Exploiting Co-Transcriptional Folding and Processing of Nascent Messenger RNA for Modulating...
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1437-Pos Board B167 The Influence of the Form of tRNA on Complex Formation with Porphyrins Ishkhan Vardanyan, Yeva Dalyan. Molecular Physics, Yerevan State University, Yerevan, Armenia. In the current work we have investigated the interaction of meso-tetra-(4N- oxyethylpyridyl) porphyrin (TOEPyP4) and its Zn(II), Cu(II), -metallocom- plexes with tRNA which has 2 forms: hairpin structure and spatial reversed ‘‘L’’ structure. The interaction of tRNA from E.Coli with porphyrins is studied by UV/Vis Spectrophotometry and Circular Dichroism methods. The measurements were performed in 0.1 BPSE and 1BPSE buffers (1 BPSE = 6 mM Na2HPO4 þ 2 mM NaH2PO4 þ185 mM NaCl þ 1 mM Na2EDTA), correspondingly m = 0.02M and m = 0.2M, pH 6.57. (tRNA has hairpin form at m = 0.02M and reversed ‘‘L’’ structure, when m = 0.2M). From the spectrophotometric titration datas the Scatchard binding isotherms for porphyrin-tRNA complexes are built and binding parameters are calculated (N - the number of binding sites per molecule of tRNA, and K - the binding constant). In case of tRNA’s hairpin structure for the values of induced CD spectra (at 400-470 nm) for complexes tRNA with TOEPyP4 and CuTOEPyP4 there is an optimum concentration of porphyrins at which the anisotropy of system is maximal. For complexes of ZnTOEPyP4 with tRNA the induced CD spectra are essentially different. The induced CD spectra of complex change a sign and continue to grow (remaining negative) starting from a certain relative con- centration. This unusual ICD spectra profile is found for all three porphyrin- tRNA complexes in case of tRNA’s reversed ‘‘L’’ structure. It is possible that at high relative concentration of porphyrins the liquid crystal form may exist in the solution. The binding constants with tRNA in case of hairpin form are an order of magni- tude greater than in case of reversed ‘‘L’’ structure. It means that this porphyrin interacts stronger with tRNA when it has hairpin form. 1438-Pos Board B168 Studying Dynamics and Conformational Changes in the Glycine Riboswitch using Electron Paramagnetic Resonance Spectroscopy Jacqueline M. Esquiaqui 1 , Gail E. Fanucci 1 , Jingdong Ye 2 . 1 Chemistry, University of Florida, Gainesville, FL, USA, 2 Chemistry, University of Central Florida, Orlando, FL, USA. Electron Paramagnetic Resonance (EPR) spectroscopy is used to study dynamics and conformational changes in the RNA glycine riboswitch. The dynamic role of the leader-linker interaction within glycine riboswitch conserved sequences is probed through site directed spin labeling and continuous wave EPR. Inter- aptamer and aptamer-expression platform interactions are elucidated through double electron-electron resonance spectroscopy. Incorporation of spin labels is achieved through optimized ligation methodologies allowing synthetically modified RNA to be joined to larger RNA sequences. Expected folding and burial of riboswitch elements will lead to restricted motion of the spin label and, addi- tionally, pulsed EPR experiments yield distance distribution profiles indicating conformational exchange between states in the absence and presence of glycine. 1439-Pos Board B169 Theoretical and Experimental Study of the Conformational Structure of HIV RNA Xiao Fan 1 , Yanyan Li 1 , Yingya Liu 1 , Po Wang 1 , Haitao Li 1,2 . 1 Jiangsu Normal University, Xuzhou, China, 2 University of Cambridge, Cambridge, United Kingdom. HIV-1 genomic RNA has a non-coding 5’ region containing sequential conserved structural motifs that controls many parts of the lifecycle. Very limited data exist on their 3-dimensional conformation and then how they work structurally. Recently the novel 3-D structural information of this most highly conserved region of the virus was reported on a promising therapeutic target 1 . In order to learn more on the structure of this RNA, we use single mole- cule fluorescence, anisotropy imaging microscopy and RNAstructure model- ling 2.3 together to monitor its conformational dynamics in physiological condition. We aim to understand with implications for RNA dimerization and protein binding during regulatory steps. References: 1. J. D. Stephenson, H. Li, J. C. Kenyon, M. Symmons, D. Klenerman, and A. M.L. Lever, (2013) Three-Dimensional RNA Structure of the Major HIV-1 Packaging Signal Region. Structure 21, 1-12. 2. D. A. Alves, H. Li, R. Codrington, X. Ren, D. Klenerman, and S. Balasubra- manian, (2008) Single-Molecule analysis of human Telomerase monomer Nature Chemical Biology, 287-289. 3. H. Li; L. Ying; J. Green; S. Balasubramanian; D. Klenerman, (2003) Ultra- sensitive Coincidence Fluorescence Detection of Single DNA Molecules. Anal. Chem.1664-1670. 