentiation, whereas its stable overexpression from a heterologous promoter increases endogenous MyoD, Myogenin and Myh3 mRNA. Knockout of MUNC (in the DRR-/- mice) also impairs differentiation of primary myoblasts in vitro. Using in vivo muscle regeneration model, we observed that knockdown of MUNC inhibits the regeneration of mice limb muscle. We also discovered a human homolog of MUNC, which is induced during differentiation of myoblasts and whose knockdown decreases differentiation. By overexpression of human sequence in murine cells we induced transcription of myogenic markers showing the cross-species conservation of MUNC’s function.

To test whether MUNC acts as a simple eRNA for MYOD gene, we overexpressed MUNC in MYOD-/- C2C12 cells. Surprisingly, MUNC overexpression in MYOD-/- C2C12 cells induces myogenic transcripts in the complete absence of MyoD protein. Genome wide analysis shows that while some of the MUNC-regulated gene expression is dependent on MyoD, there is a core set of genes that are regulated by MUNC, both upwards and downwards, independent of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Consistent with the idea that MUNC acts more like a lncRNA than a classic eRNA, there are at least two independent functional sites on the MUNC lncRNA, exon 1 being more active than exon 2, with very little activity from the intron. These results show that although MUNC is an eRNA, it regulates expression of many genes as a trans-acting lncRNA.

Long noncoding RNA MUNC involved in skeletal muscle differentiation

Magdalena Cichewicz Biochemistry and Molecular Genetics Department University of Virginia

41


Poster 11Mary C lay

The highly dynamic nature of RNA three-dimensional structures is critical to regulations of the diverse range of cellular processes including ribonucleoprotein complex formation, viral replication, gene expression, and catalysis. Advances in biophysical methods, including NMR, have exposed a variety of reoccurring motional modes in RNA including local motions of unpaired nucleotides on the ps-ns timescale, collective motions of helical domains at timescales slower than ns, transitions in secondary structure that range from localized base pair (bp) reshuffling on the μs-ms timescale, to slower (> ms) large-scale changes in secondary structure.

Resolving Slow Sugar Repuckering Dynamics

in RNA

Mary Clay Biochemsitry

Duke University

42


Poster 12Richard C layton

The advent of next generation sequencing combined with careful analysis of chimeric reads has identified ubiquitous circular RNAs. Their function, purpose, and evolutionary origins however, remain largely unknown. One model for Circular RNA formation is back splicing, and it is generally believed that this requires the two introns to have complimentary base pairs, causing them to pair and facilitate back splicing. Interestingly, circular RNAs are found in every mammal's genome; one question is whether they co-evolved, which would suggest conserved function. Since there is so much that is unknown about circular RNAs, there is a need to create new methods to quickly parse data currently available data and make sense of it. We used BED files that contain data for specific coordinates in related mammalian genomes in to identify conserved regions in where complimentary repeat elements capable of base-pairing occur. We have developed computational tools and code that can take the data from these BED files and arrange them so that it shows how circular RNAs in the human and mouse genomes are related. The code will be developed using python, LINUX and the bedtools library.

An analysis of conserved intronic repeat elements in back splicing leading to circularization

Richard ClaytonBiological & Genome SciencesUniversity of North Carolina at Chapel Hill

Richard C. Clayton, Alain Laederach, Bill Marzluff


43


Poster 13Aazt l i Cor ia

SAMMSON, also known as survival associated mitochondrial melanoma-specific oncogenic noncoding RNA, is a long-noncoding RNA (lncRNA) expressed exclusively in malignant melanoma cell lines and metastatic melanoma tissue samples. Upon depletion of SAMMSON, melanoma cells undergo apoptosis, suggesting this lncRNA is essential for melanoma cell survival. It is crucial to find a cure for this cancer; the incidence rate of melanoma has doubled since the 1970’s. SAMMSON presents an attractive target for its therapeutic potential but very little is known about SAMMSON. What is known is that SAMMSON interacts with p32, a protein necessary for proper mitochondrial maintenance. In order to pursue the potential therapeutic properties of SAMMSON it is necessary to understand the secondary structural ensembles SAMMSON adopts in vivo and its interaction with p32.

The nascent field of lncRNA biology has already shown that lncRNAs are implicated in a multitude of cellular processes, but the structures of very few lncRNAs are known. Of the known lncRNA structures, it has often been observed that the secondary structure is essential for the RNA to carry out its function. Knowing that structure and function are linked in other lncRNAs, we will model the unique structural architecture of SAMMSON and use biochemical assays to uncover the necessary features for SAMMSON: p32 interaction.

Elucidating the Functional and Structural

Domains of the LncRNA SAMMSON

Aaztli Coria Biology Department


44


Poster 14Anita Don l ic

Long non-coding RNAs (lncRNAs) are functional yet non-protein-coding RNAs that have crucial roles in many oncogenic processes. Characterization of select transcripts has provided insights into intricate tertiary structures that are crucial for their function. For example, the 3’-terminal RNA triple helix in the lncRNA MALAT1 is found to be pivotal in stabilization and nuclear retention of this transcript, which is proposed to lead to an increase in cancer- associated processes. To provide tools for disrupting this triple helix and studying its role in cancer progression, we first rationally designed and synthesized a 33-member library of small molecule probes based on a diphenylfuran scaffold (DPF). In addition, we are developing a screening platforms for quick identification of ligands that bind and destabilize the MALAT1 triple helix. Preliminary screening results indicate that current library members have a range of low- to sub-micromolar affinities to the MALAT1 stem loop and are selective for this construct over tRNA, DNA, and other stem loop constructs. Immediate future work includes biophysical characterization of these interactions, their effect on the triple helix stability, and cell-based studies. The development of chemical probes for the MALAT1 triple helix will provide valuable insight into the occurrence and function of this unique triple helix and its role in several human cancers.

Development of chemical probes and screening strategies to target an oncogenic RNA triple helix

Anita Donlic Chemistry Duke University

45


Poster 15Nich las Enge ls

Alternative splicing is a posttranscriptional mechanism that produces multiple protein isoforms from a single gene. Trafficking genes, such as, Clathrin Heavy Chain (Cltc) and Clathrin Light Chain A (Clta) employ this mechanism to regulate splice variants specifically in striated muscle1. Cltc and Clta work in conjunction in the cell to mediate endosomal sorting and endocytosis2. The Cltc gene has a splice variant that includes a 21-nucleotides (nt) exon near the C-terminus of the protein. Likewise, the Clta gene contains two alternatively spliced exons (36 nt and 54 nt) located between the Cltc binding region and the calmodulin- binding domain2. Our aim is to identify the physiological roles of the regulation of these exons by alternative splicing in a developmental-stage and tissue-specific manner. Using CRISPR/Cas9 technologies, we can address this question by blocking fetal-to-adult splicing transitions in a mouse model. We generated mice where the alternative spliced exons are deleted at the genomic level. Thus, homozygous mice can only express the fetal isoforms, while their wild type littermates gradually include the alternatively spliced exons (Giudice, Koushiuk, Blue, Cooper, in prep). We first characterized the splicing patterns of these Clathrin specific exons during development in different tissues including heart, various skeletal muscles, liver, brain, pancreas, intestine, spleen, lung, and kidney (unpublished data). These experiments revealed that Cltc (exon 21nt) was spliced out during neonatal stages and included in adulthood specifically in heart, skeletal muscles, and brain. Additionally, our data showed that inclusion of Clta (exon 54nt) is brain specific, whereas Clta (exon 36nt) is gradually included during development of the skeletal muscles, heart, and brain. At the physiological level our first observations indicate that Cltc CRISPR homozygous mice exhibit protection to cardiac hypertrophy induced by transverse aortic constriction, while also showing a slight increase in muscle weights in comparison with their wild type littermates. This difference in muscle weight may be associated with the increase in the myofiber area isolated from the flexor digitorum brevis (FDB) muscles of homozygous vs wild type littermates. We intend to fully characterize these animals in terms of their physiology. We also aim to explore the cell and molecular biology angle of this project because it will allow us to learn new aspects of Cltc and Clta functions (and the different splice variants) in muscle and neuronal contexts.

1. Giudice, J. et al. Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development. Nature Communication 5, 3603. doi: 10.1038/ncomms4603. (2014).2. Brodsky, F.M. Diversity of Clathrin Function: New Tricks for an Old Protein. Annual Review of Cell and Developmental Biology 28(1), 309-336 (2012).

Studying the functions of alternative splicing regulation of Clathrin

Heavy Chain (Cltc) and Clathrin Light Chain

A (Clta) in muscle development using

CRISPR mice

Nichlas Engels Cell Biology and Physiology


Nichlas Engels*1, R. Eric Blue*1, Eunice Y Lee1, Jimena Giudice1,2

1. Department of Cell Biology & Physiology, School of Medicine, University

of North Carolina at Chapel Hill.

2. McAllister Heart Institute, University of North Carolina at Chapel Hill

*equal contribution and presenter authors

46


Poster 16Shannon Farr is

Many neuronal functions, including memory storage, are thought to require de novo transcription and translation. In neurons, local regulation of RNA affords tight spatial and temporal control over gene expression so that in response to neuronal activity or injury, proteins can be rapidly synthesized to modify neuronal connections that are often hundreds of microns away from the cell body. However, the molecular mechanisms regulating RNA in specific cell-types in vivo are lacking. Furthermore, how RNA regulation may be contributing to cell-type specific functions is completely unknown.

In general, hippocampal neurons are extremely vulnerable to seizure, ischemic insult and trauma. However, CA2 neurons are known to be impervious to these insults in both humans and in animal models. Gene ontology analyses of RNA-Seq data comparing CA2 neurons to the susceptible neighboring neurons in CA1, CA3 and DG revealed that genes related to mitochondrial function are enriched in area CA2. Given the well-established link between mitochondria and programmed cell death, we hypothesized that CA2 neurons have robust mitochondrial function that renders them resistant to cell death relative to the neurons in neighboring subregions.

Specifically, we discovered that CA2 neurons have the highest levels of mitochondrial RNA compared to neighboring subregions, suggesting that CA2 neurons have either more mitochondria or greater levels of mitochondrial transcription. Indeed, we found that CA2 neurons have a greater mitochondrial DNA (mtDNA) copy number than neighboring neurons, indicating that CA2 neurons have more mitochondria. Moreover, nuclear-encoded mitochondrial genes including the mitochondrial calcium uniporter, Mcu, and its regulatory proteins, Mcur1 and Micu1, are also enriched in CA2, strengthening the case that mitochondria function, and in particular mitochondrial calcium signaling or sequestration, is enhanced in CA2 relative to neighboring subregions. These data provide a possible mechanism for previous results from our lab showing faster calcium extrusion rates in CA2 neurons compared with those in CA1. Lastly, we found that CA2 neurons have higher dihydroethidium (DHE) labeling, consistent with higher levels of superoxide production likely stemming from increased mitochondrial respiration. Future studies assessing the role of mitochondria in CA2 after injury may identify novel strategies to promote central nervous system (CNS) repair.