1440-Pos Board B170 Exploiting Co-Transcriptional Folding and Processing of Nascent Messenger RNA for Modulating Specific Exon Splicing Jing Lin 1 , Keng Boon Wee 1 , Zacharias Aloysius Dwi Pramono 2 , Uttam Surana 3,4 . 1 Institute of High Performance Computing, A*STAR, Singapore, Singapore, 2 National Skin Center, Singapore, Singapore, 3 Institute of Molecular and Cell Biology, A*STAR, Singapore, Singapore, 4 Bioprocessing technology Institute, A*STAR, Singapore, Singapore. RNA splicing, which involves intron removal and exon ligation, is a key step that transforms eukaryotic nascent messenger RNA (pre-mRNA) into mature mRNA. The ability to modulate inclusion or exclusion of specific exons to elicit switching between alternatively spliced variants, restoration or disrup- tion codon-reading frame, and correcting aberrant splicing has applications for both biomedical research and therapy. However, the identification of potent pre-mRNA sites, that when targeted or masked via therapeutics effi- ciently induce specific exon splicing, remains a challenge. As the transcrip- tion elongation of a pre-mRNA, exon recognition and exon splicing occur simultaneously, we hypothesized that the dynamics of co-transcriptional pre-mRNA folding could be a key factor in determining the accessibility of target sites. By analyzing all possible optimal and sub-optimal local second- ary structures of the pre-mRNA at each step of transcriptional analysis, we predicted the co-transcriptional accessibility profile and identified possible target sites that have the highest probability of being accessible throughout transcription process. To test the hypothesis, we used glycine decarboxylase (GLDC), an ‘oncogenic protein’ overexpressed in metastatic lung cancer cells, as a test case. Ten target sites each to induce specific exclusion of one of the two targeted exons of the GLDC gene, were identified to disrupt the codon reading frame to induce nonsense-mediated decay. In our wet ex- periments on NSCLC (non-small cell lung carcinoma) cell lines, specific exon exclusion were observed when each of the ten sites was masked; of these, three sites induced exon exclusion in more than 80% of total pre- mRNA. Notably, two sites predicted to be ineffective were validated to be so. Our results suggest that co-transcriptional binding accessibility can be used to aid the rational identification of potent target sites for modulating spe- cific exon splicing. 1441-Pos Board B171 Structural Polymorphism of (Cag)N Repeat RNA Elucidated using Single Molecule Nanomanipulation William T. Stephenson 1 , Sean Keller 2 , Scott A. Tenenbaum 1 , Michael Zuker 3 , Pan T.X. Li 2 . 1 SUNY College of Nanoscale Science and Engineering, Albany, NY, USA, 2 University at Albany SUNY, Albany, NY, USA, 3 Rensselaer Polytechnic Institute, Troy, NY, USA. Abnormal expansions of CAG trinucleotide repeats are responsible for 9 he- reditary human disorders including Huntington’s disease and a variety of Spinocerebellar ataxias. Disease symptoms typically manifest when the number of repeats exceeds a given threshold (typically 35þ repeats). It has been hypothesized that genetic instability and RNA toxicity arise due to the structurally polymorphic nature of expanded repeats. However, it is technically difficult to study the structure and folding of a heterogeneous population of RNAs. To overcome this problem, we applied a single- molecule approach to monitor folding of individual RNAs with up to 100 CAG repeats using optical tweezers. Compared to RNAs with non- repetitive sequences, a (CAG)n RNA folds slowly and non-cooperatively in multiple back-and-forth small steps, suggesting that the molecule un- dergoes an extensive conformational search. Most surprisingly, as a (CAG)100 RNA is extended by >100 nm, tension on the molecule remains largely unchanged at ~13.550.4 pN, which is not expected for an elastic polymer. This unusual viscoelasticity implies that force is unevenly distrib- uted in the molecular structures of the (CAG)n RNAs which possibly take the form of multi-branched, similarly-sized hairpins. The relatively constant unfolding force indicates that the folding energy landscape is almost flat at the force, and the small force fluctuation suggests that the energetic barriers between different conformations are very low. When a (CAG)n RNA is nanomanipulated to be partially unfolded, the molecule is trapped in a subset of conformations, as evident by the constant value of the mean extension. This observation leads to a hypothesis that the folding of (CAG)n RNAs is dominated by topological constraints, and the existing conformation is affected by preceding structures. Our findings support the structural poly- morphism hypothesis for the (CAG)n RNAs and provide evidence for sequential folding of repeated sequences. 282a Monday, February 17, 2014
Transcript of Exploiting Co-Transcriptional Folding and Processing of Nascent Messenger RNA for Modulating...