Transcriptome Profiling of Hippocampal Subregions Reveals a Role for Mitochondria in CA2 Physiology and Function

Shannon Farris Neurobiology Laboratory NIEHS/NIH

47


Poster 17Nate Fry

The mRNA modification, N6-methyladenosine (m6A), has recently been shown to be involved in many post-transcriptional regulation processes including mRNA stability and translational efficiency. While understanding these mechanisms are useful, it is also imperative to correlate these processes with phenotypic outputs. Here we report that m6A levels are decreased in genetically defined immortalized and oncogenically transformed human mammary epithelial cells (HMECs) as compared with their primary cell predecessor. Interestingly, after 24 hours of hypoxic exposure, m6A levels in the immortal and transformed cells were increased back to primary cell levels. It was also found that the m6A methyltransferase, Mettl3, is decreased and the demethylase, Alkbh5, is increased in the immortalized and transformed cell lines, possibly explaining the decrease in m6A in those cells. However, changes in Mettl3 and Alkbh5 do not explain the increase in m6A in hypoxia. m6A levels appear increased in hypoxic conditions not because of changes in methyltransferase or demethylase levels, but rather because of a build-up of stabilized m6A mRNA. At first glance, it would appear that transformation of the HMECs reduces m6A levels possibly to obtain a more progressive phenotype. In actuality, increasing m6A levels through overexpression of Mettl3 and Mettl14 increased proliferation, migration, and invasion of the transformed cells. Remarkably, overexpression of Mettl3 and Mettl14 had little effect on the immortalized cells, suggesting that the m6A modification may regulate migration and invasion differently in cells depending on their oncogenic potential.

The role of N6-methyladenosine in hypoxia and cellular

transformation

Nate Fry Biochemistry and Molecular Biology

Brody School of MedicineEast Carolina University

48


Poster 18Laura Ganser

Dynamic ensembles that capture the flexibility of RNA three-dimensional structures hold great promise in advancing RNA-targeted drug discovery. Here, we experimentally screened the transactivation response element (TAR) RNA from human immunodeficiency virus type-1 (HIV-1) against ~100,000 small molecules. We used this dataset, along with 170 previously reported TAR binders, to evaluate virtual screening (VS) against a high-resolution TAR ensemble determined by combining NMR spectroscopy and molecular dynamics simulations. Ensemble-based VS (EBVS) robustly enriches for true hits with an area under the receiver operator characteristic curve (ROC AUC) between ~0.80-0.95 and with ~20-67% of all hits falling within the top 2% of scored molecules, depending on the selection criteria used to define hits and non-hits. EBVS also predicts binding modes similar to bound NMR structures for 5 out of six molecules. Finally, the prediction accuracy decreased significantly with decreasing accuracy of the target ensemble or when docking against a single RNA structure. These results demonstrate that experimentally determined ensembles can significantly enrich libraries with structure-specific RNA binders as well as motivate the continued development of methods for improving ensemble accuracy.

High Performance Virtual Screening by Targeting a High-resolution RNA Dynamic Ensemble

Laura Ganser Biochemistry Department Duke University Medical Center

Laura R. Ganser1, Janghyun Lee2, Atul Rangadurai1, Aman D. Kansal1, Dawn K. Merriman3, Bharathwaj Sathyamoorthy1, and Hashim M. Al-Hashimi*1,3

1 Department of Biochemistry, Duke University School of Medicine

2 Department of Chemistry, University of Michigan

3 Department of Chemistry, Duke University

49


Poster 19Nandan Gokha le

The RNA modification N6-methyladenosine (m6A) plays an important role in the post-transcriptional regulation of eukaryotic mRNA and dynamically modulates important biological processes. We have found that m6A also regulates infection by RNA viruses in the Flaviviridae family such as hepatitis C virus (HCV). Depletion of the m6A-methyltransferases METTL3 and METTL14 increased HCV particle production, while depletion of the m6A-demethylase FTO had the opposite result, without affecting HCV RNA replication. The m6A-binding YTHDF proteins, which are the effectors of m6A function, relocalized to subcellular sites of HCV particle assembly at lipid droplets, and their depletion also led to increased viral particle production. We mapped the sites of m6A modification at multiple regions on the HCV RNA genome. Mutating an m6A site to prevent methylation increased viral titer without impacting RNA replication by regulating viral RNA-protein interactions involved in particle production. We have also mapped m6A on the RNA genomes of several other important Flaviviridae members including dengue virus, Zika virus, yellow fever virus, and West Nile virus and identified conserved regions of the viral genomes modified by m6A. Together, our work provides the first example of cytoplasmic RNA modified by m6A and reveals that m6A is a conserved regulatory mark on the RNA genomes of viruses with the Flaviviridae family. Interestingly, our work reveals that RNA virus infection also induces broad changes in the m6A “epitranscriptome” of host mRNAs, likely controlling the translation or stability of the affected transcripts, suggesting that m6A is an additional layer of post-transcriptional regulation of host gene expression following viral infection.

RNA modifications go viral!

Nandan Gokhale Department of Molecular Genetics

and Microbiology Duke University

50


Poster 20Kevin Gos len

Las1 is a recently identified endoribonuclease essential for processing eukaryotic precursor ribosomal RNA (pre-rRNA). The polycistronic pre-rRNA contains spacer regions (5’ ETS, ITS1, ITS2, 3’ ETS) that must be removed to liberate the functional 18S, 5.8S and 25S rRNA. Las1 catalyzes the cleavage of the C2 site to initiate degradation of the ITS2. Cleavage is achieved through the action of the catalytic Higher Eukaryote and Prokaryote Nucleotide binding domain (HEPN) of Las1, a metal-independent nuclease domain found in a variety of RNA-binding and cutting enzymes. Catalytic HEPN domains harbor a conserved Rx4-6H motif (where x is any amino acid) responsible for catalysis. Aside from the invariable arginine and histidine residues, the intervening sequence differs across different HEPN nucleases. Although variable, these intervening residues are critical for function, as replacing the motif with the HEPN motif from other nucleases eliminates catalytic activity. It is currently unclear how each Las1 HEPN motif residue contributes to substrate specificity and catalysis. To address this gap, we aimed to characterize the active site motif of the HEPN domain from Saccharomyces cerevisiae Las1 by in vivo and biochemical studies. Residues in the S. cerevsiae Las1 HEPN motif were targeted for mutagenesis. Las1 mutants were expressed from ARS1-CEN4 YCplac vectors transformed into a tet-off strain to repress endogenous LAS1 expression. Las1 mutants caused a severe defect in cell proliferation, suggesting the essential Las1 nuclease was compromised. Las1 forms a stable tetramer with its binding partner, Grc3, which is required for Las1 nuclease activity. Stability of the complex formed between Las1 mutants and Grc3 was measured by gel filtration. To verify whether these Las1 mutations had an effect on nuclease function, an ITS2 RNA substrate mimic was incubated with recombinant Las1 variants. Using sucrose gradients to profile ribosome production in vivo, we also show that the Las1 HEPN motif is essential for ribosomal assembly. Additionally, Northern blots were performed to reveal mutants’ effects on pre-rRNA processing. These data identify the importance of the Rx4-6H HEPN active site motif, which is critical for Las1’s function in pre-rRNA processing. Together, our work offers new insight into the role of the HEPN motif of the essential endoribonuclease Las1.

Characterization of the Essential Las1 HEPN Nuclease in Ribosomal RNA Processing

Kevin Goslen Signal Transduction Laboratory National Institute of Environmental Health Sciences

Kevin H. Goslen, Monica C. Pillon, Mack Sobhany, Robin E. Stanley

51


Poster 21Kelsey Gray

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by homozygous loss-of-function mutations in the human survival motor neuron 1 (SMN1) gene, which leads to reduced levels of functional survival motor neuron (SMN) protein. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of SMN isoforms are poorly understood.

We generated transgenic Drosophila melanogaster that express only flag-tagged wild-type SMN using the endogenous promoter. We collected embryos from these animals and analyzed Flag-purified lysates by ‘label-free’ mass spectrometry. We identified Flag-SMN, along with other core SMN complex components such as the Sm proteins and the Gemins.

In addition, we identified the SCF-Slmb ubiquitin E3 ligase complex as a novel SMN binding partner. Each component: supernumerary limbs (Slmb), SkpA, and Cullin 1 was highly enriched (at least 10 fold) in Flag-SMN samples as compared to the control sample. SCF-Slmb interacts with a phospho-degron embedded within the human and fruitfly SMN YG-box oligomerization domain. Substitution of a conserved serine (S270A) interferes with SCF-Slmb binding and stabilizes SMNΔ7. Proteins containing SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

SCF-Slmb mediates degradation of Survival

Motor Neuron (SMN) protein

Kelsey Gray Integrative Program for Biological

& Genome Sciences (iBGS) University of North Carolina

at Chapel Hill

52


Poster 22Tor in Greenwood

An RNA sequence is typically predicted to fold to a single minimum free energy conformation. But, an increasing number of RNA molecules are now known to fold into multiple stable structures. Adding experimental data as auxiliary information into structure prediction methods improves accuracy when there is one dominant conformation. However, adapting the methods for multimodal structural distributions is challenging. In this work, we analyze how existing structure prediction models would behave in the presence of auxiliary data derived from multimodal structural distributions, and we discover several shortcomings. Additionally, we identify how experimental data must be improved to deconvolve multimodal signals, by analyzing experimentally-derived data distributions with a statistical framework.

Using Experimental Data to Deconvolve Structural Signals

Torin Greenwood School of Mathematics Georgia Institute of Technology

53


Poster 23Hsiang-Ting Ho

C/D box small nucleolar RNAs (snoRNA) are a multifunctional family of non-coding RNA that play a critical role in guiding 2'-O-methylation of ribosomal RNA (rRNA). In addition to their rRNA targets, current work from our laboratory suggests that snoRNAs of the Rpl13a locus (Rpl13a snoRNAs) direct methylation of distinct mRNA species, further regulating protein translation in vitro and in vivo. Interestingly, we discovered that germline knockout of four C/D box Rpl13a snoRNAs (U32a, U33, U34 and U35a) in adolescent mice produced developmentally smaller hearts. Similarly in H9C2 rat cardiomyoblasts, locked nucleic acid (LNA) antisense oligonucleotide knockdown of Rpl13a snoRNAs significantly reduced H9C2 cell size. Based on the findings, we hypothesized that Rpl13a snoRNAs may influence cardiac cell size through 2'-O-methylation. Interestingly, both mRNA and protein expression of a central regulator of cellular growth were significantly reduced in hearts from Rpl13a snoRNA knockout mice. Further, LNA antisense knockdown of Rpl13a snoRNAs in H9C2 cardiomyoblasts also significantly reduced mRNA expression of this particular regulator. Taken together, these results suggest that Rpl13a snoRNAs regulate cardiac cell size mediated through a central cell growth pathway.

Regulation of cell size by Rpl13a snoRNAs

Hsiang-Ting Ho Department of Medicine

Duke University Medical Center

54


Poster 24Isabe l Hoerr

Alternative splicing is a post-transcriptional RNA processing mechanism that allows one gene to code for multiple protein isoforms. Over 90% of human genes undergo alternative splicing, allowing for extensive protein diversity from a limited number of genes. Coordinated transcriptional and posttranscriptional networks regulate organ development and alternative splicing plays a role in developmental processes. The heart, skeletal muscle, and brain are the organs that have the most tissue-specific and evolutionarily conserved alternative splicing. This brings us to question how developmental and tissue-specific splicing affects protein function and physiology. We have previously found that during heart development, membrane trafficking genes undergo alternative splicing transitions. We found that trafficking genes are mis-spliced in heart failure in mice and we observed that the alternative exons are progressively included not only in the heart but also in different types of skeletal muscles. In contrast, most of these developmental changes do not occur in other tissues. This evidence demonstrates that splicing regulation of membrane trafficking genes is striated muscle specific, suggesting functional roles associated with the functions of these contractile tissues. We confirmed that the mRNA splicing transitions are translated into the expression of different protein variants during heart and skeletal muscle development.

Understanding the interplay between splicing and trafficking at the cell biology and mechanistic level is the knowledge gap inspiring our new research. We hypothesize that alternative splicing mechanisms generate specific trafficking isoforms that contribute to the biogenesis and maintenance of cell architecture and are important for cell type –specific functions.

We are now aiming to identify the roles of each of four trafficking splicing events (Trip10, Snap23, Cltc, Tmed2). Depletion of specific trafficking proteins dramatically reduces cell viability and differentiation, substantiating their importance. Furthermore, blocking splicing transitions by morpholinos in Trip10 (Cdc42- interacting protein-2) or Snap23 (SNARE-complex member) genes increased myoblast fusion and produced myotube hypertrophy, which suggests that precise splicing contributes to normal myotube formation.

Our results reveal previously unidentified roles of trafficking proteins in muscle homeostasis and intracellular architecture and highlight specific prerequisites for adult splice variants.

Roles of trafficking proteins in muscle homeostasis and myofiber structure

Isabel Hoerr Cell Biology and Physiology University of North Carolina at Chapel Hill Medical School

Blue RE1, Engels N1, Hoerr I1,Harrison J1, Lee EY1, Giudice J1,2

1. Department of Cell Biology & Physiology, School of Medicine, University of North Carolina at Chapel Hill

2. McAllister Heart Institute, University of North Carolina at Chapel Hill

55


Macromolecular evolution in the crowded intracellular environment has given rise to the existence of weak but important protein interaction networks. For membranes, the added two-dimensional constraint decreases the affinity required to maintain stable protein contact leading to the development of weakly-interacting, multicomponent complexes where overall protein proximity, not just immediate interactions, are functionally important. Key examples include cytoskeletal assembly of signaling proteins, synaptic vesicle assembly and protein processing on the rough endoplasmic reticulum (RER). The last topic has become controversial with the emergence of data demonstrating many processes including mRNA silencing, autophagosome assembly and global translation of the transcriptome, occur on this single ER domain. To determine the mechanisms by which global mRNA translation localizes to the ER, more must be known about the subcellular networks creating the RER. Recently developed proximity labeling techniques now provide an experimental methodology for extended mapping of the ER. The following experiments use BioID as a method to determine macromolecular organization of the RER using several proteins that have been validated as RER residents by their association with ribosomes. Mass spectrometry results show each protein exists in a distinct micro-environment supporting the hypothesis that organization of the ER extends from the ultrastructure to the smaller protein components. Current experiments are focused on identifying the translational fingerprints of different ribosome-linked domains.

Poster 25Alyson Hoffman

Neighborhoods of the Rough Er By Street View:

Using Bioid to Discover the Translational

Landscape of a Multitasking Membrane

Environment

Alyson Hoffman Biochemistry

Duke University

Hoffman AM1, Nicchitta CV1,2*

Departments of Biochemistry1 and Cell Biology2, Duke University Medical Center

56


Poster 26Chr istopher Ho lmquist

Cells must synthesize large amounts of histone during S-phase to package newly replicated DNA. Cells must coordinate histone protein synthesis with DNA replication, and limit histone synthesis to S-phase. For mammalian cells, much of this histone protein regulation is post-transcriptional, via pre-mRNA processing and regulation of histone mRNA half-life. Histone mRNAs differ from all other cellular mRNAs, since they don’t end in polyA tail. In metazoans, processed histone mRNAs end in a unique stem-loop structure, rather than polyA tail. A single protein, stem-loop binding protein, binds the stem loop and participates in all steps of histone mRNA metabolism. A major regulatory step is regulation of histone mRNA half-life. Histone mRNAs are rapidly degraded at the end of S-phase or when DNA is inhibited, and the half-life is mediated by the stem-loop at the 3’ terminus. A second protein, the 3’ to 5’ exonuclease, 3’hExo, binds the 3’ end of histone mRNA together with SLBP. 3’hExo trims the histone mRNA formed by processing, removing 2 nts and leaving a 3 nt tail after the stem loop. Our lab has found the uridylation of histone mRNA 3’ termini is essential for maintaining proper length and for rapid degradation of the mRNA during S-phase (1,2), and Hoefig et al (3) showed that 3’hExo is necessary for rapid degradation of histone mRNAs. To determine which terminal uridyl transferase is required I used CRISPR-Cas9 genome editing to knockout both a TUTase and 3’hExo. KO’s of either TUT7 or 3’hExo prevented rapid degradation of histone mRNA when DNA replication is inhibited in S-phase. Using high-throughput sequencing, we previously defined the major degradation intermediates as uridylations that occur on RNAs partially degraded into the stem by 3’ hExo. Knockout of 3’hExo prevented formation of these intermediates and very little histone mRNA was degraded in 60 minutes after blocking DNA replication. In addition the histone mRNAs in S-phase were longer than in control cells, and many contained short uridine tails. In contrast, in the TUT7 knockout, there was degradation into the stem by 3’hExo, but no uridylation, and these intermediates accumulated in large amounts, and histone mRNA was only slowly degraded. In contrast KD of the other major uridyl transferase, TUT4, had no effect on histone mRNA degradation. We conclude that TUT7 likely interacts specifically with the 3’ end of histone mRNP, either with SLBP or 3’hExo, to mediate histone mRNA degradation.

1. Mullen, T.E. and Marzluff, W.F. 2008. Degradation of histone mRNA requires oligouridylation followed by decapping and simultaneous degradation of the mRNA both 5’ to 3’ and 3’ to 5’. Genes & Development. 22, 1 (2008), 50–65.

2. Lackey, P.E. et al. 2016. TUT7 catalyzes the uridylation of the 3’ end for rapid degradation of histone mRNA. RNA (New York, N.Y.). (2016), 1–16.

3. Hoefig, K.P. et al. 2013. Eri1 degrades the stem-loop of oligouridylated histone mRNAs to induce replication-dependent decay. Nature structural & molecular biology. 20, 1 (2013), 73–81.

CRISPR Knockout studies show that the Uridiyl Transferase TUTase 7 and the 3’ to 5’ Exonuclease 3’hExo are both required for Histone mRNA Degradation

Christopher Holmquist Eshelman School of Pharmacy, Division of CBMC University of North Carolina at Chapel Hill

Holmquist, C.1, Marzluff, W.F.1,2

1 Eshelman School of Pharmacy CBMC, University of North Carolina at Chapel Hill

2 Dept. of Biochemistry and Biophysics, University of North Carolina at Chapel Hill

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Poster 27Maryam Hosse in i

Mammalian mitochondrial (mmt) ribosomes evolved from ancestral bacterial ribosomes by large-scale reduction of ribosomal RNAs (rRNAs), loss of guanosine (from 31% to 18% G) and increase of ribosomal protein (rProtein) content. Mmt ribosomes translate just 13 polypeptides of the electron transport chain (ETC); they are exposed to reactive oxygen species (ROS) generated by the ETC, and appear to turn over every 4-5 hours, at a rate ~24 times faster than for cytosolic ribosomes.1 Therefore, they are subject to strong selection pressure favoring structural changes that reduce or limit oxidative damage. Consistent with this, we find that far fewer Gs in mmt are significantly exposed to solvent than in bacteria. Only the functionally most indispensable Gs, those that interact directly with substrates, are retained at sites significantly exposed to solvent. We will present results of detailed structure-correlated analysis of high-quality rRNA sequence alignments.2 illustrating how mmt ribosomes effectively deploy a relatively small number of Gs to stabilize the secondary and tertiary rRNA structure and ensure correct folding, while minimizing solvent exposure of Gs.

1. Gelfand, R. and Attardi, G. (1981) “Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable.” Mol Cell Biol. 1981; 1(6): 497–511.

2. Cannone, J.J., Sweeney, B.A., Petrov, A.I., Gutell, R.R., Zirbel, C.L., and Leontis, N. (2015) “R3D-2-MSA: the RNA 3D structure-to-multiple sequence alignment server.” Nucleic Acids Res. 43(W1):W15-23.