282a Monday, February 17, 2014
1437-Pos Board B167The Influence of the Form of tRNA onComplex Formation with PorphyrinsIshkhan Vardanyan, Yeva Dalyan.Molecular Physics, Yerevan State University, Yerevan, Armenia.In the current work we have investigated the interaction of meso-tetra-(4N-oxyethylpyridyl) porphyrin (TOEPyP4) and its Zn(II), Cu(II), -metallocom-plexes with tRNA which has 2 forms: hairpin structure and spatial reversed‘‘L’’ structure. The interaction of tRNA from E.Coli with porphyrins is studiedby UV/Vis Spectrophotometry and Circular Dichroism methods.The measurements were performed in 0.1 BPSE and 1BPSE buffers (1 BPSE =6 mM Na2HPO4þ 2 mM NaH2PO4 þ185 mM NaCl þ 1 mM Na2EDTA),correspondingly m = 0.02M and m = 0.2M, pH 6.57. (tRNA has hairpin format m = 0.02M and reversed ‘‘L’’ structure, when m = 0.2M).From the spectrophotometric titration datas the Scatchard binding isotherms forporphyrin-tRNA complexes are built and binding parameters are calculated (N -the number of binding sites per molecule of tRNA, and K - the binding constant).In case of tRNA’s hairpin structure for the values of induced CD spectra (at400-470 nm) for complexes tRNA with TOEPyP4 and CuTOEPyP4 there isan optimum concentration of porphyrins at which the anisotropy of system ismaximal. For complexes of ZnTOEPyP4 with tRNA the induced CD spectraare essentially different. The induced CD spectra of complex change a signand continue to grow (remaining negative) starting from a certain relative con-centration. This unusual ICD spectra profile is found for all three porphyrin-tRNA complexes in case of tRNA’s reversed ‘‘L’’ structure. It is possiblethat at high relative concentration of porphyrins the liquid crystal form mayexist in the solution.The binding constants with tRNA in case of hairpin form are an order of magni-tude greater than in case of reversed ‘‘L’’ structure. It means that this porphyrininteracts stronger with tRNA when it has hairpin form.
1438-Pos Board B168Studying Dynamics and Conformational Changes in the GlycineRiboswitch using Electron Paramagnetic Resonance SpectroscopyJacqueline M. Esquiaqui1, Gail E. Fanucci1, Jingdong Ye2.1Chemistry, University of Florida, Gainesville, FL, USA, 2Chemistry,University of Central Florida, Orlando, FL, USA.Electron Paramagnetic Resonance (EPR) spectroscopy is used to study dynamicsand conformational changes in the RNAglycine riboswitch. The dynamic role ofthe leader-linker interaction within glycine riboswitch conserved sequences isprobed through site directed spin labeling and continuous wave EPR. Inter-aptamer and aptamer-expression platform interactions are elucidated throughdouble electron-electron resonance spectroscopy. Incorporation of spin labelsis achieved through optimized ligation methodologies allowing syntheticallymodifiedRNA to be joined to largerRNAsequences. Expected folding andburialof riboswitch elements will lead to restricted motion of the spin label and, addi-tionally, pulsed EPR experiments yield distance distribution profiles indicatingconformational exchange between states in the absence and presence of glycine.
1439-Pos Board B169Theoretical and Experimental Study of the Conformational Structure ofHIV RNAXiao Fan1, Yanyan Li1, Yingya Liu1, Po Wang1, Haitao Li1,2.1Jiangsu Normal University, Xuzhou, China, 2University of Cambridge,Cambridge, United Kingdom.HIV-1 genomic RNA has a non-coding 5’ region containing sequentialconserved structural motifs that controls many parts of the lifecycle. Verylimited data exist on their 3-dimensional conformation and then how theywork structurally. Recently the novel 3-D structural information of this mosthighly conserved region of the virus was reported on a promising therapeutictarget1. In order to learn more on the structure of this RNA, we use single mole-cule fluorescence, anisotropy imaging microscopy and RNAstructure model-ling2.3 together to monitor its conformational dynamics in physiologicalcondition. We aim to understand with implications for RNA dimerizationand protein binding during regulatory steps.References:1. J. D. Stephenson, H. Li, J. C. Kenyon, M. Symmons, D. Klenerman, and A.M.L. Lever, (2013) Three-Dimensional RNA Structure of the Major HIV-1Packaging Signal Region. Structure 21, 1-12.2. D. A. Alves, H. Li, R. Codrington, X. Ren, D. Klenerman, and S. Balasubra-manian, (2008) Single-Molecule analysis of human Telomerase monomerNature Chemical Biology, 287-289.3. H. Li; L. Ying; J. Green; S. Balasubramanian; D. Klenerman, (2003) Ultra-sensitive Coincidence Fluorescence Detection of Single DNAMolecules. Anal.Chem.1664-1670.