The evolutionary path of the mammalian

mitochondrial ribosome: How to fold a functional

RNA with fewer Guanines

Maryam Hosseini Chemistry

Bowling Green State University

58


Poster 28Megan Ke l ly

Noncoding RNAs (ncRNAs) have been implicated in virtually every type of human disease, and have been found to be essential in many important human pathogens. However, targeting these RNA structures with traditional small molecule drug development approaches has proven exceptionally challenging. RNA-based therapeutics such as anti-sense oligonucleotides has proven difficult due to delivery limitations and inadequate target selectivity. An alternative approach is using cell-permeable small molecules that bypass delivery limitations. However, due to their unique structural properties and mechanisms of action, there has been little success in applying the conventional protein drug discovery paradigm to ncRNAs. Compound libraries used in high throughput screens are biased to small molecules that optimally bind to the deep and hydrophobic pockets in proteins; in contrast, ncRNAs tend to have more polar and solvent exposed pockets that lack uniqueness. As a result, while there has been great progress over the past decades in improving the potency of therapeutic agents that target viral RNA (HIV in particular), none have proven sufficiently efficacious to reach the clinic. Computational docking and virtual screening can address many of these challenges by allowing broad examination of chemical space in search of unique small molecule scaffolds that target the complex RNA conformations adopted by many viral genomes. This project attempts to address these challenges through the development of RNA-ligand databases and new approaches for ensemble-based screening of large complex viral RNAs. I have developed a diversity-oriented small molecules library for virtual screening. I have applied this to docking of experimentally selected ensembles of small hairpin-loop RNAs in HIV1, and preliminarily found novel RNA-binding chemical scaffolds.

Targeting HIV1 ncRNA with dynamic ensembles and virtual screening

Megan KellyBiochemistry Duke University

59


Poster 29Taej in K im

Utilizing small interfering RNA (siRNA) for gene therapy has been widely studied based on its gene silencing capability. However, unmodified naked siRNA delivery to target cells is challenging because of short half-lives in blood serum and difficulty in uptake due to its negatively charged surface. To bypass these biological barriers in siRNA delivery, we investigated the use of cationic bolaamphiphile (bola) vesicles, GLH-19, GLH-20, and the mixture of two, GLH-19/GLH-20. GLH-19 has an acetylcholine head group while GLH-20 has an acetylcholine ester head group which can be hydrolyzed by choline esterase (ChE) after it penetrates the brain blood barrier (BBB). Using in-silico, in-vitro, and in-vivo studies, we characterized the delivery of siRNA/bola complexes in terms of stability, protection, transfection, and gene silencing. From our in-vitro experiments and atomic level in silico studies, we found that the GLH-19 vesicles have not only higher stability but also better protection and higher transfection efficiency than the GLH-20 vesicles. On the other hand, both of the GLH-19/siRNA and GLH-20/siRNA complexes showed similar efficiency of eGFP silencing in MDA-MB-231 cells. We also investigated the in-vivo distribution of siRNA/GLH-19 and siRNA/GLH-19/GLH-20 complexes in the liver, tumor, and brain. We found that the siRNA/GLH-19 complexes were delivered into tumors while no major accumulation was observed in the liver. We also found that siRNA/GLH-19/GLH-20 complexes were efficiently delivered into the brain. Therefore, our in-silico, in-vitro, and in-vivo studies showed that both siRNA/GLH-19 and siRNA/GLH-20 complexes can be used for siRNA delivery with different characteristics. When the two bolas are mixed together, GLH-19 provides enhanced stability, protection, and transfection, while GLH-20 provides efficient BBB penetration and siRNA release due to the hydrolyzation of GLH-20 head group by ChE.

In-Silico, In-Vitro, and In-Vivo Studies of siRNA

Delivery Using Cationic Bolaamphiphile Vesicles

Taejin Kim National Cancer Institute

National Institutes of Health

60


Poster 30Manjar i K i ran

Low- -grade glioma (LGG) develops in the supporting glial cLow- -grade glioma (LGG) develops in the supporting glial cells of brain. A more aggressive form of brain cancer is known as glioblastoma multiforme (GBM). With 5- -year relative survival rate of 43% for LGG and 6% for GBM, it is estimated that more than 16,000 adults in the USA will die from primary brain cancer this year. The poor survival rate of LGGs indicates that there is a need for a better prognostic marker that will enable the physician to give more aggressive therapy at the outset even for LGGs. Long non- -coding RNAs are gaining widespread attention as a potential biomarker for cancer diagnosis and prognosis. In our previous study, we determined the expression of many known and novel lncRNAs in over 750 brain cancer and normal brain tissue RNA- -seq dataset from the Cancer Genome Atlas (TCGA) and other publicly available dataset. We reported over a thousand lncRNAs induced or repressed in glial tumors relative to normal brains (Reon et al, PLoS Medicine 2016).

We have now developed a computational model for prognosis of low- -grade gliomas by combining lncRNA expression; Cox regression and L1- -LASSO penalized method. We trained the model on a set of 267 patients with gene expression data and clinical information and identified 10 lncRNAs that could act as a prognostic indicator independent of grade, patient age or IDH1/2 mutational status to predict the overall survival of patients. Patients in the training set, as well as a separate validation set, could be dichotomized according to their risk score calculated by adding the lncRNA expression and the coefficient obtained by multivariate cox regression done for the 10 lncRNA (Hazard Ratio (HR)=4.53, p=3.3e- -08 for training set (n=267), HR=6.09, p=5e- -07 for validation set (n=179)). To test whether this model is useful on a completely different cohort of patients, gene expression data from the Chinese Glioma Genome Atlas (CGGA) for 274 Chinese LGG and GBM patients was used. We can also cluster this new set of gliomas patients into two clusters with the same method established from the TCGA training set. The patients in low risk score group have significantly higher survival than the patients in high- - risk group (HR=1.45, p=0.024). Thus, there is a clear prognostic signature based on expression level of 10 lncRNA that can be applied to diverse populations of glioma patients. Besides their use as a biomarker, these lncRNAs need to be studied in detail to determine how they are affecting patient outcome.

Long-Noncoding RNA Based Prognostic Signature for Gliomas

Manjari Kiran Department of Biochemistry and Molecular Genetics University of Virginia

Manjari Kiran1, Daniel Keenan2, Anindya Dutta1.

1 University of Virginia, School of Medicine, Biochemistry and Molecular Genetics

2 University of Virginia, School of Medicine, Department of Statistics

61


Poster 31Jayashree Kumar

Non-coding intronic regions constitute approximately 95 % of all messenger RNAs (mRNAs). However, the study of human gene mutations has thus far been primarily focused on the coding exonic regions of mRNA and far less attention has been paid to ones found in the introns. The Human Genome Mutation Database (HGMD), which is a repository of manually curated disease-associated rare variants identified in clinical studies, contains approximately 15,000 single-base mutations located within the introns. HGMD classifies these intronic mutations as splicing mutations and in most cases these are thought to be causative of the genetic phenotype. While a large majority of HGMD’s splice mutations are found within the splice sites located in exon-intron boundaries, 7% are located within the introns themselves and it is unclear how these mutations result in deleterious effects. Intronic regions are more rarely sequenced in standard clinical studies so this number is likely underestimated. Intronic splice enhancers and silencers are known to influence the use of nearby splice sites, however only 40% of intronic variants map directly to these sites. RNA secondary structure has been shown to modulate splicing regulatory protein access. We hypothesize that some of the intronic disease-associated mutations are altering the local secondary structure of the RNA and hence introducing changes in splicing. We developed a computational pipeline to identify these single base disease-associated intronic mutations and identified 85 potential mutations predicted to modify local structure. Furthermore, we categorized these by whether they affect the accessibility of known intronic splice motifs. In 62% we identified a motif affected by the structure change, suggesting variant induced conformational rearrangement of introns is likely an important and understudied mechanism of human genetic disease.

RiboSPLitches: Intronic point mutations that

alter splicing through local RNA structural

changes

Jayashree Kumar Bioinformatics and

Computational Biology University of North Carolina

at Chapel Hill

62


Poster 32Lela Lackey

The relationship between mRNA levels and protein levels is non-linear. Post-transcriptional regulatory steps, especially translational control, govern how efficiently protein is produced. RNA structure is an important element in translational regulation. We hypothesize that RNA structure is a dynamic projection of the cellular environment and actively contributes to gene regulation by combining sequence-based and environment-induced structures to influence post-transcriptional regulation. We focus on cell cycle regulation as a common, dynamic cellular process where the translational efficiency of many different mRNAs has been shown to change from one phase to the next. We have determined the secondary structures of several translationally regulated 5’UTRs using SHAPE-MaP (Selective 2’ Hydroxyl Acylation by Primer Extension and Mutational Profiling). This chemical structure probing technique is unique in that it reads through chemical modifications during reverse transcription and can be coupled to next generation sequencing and incorporated into RNA structural modeling. Our data show differences in 5’UTR structure between primarily G1 phase cells and primarily S phase cells. These results suggest that mRNA structure is a dynamic attribute that may play a role in translational regulation. We will also identify additional functional 5’UTRs that impact cell-cycle dependent translational regulation for secondary structure determination. To do so, we will analyze populations of cells with flow cytometry using new cell cycle phase dyes and a GFP-degron construct, which will allow us to quantify rapid changes in protein production.

The role of dynamic mRNA structure in translational regulation

Lela Lackey Biology Department University of North Carolina at Chapel Hill

63


Poster 33Br ittany Law

Small nucleolar RNAs (snoRNAs) have been canonically described to guide site-specific modifications to snRNA and rRNA. However, we and others have reported novel snoRNA-driven phenotypes that are not linked to modification of these targets. For example, we have shown that four box C/D snoRNAs from the Rpl13a locus (SNORD32A/33/34/35A) have a role enhancing reactive oxygen species in response to stress. Since this response was shown to be independent of snoRNA-guided 2’-o-methylation (2’OMe) of rRNA, we hypothesized that snoRNAs might also guide 2’OMe modification of mRNAs involved in the oxidative stress response. Building on available CLIP-seq data, we now have identified and validated mRNA targets of Rpl13a snoRNAs. Utilizing an RT-qPCR approach in which reverse transcriptase is sensitive to 2’OMe, we have been able to detect changes in target mRNA methylation under conditions lacking the relevant snoRNA or fibrillarin. Loss of 2’OMe is associated with alterations in target mRNA and protein levels in both cells and animals. These results support a unique role for snoRNAs in influencing the regulation of mRNA and protein both in vitro and in vivo.

Box C/D small nucleolar RNAs regulate gene

expression by guiding 2 O-methylation

of mRNA

Brittany Law Medicine - Cardiology

Duke University

64


Poster 34Noah Lega l l

Circular RNAs (circRNA) are structural non coding RNAs that form by back-splicing: the covalent linking of flanking introns surrounding an exon or groups of exons. Back-splicing creates a circular structure that contains exons that are then are not processed any further, but accumulate a stable RNAs in the cytoplasm. Although the ultimate goal of circRNA function is mysterious and not fully understood, their post transcriptional regulation of exons makes them a strong candidate for study in order to combat protein mediated diseases.

Inference can be made that the introns that exhibit back-splicing potential must be fundamentally different than introns associated with canonical mRNA biogenesis. In an effort to better understand the elements that facilitate the biogenesis of circRNA, a bioinformatic study was conducted in order to infer features among flanking introns of circRNA that would suggest increased efficiency of back-splicing. Hybridization free energy, intron ALU element content, and intron-to-intron complementation were some of the features that were computationally derived for all annotated circular RNAs. Over 70% on the introns in the human genome involved in backsplicing contained inverted Alu elements. We altered the distance of these inverted Alu elements from the circularized exons, and found thayt both repeat elements must be closer than 1 kB to the backspliced exons for efficient circularization.

Computational Analysis of Circular RNA Formation And Secondary Structure Inference

Noah Legall Department of Biology University of North Carolinaat Chapel Hill

Noah Legall, Josh Welch, Rita Meganck, Alain Laederach and William F. Marzluff


65


Poster 35Yu-Hua Lo

Ribosome assembly is a complex process that requires hundreds of essential assembly factors, including Rix7 (NVL2 in mammals) and Nsa1 (WDR74 in mammals). Rix7 is a type II double ring, AAA-ATPase, which is closely related to the well-known Cdc48/p97. Previous studies in Saccharomyces cerevisiae suggest that Rix7 mediates the release of Nsa1 from nucleolar pre-60S particles however the underlying mechanisms of this release are unknown. Through multiple structural analyses we show that S. cerevisiae Nsa1, is composed of an N-terminal seven bladed-WD40 domain followed by a lysine rich C-terminus that extends away from the WD40 domain and is required for nucleolar localization. Co-immunoprecipitation assays with the mammalian homologues identified a well-conserved interface within WDR74 that is important for its association with NVL2. We further show that WDR74 associates with the D1 AAA domain of NVL2, which represents a novel mode of binding of a cofactor with a type-II AAA-ATPase.

Structural Analysis Reveals Features of Ribosome Assembly

Factor Nsa1/WDR74 Important

for Localization and Interaction with

Rix7/NVL2

Yu-Hua Lo Signal Transduction Laboratory

National Institute ofEnvironmental Health Sciences,

National Institutes of Health

66


Poster 36Michae l McFadden

Gene expression during the type I interferon response must be strictly regulated to allow for both efficient production of antiviral effectors, and controlled shut-off of inflammatory factors to avoid tissue damage and autoimmunity. RNA base modifications represent an emerging paradigm of gene regulation, which occurs at the post-transcriptional level. N6-methyladenosine (m6A) and the related modification m6Am are reversible modifications whose patterns on mRNA can change during cellular stress. We mapped m6A and m6Am modifications during the type I interferon response and show dynamic changes to many innate immune-related genes. Additionally, we show that perturbation of the cellular m6A and m6Am-related machinery affects production of the antiviral effector protein IFITM1. Depletion of the demethylase FTO increases protein levels of IFITM1 through increasing the stability of IFITM1 mRNA in an IFN-dependent fashion. Conversely, depletion of the methyltransferase-like protein METTL14 decreases protein abundance of IFITM1. We have previously shown that depletion of FTO limits Hepatitis C virus replication, while depletion of METTL14 increases HCV replication. The contribution of m6A in gene regulation of antiviral factors such as IFITM1 warrants further investigation. In future studies, we will knockout IFITM1 to examine its contribution in limiting flavivirus replication in the context of FTO depletion. Further, we will determine the mechanisms by which FTO controls the stability of IFITM1 mRNA, and by which METTL14 regulates IFITM1 protein abundance. These studies will identify a role for RNA modifications and the RNA methylation machinery in antiviral innate immunity.

RNA Modifications in Type I Interferon Response Regulation

Michael McFadden Molecular Genetics & Microbiology Duke University

67


Poster 37Rita Meganck

Circular RNAs (circRNAs) are a class of mammalian RNAs whose prevalence has recently been recognized. Most circRNAs are generated by exon circularization. This is performed by the spliceosome via backsplicing, in which the splice donor site is spliced to an upstream acceptor site (the reverse of the normal splicing process). Endogenously circularized exons often contain inverted repeats in the flanking introns, which promote backsplicing by bringing the splice junctions close together. However, in different tissues, circularization of the same RNA occurs to different extents, suggesting that there is tissue-specific regulation. There are also differences in the ratio of linear to circular RNAs, and in some cases, the circRNA is the predominant product. CircRNAs are extremely stable; since they have no free ends, they are resistant to cellular exonucleases. Accordingly, circRNAs are more long-lived than their linear counterparts, and may accumulate over time in non-dividing tissues. Previously, we created an inducible circRNA reporter system, which we termed circGFP-CD (Csy4-Dependent). Circularization from this construct is dependent on Csy4, a Class 1 Type 1F CRISPR endoribonuclease. The reporter contains a split GFP motif, an intron with a Csy4 hairpin, and a competing splice acceptor leading to a dsRed ORF. In the absence of Csy4, forward splicing occurs and dsRed is produced; in the presence of Csy4, the intron is cleaved at the Csy4-targeted hairpin, allowing backsplicing to occur, and joining the split GFP fragments into a full length ORF whose translation is driven by an EMCV IRES. In the current study, we have tested the utility of circRNA expression constructs in vivo by injecting recombinant AAV vectors encoding a split GFP cassette (which produces GFP only upon circularization) into mice. The cassettes tested include different endogenous intron pairs to drive circularization. Multiple organs were harvested and analyzed for direct circRNA expression as well as GFP expression. In multiple cell types and tissues, the circRNAs expressed more GFP than a linear control, presumably due to their increased stability. Thus, the properties of circRNAs make them a candidate for expressing protein in non-dividing cells.

Development of protein-expressing circular

RNA vectors

Rita Meganck Biochemistry


68


Poster 38Dawn Merr iman

RNA bulge motifs are essential building blocks of RNA structure, and recognition. They help to define the orientation and dynamics of helical elements and are often sites of tertiary contacts, and recognition of functionally important small molecules and proteins. Here, we utilize the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) as a model system to investigate how interhelical stacking dynamics of poly-pyrimidine bulge motifs vary as a function of length. Previous work exemplified TAR as a simple two-state system in which helices linked by a tri-pyrimidine bulge motif are either co-axially stacked or a flexible ensemble of bent conformations. Utilizing chemical shift titrations of various length bulge motifs with Mg2+, we found a small difference in the energetics of the stacking equilibrium between 2 and 7 poly-pyrimidine nucleotide bulge motifs (DDG 0.68 ± 0.02 kcal/mol), and that single bulge motifs do not undergo a stacking transition. This was further investigated with residual dipolar couplings, providing strong evidence poly-pyrimidine bulges > 2 nucleotides undergo the same stacking transition with a significant increase in the interhelical degree of order occurring when transitioning from low (25mM NaCl, 0 Mg2+) to high (25mM NaCl, 3mM Mg2+) salt conditions (Jint-U7 0.34 -0.84). To explain the small energetic penalty of interhelical stacking we compared measured data to coarse-grained molecular dynamic simulations (TOPRNA) to reveal considerable interhelical twist dynamics in the stacked state; alluding to twisting dynamics compensating for the entropic cost of stacking bulge motifs. Finally, relaxation dispersion measurements allowed for the estimation of the interhelical stacking rate to be ~ 16,000 s-1 at 25°C. This data set can be incorporated in to 3D RNA folding algorithms to better predict folding energetics of complex RNA systems.

Energetics of Two-state Stacking Dynamics in Poly-pyrimidine RNA Bulge Motifs Exhibit a Weak Dependence on Bulge Length

Dawn Merriman Chemistry Duke University

Dawn K. Merriman1, Jiayi Yuan1,2, Antony M. Mustoe3, Honglue Shi1, Nicole Orlovsky1, and Hashim M. Al-Hashimi*1,4

1 Department of Chemistry, Duke University

2 Department of Biology, Duke University

3 Department of Chemistry, University of North Carolina at Chapel Hill

4 Department of Biochemistry, Duke University Medical Center

69


Poster 39Angelo Moreno

Aptamers are single stranded RNAs and DNAs that bind with high affinity to defined molecular targets and are easily reversed by complimentary oligonucleotides, making them attractive candidates for clinical applications. Though they exhibit potent in vivo effects, aptamers are often conjugated with carrier molecules to increase their circulating half-life. Many formulations utilize polyethylene glycol (PEG), an easily conjugated polymer common to medicaments, and currently there is one FDA-approved aptamer that uses PEG. An RNA aptamer designed in our laboratory targeting coagulation Factor IX/IXa exhibiting potent anticoagulant activity, was evaluated in patients undergoing percutaneous coronary intervention (PCI), previously known as angioplasty. Phase I and II clinical trials were promising. However, severe adverse allergic reactions were observed in 10/1605 (<1%) patients who were given intravenous administration of the RNA aptamer, forcing premature termination of the Phase III trial. Preliminary investigation correlated the severity of the allergic reactions to the presence of pre-existing anti-PEG antibodies in these patients. In this study, we examine the interface between the immune system and aptamers and demonstrate that anti-PEG antibodies inhibit aptamer function both in vitro and in vivo shedding light on the PEG-sensitivity phenomenon and how it relates to oligo-based therapies. Considering the ubiquity of PEG in both commercial and medical products it is clear that further analysis of this anomaly is warranted. These data will help inform not only the design of oligo-based therapies but drug development as a whole.