1440-Pos Board B170Exploiting Co-Transcriptional Folding and Processing of NascentMessenger RNA for Modulating Specific Exon SplicingJing Lin1, Keng Boon Wee1, Zacharias Aloysius Dwi Pramono2,Uttam Surana3,4.1Institute of High Performance Computing, A*STAR, Singapore, Singapore,2National Skin Center, Singapore, Singapore, 3Institute of Molecular and CellBiology, A*STAR, Singapore, Singapore, 4Bioprocessing technologyInstitute, A*STAR, Singapore, Singapore.RNA splicing, which involves intron removal and exon ligation, is a key stepthat transforms eukaryotic nascent messenger RNA (pre-mRNA) into maturemRNA. The ability to modulate inclusion or exclusion of specific exons toelicit switching between alternatively spliced variants, restoration or disrup-tion codon-reading frame, and correcting aberrant splicing has applicationsfor both biomedical research and therapy. However, the identification ofpotent pre-mRNA sites, that when targeted or masked via therapeutics effi-ciently induce specific exon splicing, remains a challenge. As the transcrip-tion elongation of a pre-mRNA, exon recognition and exon splicing occursimultaneously, we hypothesized that the dynamics of co-transcriptionalpre-mRNA folding could be a key factor in determining the accessibility oftarget sites. By analyzing all possible optimal and sub-optimal local second-ary structures of the pre-mRNA at each step of transcriptional analysis, wepredicted the co-transcriptional accessibility profile and identified possibletarget sites that have the highest probability of being accessible throughouttranscription process. To test the hypothesis, we used glycine decarboxylase(GLDC), an ‘oncogenic protein’ overexpressed in metastatic lung cancercells, as a test case. Ten target sites each to induce specific exclusion ofone of the two targeted exons of the GLDC gene, were identified to disruptthe codon reading frame to induce nonsense-mediated decay. In our wet ex-periments on NSCLC (non-small cell lung carcinoma) cell lines, specificexon exclusion were observed when each of the ten sites was masked; ofthese, three sites induced exon exclusion in more than 80% of total pre-mRNA. Notably, two sites predicted to be ineffective were validated to beso. Our results suggest that co-transcriptional binding accessibility can beused to aid the rational identification of potent target sites for modulating spe-cific exon splicing.
1441-Pos Board B171Structural Polymorphism of (Cag)N Repeat RNA Elucidated using SingleMolecule NanomanipulationWilliam T. Stephenson1, Sean Keller2, Scott A. Tenenbaum1,Michael Zuker3, Pan T.X. Li2.1SUNY College of Nanoscale Science and Engineering, Albany, NY, USA,2University at Albany SUNY, Albany, NY, USA, 3Rensselaer PolytechnicInstitute, Troy, NY, USA.Abnormal expansions of CAG trinucleotide repeats are responsible for 9 he-reditary human disorders including Huntington’s disease and a variety ofSpinocerebellar ataxias. Disease symptoms typically manifest when thenumber of repeats exceeds a given threshold (typically 35þ repeats). Ithas been hypothesized that genetic instability and RNA toxicity arise dueto the structurally polymorphic nature of expanded repeats. However, it istechnically difficult to study the structure and folding of a heterogeneouspopulation of RNAs. To overcome this problem, we applied a single-molecule approach to monitor folding of individual RNAs with up to 100CAG repeats using optical tweezers. Compared to RNAs with non-repetitive sequences, a (CAG)n RNA folds slowly and non-cooperativelyin multiple back-and-forth small steps, suggesting that the molecule un-dergoes an extensive conformational search. Most surprisingly, as a(CAG)100 RNA is extended by >100 nm, tension on the molecule remainslargely unchanged at ~13.550.4 pN, which is not expected for an elasticpolymer. This unusual viscoelasticity implies that force is unevenly distrib-uted in the molecular structures of the (CAG)n RNAs which possibly takethe form of multi-branched, similarly-sized hairpins. The relatively constantunfolding force indicates that the folding energy landscape is almost flat atthe force, and the small force fluctuation suggests that the energetic barriersbetween different conformations are very low. When a (CAG)n RNA isnanomanipulated to be partially unfolded, the molecule is trapped in a subsetof conformations, as evident by the constant value of the mean extension.This observation leads to a hypothesis that the folding of (CAG)n RNAsis dominated by topological constraints, and the existing conformation isaffected by preceding structures. Our findings support the structural poly-morphism hypothesis for the (CAG)n RNAs and provide evidence forsequential folding of repeated sequences.