A Lesson in Drug Design: RNA Aptamer Function is Inhibited by Pre-Existing Anti-PEG Antibodies in a

Human Clinical Trial

Angelo Moreno Surgery / Molecular Genetics

and Microbiology Duke University

Angelo Moreno, George Pitoc, Juliana Layzer, Nancy Ganson, Michael Hershfield,

Alice Tarantal, and Bruce Sullenger

70


Poster 40Br ittany Morgan

The ENCODE project revolutionized RNA biology by identifying thousands of non-coding RNAs that are essential in important cellular processes and are highly misregulated in numerous diseases. Despite this discovery, less than 35 RNA elements have been targeted with chemical probes, and only the ribosome has been targeted with FDA-approved drugs. To date, most RNA-based small molecule screens have utilized commercially available libraries, which result in low hit rates for RNA and the identification of promiscuous ligands with limited efficacy in vivo. It is proposed that these libraries are biased with protein-binding chemotypes, and therefore, the goal of this work is to identify RNA-privileged physicochemical, structural, and spatial properties, design an RNA-biased library, and synthesize RNA-targeted small molecules. To begin, the RNA-targeted BIoactive ligaNd Database (R-BIND) was compiled from the literature, which includes ligands that have activity in cell culture and/or mouse models. Chemoinformatic parameters and principal moments of inertia vectors were calculated and compared to FDA-approved drugs, general RNA-binding ligands, and screening libraries to distinguish key two-dimensional properties and the molecular shape of the ligands, respectively. Three key guiding principals emerged for bioactive RNA ligands: i) compliance to medicinal chemistry rules; ii) an increase in nitrogen atom count and ligand rigidity; and iii) a statistically significant shift in rod-like character. To incorporate these properties into a synthetic combinatorial library, REtrosynthetic Combinatorial Analysis Procedure was used to fragment R-BIND into building blocks, which inspired the selection and purchase of amine-based subunits. Using these subunits, a theoretical library of 2500 small molecules was generated based on a designed oxazolidinone scaffold, a RNA-privileged core with known biological activity. The small molecules that reflected the identified guiding principals were selected for synthesis by using a Nearest Neighbor algorithm. Then, the novel scaffold was synthesized from 3-butene-1,2-diol, where the primary alcohol is first protected and the secondary alcohol is then converted to a trichloroacetyl carbamate. A copper-assisted palladium cyclization yields the oxazolidinone-based scaffold, where both protecting groups are removed under mild conditions. Utilizing the subunits, orthogonal substitution is achieved at two positions, as well as oxidation followed by amide coupling for synthesis of the selected library. The methodology developed in this work can be applied to additional RNA-privileged scaffolds to synthesize diverse RNA-targeted libraries for the discovery of novel RNA-based chemical probes and therapeutics.

Chemoinformatic-driven design and synthesis of an RNA-targeted small molecule library

Brittany Morgan Chemistry Duke University

71


Poster 41Anthony Mustoe

Messenger RNAs (mRNAs) can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, structure remains similar between in-cell and cell-free RNA, indicating broad potential for structure-mediated gene regulation. We find that translation efficiency of endogenous genes is predominantly regulated by unfolding kinetics of the ribosome binding site. We discover conserved structured elements in 35% of untranslated regions, several of which we validate as novel protein binding motifs. Our study establishes RNA structure as a pervasive regulator of gene expression and implies that most functional structures remain to be discovered.

Pervasive and diverse regulatory functions of

mRNA structure revealed by high-resolution

SHAPE probing

Anthony Mustoe Department of Chemistry

University of North Carolinaat Chapel Hil

Anthony M. Mustoe1, Steven Busan1, Greggory M. Rice1,2, Christine E. Hajdin2,

Brant K. Peterson2, Vera Ruda2, Neil Kubica2, Jeremy L. Baryza2, and

Kevin M. Weeks1

1 Department of Chemistry, University of North Carolina,

at Chapel Hill

2 Novartis Institutes for Biomedical Research, Inc.

72


Poster 42Chid inma Nnadi

The twister RNA motif was discovered during a bioinformatics search of non-coding RNA databases. Twister ribozymes form a class of small site-specific self-cleaving nucleolytic ribozymes. A member of the twister class, environmental 22, was identified from a sequence derived from an unknown organism. We report that the env22 twister ribozyme cleaves inefficiently in the presence of magnesium with a midpoint of cleavage of 8.9(±1.5) mM MgCl2. This is comparatively higher than the midpoint of cleavage for the Oryza sativa twister ribozyme (K1/2 = 135(±12) μM MgCl2) reported by Panja et al. A unique characteristic of the O. sativa twister ribozyme is that lower concentrations of transition metal ions than magnesium ions are required to activate the ribozyme’s self-cleavage activity (Panja et al.). The env22 twister ribozyme exhibited activity in the presence of magnesium or calcium ions but showed little to no activity with transition metal ions. The env22 twister ribozyme also appeared to require higher RNA strand concentration for cleavage efficiency compared to O. sativa. Our findings support the phenomena that intrinsic stability and divalent metal ion selectivity varies among the twister ribozyme family members. This observation has motivated current experiments involving the elongation and manipulation of the P3 helical stem of the O. sativa twister ribozyme to test how sequence variation affects the ribozyme’s folding and catalytic activity.

Panja, S., Hua, B., Zegarra, D., Ha, T., Woodson, S. (2017). Metals induce transient folding and activation of the twister ribozyme. Nat. Chem. Biol. 13, 1109–1114.

Rice and Seawater: Stability and Metal Ion Selectivity Varies Among Twister Ribozymes

Chidinma Nnadi T.C. Jenkins Department of BiophysicsJohns Hopkins University

Chidinma Nnadi, Subrata Panja, &Sarah A Woodson

73


Poster 43John Noto

Circular RNAs (circRNAs) have become an increasingly popular subject in molecular biology. New techniques have allowed researchers to characterize their widespread occurrence, evolutionary conservation, unique cellular stability, and functional relevance. The relative stability of circRNAs makes them an interesting candidate for therapeutic use, and several groups have devised methods for expressing “designer” circRNAs. Our lab has used the process of tRNA splicing, where removed introns are ligated to form circles, to robustly express designed circular RNAs. Our data suggest that this method is capable of generating higher yields of expression than those utilizing back-splicing of Pol II transcripts, another mode of circular RNA production. We also characterize transgenic expression of a fluorescent circRNA in living fruit flies, and detail its age-dependent accumulation.

Engineered expression of tRNA intron-derived

circular RNAs in cell culture and in animals

John Noto Genetics & Molecular Biology


74


Poster 44Lorena Par lea

Despite the medical advances of diagnosis and treatment, cancer remains one of the most challenging “diseases” to treat, due to peculiarities from cell to cell, tissue to tissue, organ to organ, and even individual to individual. Largely, cancer is progressive and invasive, characterized by a high cell proliferation rate and elevated expression of chromosomal instability associated proteins, certain kinases and some key regulator proteins. Therapeutic treatments targeting these proteins, specifically the proteins involved in cell cycle and apoptosis, hold great promise for treatment of solid tumor cancers. Downregulating proteins that promote proliferation and evading programmed cell death is possible through RNA Interference pathways. Such nucleic acid-based therapeutics can target various molecular processes, and present high biocompatibility, low cytotoxicity and controlled immunogenicity. Moreover, nucleic acid structures can be strategically programmed to exhibit controlled sizes, shapes, stoichiometries, and functions, and their chemical and enzymatical synthesis is simple and straightforward. In addition, nucleic acid constructs can incorporate single or combinations of functional moieties, such as silencing RNAs (siRNAs) that downregulate the expression of genes, fluorescent labels for imaging, aptamers for direct molecular or cellular targeting, proteins as diagnostic and prognostic markers, and small molecules or drugs as anticancer chemical agents. Additionally, nucleic acid constructs have multivalence potential, and can be modulated to target multiple biological processes simultaneously. To date only handful of RNA-based constructs have been nanoengineered for therapeutic and/or in vivo applications. Here, we present the use of linear RNA/DNA hybrid constructs functionalized with Dicer substrate siRNAs (DsiRNAs) against Polo-like kinase 1, B-cell lymphoma 2, and Survivin to knock down the gene expression, arrest cell cycle and restores apoptosis in epithelial-derived solid tumor cancers. Furthermore, we hypothesize that targeting simultaneously different stages of the apoptotic pathway should enhance the results and have a synergistic therapeutic effect.

Hybrid DNA/RNA constructs targeting anti-apoptotic proteins in epithelial-derived solid tumor cancers

Lorena Parlea RNA Biology Laboratory National Cancer Institute/National Institutes of Health

75


Poster 45Miche l le Potter-B irr ie l

Histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated but instead end in a conserved 3’ stem-loop. The 3’ stem-loop participates in cell cycle regulation of histone mRNAs, with both processing and stability of the mRNA, being tightly regulated. Histone mRNA biogenesis requires multiple factors to ensure the homeostasis of the cell and the stem-loop binding protein (SLBP) is a key factor in this process. SLBP is required to properly process histone mRNAs in vivo by the binding to the stem-loop, stays bound until the mature mRNA is translocated to the cytoplasm where it is essential for translation. To study this process in the intact animal, I am using Drosophila melanogaster. dSLBP histone mRNA biosynthesis function is accomplished by 2 structural domains, a RNA binding domain (RBD) and a RNA processing domain (RPD) at the C-terminus. My aim is to investigate the structural requirements for dSLBP binding and function in vivo. In vitro studies done by our group have shown that the RBD and the C-terminus are sufficient for histone mRNA processing. However, the biological role of these sequences is not well understood. To further elucidate the biological function, I am currently using Fly-CRISPRCas9 as an approach to create a null allele of dSLBP which is larvae lethal. Genetic analyses will be done to test the ability of each mutant to rescue this phenotype. In conclusion, these approaches will provide a better understanding of the structure-function and biological role of dSLBP in histone mRNA metabolism in vivo.

Role of the Stem-Loop Binding Protein (SLBP)

in histone pre-mRNA processing in Drosophila

melanogaster

Michelle Potter-Birriel Curriculum in Genetics and

Molecular Biology, Integrative Program for Biological

& Genome Sciences (iBGS) University of North Carolina at

Chapel Hill

76


Poster 46Amanda Ra imer

Spinal muscular atrophy (SMA) is a recessive neurodegenerative disease and the leading genetic cause of death in young children. While it is known that SMA is caused by reduced amounts of functional Survival Motor Neuron (SMN) protein, the aberrant molecular mechanisms that lead to motor neuron degeneration and muscle loss are unclear. SMN’s canonical role is in snRNP biogenesis, however it has been implicated in many other functions such as mRNA transport, cytoskeleton organization, and endocytosis. To further elucidate the pathophysiology of SMA, our laboratory has developed an allelic series of human patient-derived Smn mutations in Drosophila. These missense mutations cover all three functional domains of SMN protein and exhibit disease-related phenotypes. Our objective is to use this model to compare disease phenotypes to proteomic environments that arise from mutations in the various domains and residues. To assess disease-related phenotypes we performed assays for viability and muscle function through locomotion. Viability assays show that these mutant lines cover a spectrum of severities. Crawling assays also show a spectrum of locomotor defects, but they do not always correlate with viability. Using the viability and locomotion phenotypes, we have analyzed a subset of fly lines by whole proteome analysis. These lines include mutations in all three functional domains of SMN and varying severities of viability and locomotion defects. The results of this analysis are expected to provide insight into how loss of Smn function affects other proteins and pathways, and which effects depend on specific functional domains. Overall, using characterization and proteomic analysis of these patient-derived mutation lines helps us better understand how the implicated pathways and functions of SMN fit into the disease etiology, and which domains/residues are important for these functions.

Characterizing SMA patient-derived mutations to connect proteomic environment with disease-related phenotypes

Amanda Raimer Curriculum in Genetics and Molecular Biology University of North Carolina at Chapel Hill

77


Poster 47Ramalakshmi Ramasamy

The Ink4/ARF locus (CDKN2A/B) encodes three tumor suppressor proteins: p16Ink4a (CDKN2A) , ARF (p14ARF in humans, p19ARF in mice) (CDKN2A) and p15Ink4b (CDKN2B). The CDKN2A gene that encodes p16Ink4A and ARF mRNA has three exons, two of which are shared by the two mRNAs, using different reading frames. Tumor suppressor proteins play an important role in mammalian cell cycle control. Hence the stability of mRNA of these proteins relates to the effect they have on biological functions. Based on the data available from previous mRNA stability studies in HeLa cells, it is observed that CDKN2A (p16Ink4A and p14ARF) mRNAs have a greater half-life (>24h) compared to CDKN2B (p15Ink4b) mRNA and also compared to mRNAs of tumor suppressor genes TP53 (p53), CDKN1B (p27Kip1), CDKN2C (p18Ink4c), CDKN1A (p21Cip1) and PTEN. Hence, we are interested in finding the factor(s) that confer stability to these mRNAs. We start by analyzing the CDKN2A mRNAs for the most common factors that contribute to mRNA stability, such as transcript circularity, 5’ UTR, 3’ UTR, presence of known motifs that contribute to mRNA stability, etc. Available Poly-A Tail-seq and Ribominus RNA-seq datasets from HeLa cells were also used for our analyses. The various splice isoforms of these mRNA were included in the analyses. Further, we propose a straight forward approach that uses CRISPR technology to specifically delete different regions of the three exons of CDKN2A. The differential mRNA stability between the modified transcripts and the original transcript would give us an idea of the significance of specific exonic regions of CDKN2A that contribute to the stability of it’s mRNA. This approach can further be extended to the intronic regions of CDKN2A. These results would help us better understand the factors that play a role in the stability of p16Ink4A and ARF mRNA and their effects on cancer and aging.

Stability of p16Ink4a and p14ARF mRNA in

HeLa Cells

Ramalakshmi Ramasamy Department of Biology, Lineberger

Comprehensive Cancer Center University of North Carolina

at Chapel Hill

Ramalakshmi Ramasamy, Krishnamurthy Janakiraman, Norman Sharpless and

Alain Laederach*

78


Poster 48Atu l Kaush ik Rangadura i

Watson-Crick (WC) base pairs in B-DNA exist in dynamic equilibrium with Hoogsteen (HG) base pairs which are formed by rotating the purine base about the glycosidic bond to a syn conformation. It was recently shown that in stark contrast to DNA, HG base pairs are energetically disfavored in A-RNA. In this study, we demonstrate that the instability of HG bps in RNA is caused due to geometric constraints imposed by the A-form that increase the energetic penalty associated with flipping the purine base to a syn conformation; we find that the 2’ Hydroxyl group does not contribute towards determining the Hoogsteen preferences of RNA. The findings from this study help understand the mode of action of epitranscriptomic RNA modifications such as N1-methylguanosine (m1G) and N1-methyladenosine (m1A) that act by disrupting duplex structure. Moreover, it also hints at the possibility that DNA in non-canonical environments such as RNA-DNA hybrids and chimeras would exhibit a reduced tendancy to form HG base pairs.

Insights into Hoogsteen Base Pairs in DNA and RNA

Atul Kaushik Rangadurai Biochemistry Duke University

79


Poster 49Thomas Ray

Transcript diversity via alternative splicing and alternative promoter usage are mechanisms by which a genome can maximize functional capacity on a per gene basis. These processes can potentially generate proteins from the same gene that are functionally distinct and spatially regulated. For these reasons alternative splicing of cell- surface receptors is an appealing mechanism to pursue how neurons interact during development to set up highly specific synaptic connections.

Despite mounting evidence that isoform diversity is critical to neural circuit formation, this diversity is still widely disregarded in gene expression and protein function studies, leading to inaccurate characterizations of gene function. One reason for this problem is the lack of tools for cataloging isoforms at the mRNA and protein level. While high throughput RNA sequencing is ubiquitous, the short reads generate an incomplete representation of the transcriptome, making subsequent gene isoform analyses dependent on computational assembly of transcripts—a problematic approach. Consequently, incomplete cataloguing of gene isoforms results in incomplete protein libraries, thereby impeding protein identification by mass spectrometry.

Here we describe an experimental and bioinformatic workflow to overcome these limitations. First, we analyzed RNAseq data from developing mouse retina and identified cell-surface receptors with unannotated high isoform diversity. Of these, 31 were chosen for targeted single molecule PacBio sequencing, which yields full-length transcript coverage for isoform identification. We sequenced developing and adult retina to identify isoforms that correlate with key neural developmental hallmarks. Bioinformatics tools (Iso-Seq, SQANTI, custom) were used to catalogue isoforms, and molecular tools were used to verify temporal (RNAseq, qPCR) or spatial (BaseScope in situ hybridization) expression of isoforms in the retina. The catalog of full-length PacBio-sequenced isoforms was used to generate an in silico protein library of unannotated “dark” isoforms (i.e. isoforms absent from RefSeq based libraries). Then we performed mass spectrometry on cell-surface protein preparations from retinal tissue, and searched for novel protein isoforms using our library. This pipeline proved robust in identifying novel cDNA isoforms that are translated into protein. We found a surprising number of “dark” isoforms, many of which are positioned to diversify the functions of genes known to influence retinal development and disease.

Sequencing and proteomic approach

illuminates mRNA and protein isoform diversity

and their contributions to neural development

and disease

Thomas Ray Neurobiology

Duke University

80


Poster 50Poorna Roy

Splicing in eukaryotes is a crucial step in which noncoding sequences (introns) within precursor-messenger RNAs (pre-mRNAs) are removed and the remaining coding sequences (exons) are ligated together to form mature mRNA. Pre-mRNA splicing is achieved by a multi-megadalton RNA-protein complex called spliceosome. Unlike most RNA or protein enzymes, the spliceosome does not maintain a stable composition. Rather, it is a dynamic machinery that involves five main small nuclear ribonucleoprotein (snRNP) complexes that associate, rearrange and dissociate during at least seven distinct steps of splicing, including two chemical catalysis steps. In the splicing cycle, the role of pre-mRNA is often viewed as a passive substrate. However, recent reports have indicated that pre-mRNAs can actively influence regulation and catalytic efficiency of splicing. Our goal is to elucidate the impact of introns secondary structure on splicing activity.

To this end, we use Saccharomyces cerevisiae as our model system. Using a temperature-sensitive strain carrying a TAP-tagged Cef1 protein (Prp2-1,Cef1-TAP), we have isolated the yeast Bact complex (the activated complex right before first catalytic step), as confirmed by RT-PCR and Northern blot analysis. Presence of known pre-mRNA targets and identification of new splicing substrates was achieved by isolating RNA from the complex and performing RNA sequencing (RNA-seq). Computational analysis of the data revealed significant enrichment of >90% of the known pre-mRNA targets that would be predicted to be present in the Bact complex isolated during vegetative growth. With this high-confidence list of spliceosome substrates in hand, we next plan to carry out pre-mRNA secondary structure prediction within the Bact complex using selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) analysis in collaboration with the Laederach lab at the University of North Carolina at Chapel Hill. We anticipate that a transcriptome-wide dissection of RNA conformations along the splicing cycle will inform on the roles of pre-mRNA secondary structure changes in splicing regulation.

Identity and secondary structure of pre-mRNAs in the activated yeast spliceosome

Poorna Roy College of Literature,Science and Arts University of Michigan

Matthew L. Kahlscheuer, Poorna Roy, Julia R. Widom, Lela Lackey, Alain Laederach, Michelle T. Paulsen, Mats Ljungman and Nils G. Walter

81


Poster 51Casey Schmidt

Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include removal of introns. Recently, our lab discovered a new class of circular RNAs formed by intramolecular ligation of excised tRNA introns in Drosophila melanogaster. We term these molecules tRNA intronic circular (tric)RNAs. We have found tricRNAs to be stable, highly abundant, and conserved in insects.

To investigate the mechanism of tricRNA biogenesis, we have generated a reporter that replaces the majority of the endogenous introns of two Drosophila tRNA genes with the Broccoli fluorescent RNA aptamer. Using this reporter, we investigated the cis elements required for proper tricRNA splicing. We discovered that a stem of at least eight nucleotides is necessary to separate the tRNA domain from the Broccoli domain. We also observed that disrupting the anticodon-intron base pair, which is a conserved feature of eukaryotic pre-tRNAs, results in dramatically reduced tricRNA production. Furthermore, we found that strengthening the weak base pairs that are proximal to the anticodon-intron base pair also leads to a reduction in tricRNAs. These weak base pairs are likely present to facilitate proper splicing.

In addition to these cis element studies, we are also using the Broccoli reporter to identify tricRNA processing factors. We found that several known tRNA processing factors, such as the RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis, and depleting these factors reduces proper tricRNA splicing. Interestingly, we observed that depletion of cbc, a kinase whose human homolog Clp1 has been shown to phosphorylate tRNA 3’ exons in vitro, results in increased tricRNAs, both endogenous and reporter. It has been postulated that Clp1 is an in vivo negative regulator of tRNA biogenesis, and these studies appear to support that model. Furthermore, we identified Dis3 as an endonuclease that can degrade tricRNAs, as its knockdown results in increased tricRNAs while its overexpression results in decreased tricRNAs. Taken together, we have characterized the major players in the tricRNA biogenesis pathway, and have provided evidence for a negative regulator of tRNA biogenesis.

Characterization of tRNA intronic circular (tric)

RNA biogenesis

Casey Schmidt Integrative Program for Biological

& Genome Sciences (iBGS) University of North Carolina

at Chapel Hill

82


Poster 52Ashlyn Spr ing

Spinal Muscular Atrophy (SMA) is an early-onset neurodegenerative disorder that primarily affects the neuromuscular junction (NMJ) and is caused by mutation of the Survival Motor Neuron 1 (SMN1) gene. SMA is typified by reduced motor function caused by NMJ denervation, subsequent muscle atrophy, and death early in life in its severe manifestations. In most patients, the SMN1 gene is completely deleted. More rarely, patients carry small SMN1 mutations that change a single amino acid in the SMN protein. SMN1 is highly conserved and has a single homolog of the same name (Smn) in Drosophila melanogaster. In pursuit of 1) a robust disease model of SMA in the fly and 2) an effective system in which to study the molecular mechanisms underlying SMA onset and pathogenesis, we have generated a set of fourteen transgenic lines carrying patient-derived small mutations in the fly Smn gene. Phenotypic characterization of wandering third instar larvae carrying these Smn mutations reveals reduced viability, locomotor defects, a mild decrease in bouton number, and limited to no signs of muscle atrophy. These data suggest that larvae carrying SMA-causing patient mutations can be used as a model for the pre-symptomatic and early stages of SMA pathogenesis. In the ten lines that reach the adult stage, we observe reduced longevity and impaired locomotion. For flies carrying stronger Smn mutations, altered gate, muscle weakness, and paralysis of the legs is readily observable. The manifestation of these phenotypes in the adult indicates that this stage is an effective model for post-onset characteristics of SMA.

In addition to examining phenotypes traditionally associated with SMA, we also are exploring the link between Smn and innate immune function. In all cases, larvae expressing mutant Smn develop melanotic masses in late larval stages, indicating an activated innate immune response. This appears to be a cell-autonomous role for Smn, as RNAi-mediated Smn knockdown specifically in immune tissues is sufficient to induce melanotic mass formation. Initial examination of the molecular pathways that may be responsible indicates that Smn may negatively impinge upon NF-κB signaling through physical interaction with bendless and Traf6 to modulate the Toll and IMD pathways.

Collectively, this works suggest that mutations in the fly Smn gene can be used to model many aspects of SMA and can also serve as a system for the discovery of novel Smn functions.

Comprehensive modeling of Spinal Muscular Atrophy in Drosophila melanogaster

Ashlyn Spring SPIRE Postdoctoral Fellowship Program University of North Carolina at Chapel Hill

Ashlyn M. Spring, Amanda Raimer, Christine Hamilton, Dominique Brown, Helen Dinh, Michela Schillinger, Kelsey Gray, Greg Matera

83


Poster 53Al ine Umuhire Juru

Non-protein coding RNA transcripts have been increasingly recognized as potential drug targets owing to their important roles in cellular processes. Since peptide-based and RNA-based therapeutics often exhibit poor in vivo delivery and pharmacokinetics, small molecules offer an excellent alternative tool for targeting RNA. However, due to its unique chemical and structural properties compared to protein targets, RNA has been difficult to target with drug-like small molecules. For example, despite the proven promise of targeting RNA with multivalent ligands, progress in this area has been hampered by current limitations in three-dimensional structure characterization of large RNAs. There is thus a need to develop techniques that do not rely on structure-based design. We are developing an imine-based dynamic combinatorial chemistry (DCC) technique for multivalent ligand discovery for large RNAs. In DCC, a target biomolecule is incubated with a thermodynamically-controlled dynamic library of small molecules, allowing it to select its highest affinity binders. DCC is thus superior to structure-based design since all thermodynamically stable conformations of the target participate in the ligand discovery process. To date, we have identified favorable conditions for imine formation in aqueous media, conducted comparative studies of amine reactivity towards imine formation, and begun validation studies on a known RNA binding scaffold. For validation studies, an aldehyde scaffold will be incubated with a diverse library of primary amines to discover ligands for the HIV-1 Transactivation Response (TAR) RNA. Upon validating the DCC method, we will set out to identify first multivalent ligands for a number of disease-relevant RNA targets. Ultimately, this work will provide a much-needed platform for multivalent ligand discovery for large RNAs, particularly those that have yet to be structurally characterized.

Imine-based dynamic combinatorial chemistry

for discovery of multivalent

RNA-binding ligands

Aline Umuhire Juru Chemistry

Duke University

84


Poster 54Sarah Wicks

Non-coding RNAs (ncRNAs) are a novel class of biomolecules implicated in important biological processes and are often misregulated in various diseases. To study disease-driving ncRNAs, small molecule chemical probes have been proven to be a viable avenue. Commonly, fluorescence-based labeling and immobilization techniques are used to study RNA:small molecule interactions, which hinders the types of RNAs and small molecules that can be screened. In order to fundamentally understand RNA:small molecule recognition, we propose to utilize high-throughput screening assays that can be used with a variety of small molecules and RNAs without the need for ligand or target modification. We herein describe a TO-PRO-1 displacement assay as an appropriate screening tool along with current efforts to identify and characterize RNA:small molecule interactions. To investigate several assay parameters, the HIV-1 Transactivation Response Element (TAR) was used as a model system. Using TAR, the dissociation constant of TO-PRO-1, signal-to-background ratio, and effective RNA concentration for displacement were assessed. To confirm these optimized conditions, small molecules that bind TAR were used to evaluate displacement of TO-PRO-1 and binding curves were obtained. Currently, the TO-PRO-1 displacement assay is being utilized with biologically relevant RNAs to screen diverse, RNA-biased small molecules and reveal structure-activity relationships. Derivatives of a lead RNA-privileged amiloride-based small molecule with moderate affinity for TAR have been compared to identify substituents that enhance binding. In addition, commercially available small molecules that encompass RNA-privileged chemical space are being selected and screened to identify new RNA-binding scaffolds and leads for therapeutically-relevant RNAs. Concomitantly, the physicochemical properties of small molecules are being analyzed and compared to elucidate guiding principles that bias recognition. At the conclusion of this work, hundreds of RNA:small molecule interactions will be assessed and the physicochemical properties that lead to specific interactions will be determined. Furthermore, by expanding the diversity of RNA targets, small molecules, and methods used to identify and analyze these interactions, rational approaches for targeting RNA will be developed. Such approaches can result in selective small molecule probes to study the role of RNA in disease and aid in the discovery of therapeutic leads.

Fluorescent Indicator Displacement Assay to Identify and Characterize RNA: Small Molecule Interactions

Sarah Wicks Chemistry Duke University

85


Poster 55Hannah Wiedner

Alternative splicing is an essential regulatory mechanism by which diverse transcripts and proteins are derived from a finite genome. During development, complex splicing transitions occur, leading to changes in transcriptomes and proteomes. Previously, we found evidence of extensive splicing transitions during postnatal mouse heart development1. Numerous alternatively spliced genes encode for proteins that have roles in trafficking and membrane dynamics1. Still, the coordinators of this splicing-trafficking network are unknown. Compelling evidence shows that chromatin remodeling controls the silencing of fetal genes during normal heart development2. In heart failure, chromatin markers revert to a fetal profile which allows for the re-expression of fetal genes3. By manual inspection of our own RNA-sequencing data in mouse heart development1, and chromatin immunoprecipitation-sequencing (ChIP-seq) data available from the ENCODE project4,5, we found altered chromatin marks in regions that are close to the alternative exons of specific trafficking splicing events. This observation led us to ask about the possible role of chromatin remodeling in the regulation of splicing of trafficking genes. Based on the evidence of developmental splicing transitions, dramatic changes in chromatin structure during development, and the reversion to fetal patterns in both splicing and chromatin marks during heart disease, we hypothesized that epigenetic marks might contribute to the coordination of alternative splicing in heart development. Epigenetic mechanisms have already been shown to regulate alternative splicing in cancer and neuronal cell culture models6,7. To investigate this connection in muscle context, we used the murine C2C12 cell line which we have demonstrated to robustly reproduce the splicing-trafficking transitions observed in muscle development (unpublished data). Differentiated myotubes were treated with two drugs: the histone deacetylase inhibitor, trichostatin A (TSA), and a topoisomerase I inhibitor, camptothecin (CPT). Endogenous splicing patterns were investigated by RT-PCR and polyacrylamide gel electrophoresis. The percent inclusion of each alternative exon was quantified by densitometry. In general, TSA preferentially promotes skipping of the alternative exons in trafficking genes whereas CPT treatment did not significantly impact the splicing patterns of these genes. From these experiments, we conclude that chromatin accessibility and its acetylation state contributes to the regulation of splicing of trafficking genes in muscle cell differentiation. In the future, we plan to evaluate the role of RNA-polymerase II elongation rate as well as identification of the specific chromatin marks relative to alternative exons. We are also interested on identifying RNA- binding proteins that may cooperate with epigenetic mechanisms in the regulation of splicing.

Alternative splicing of trafficking genes

respond to chromatin acetylation in

muscle cells

Hannah Wiedner Department of Cell Biology

& Physiology University of North Carolina

at Chapel Hill

Hannah Wiedner1,2, R. Eric Blue1, Jimena Giudice1,3,4

1. Department of Cell Biology & Physiology

2. Biological and Biomedical Sciences Program

3. Curriculum in Genetics & Molecular Biology

4. McAllister Heart Institute. The University of North Carolina at

Chapel Hill

86


Poster 56Bo Zhao

Riboswitches are a class of non-coding RNAs located in the 5’ UTR of mRNAs that undergo a structural transition upon ligand recognition to control gene expression. Fluoride riboswitch uniquely recognizes a single negatively charged fluoride ion and induces fluoride toxicity response in cells. A high-resolution crystal structure of the ligand-bound aptamer reveals compact pseudoknot architecture and the basis of fluoride recognition. However, a molecular understanding of the dynamic interplay between the ligand-free/bound aptamers and the expression platform that underlies gene regulation remains elusive. Here, using chemical exchange saturation transfer (CEST) NMR spectroscopy and transcription assays, we present the structural, dynamic, and functional basis for gene regulation of this riboswitch, which is achieved through a ligand-dependent, excited-state mediated mechanism.

Structural, Dynamic, and Functional Basis of a Transcriptional Riboswitch

Bo Zhao Department of Chemistry University of North Carolina at Chapel Hill

87


Poster 57Bei L iu

N-6 methyladenosine (m6A) is the most prevalent post-transcriptional modication in messenger RNA (mRNA) and long non-coding RNA in eukaryotes. It plays multiple roles in biological processes, including mRNA metabolism, translation initiation, cell fate determination, stress response and micro-RNA processing. Previous thermodynamic studies demonstrated that m6A destabilizes RNA duplexes by ~0.5-1.7 kcal/mol. This destabilization in RNA helices is caused by disruption of the A•U base pair and can promote protein binding in lncRNAs by local melting of helices. Here, we report how m6A can stabilize one nucleotide bulge RNA helix-junction-helix motif in a Mg2+ dependent manner. Specically, m6A stabilizes the A•U base pair at the bulge-helix junction and the global RNA stability only in the presence of Mg2+. The stabilization of methylated RNA provides a new mechanism for modulating RNA structure and function.

m6A stabilizes junctional A-U base

pair in a Mg2+ dependent manner:

Impact on m6A reader protein

recognition

Bei LiuDepartment of Biochemistry

Duke University

88


NOTES

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