Talk abstracts
Friday 01:00-01:12pm: Selectivity in substrate binding by the exonuclease Rrp6p
Armend Axhemi (Center for RNA Molecular Biology, Case Western Reserve University), Sukanya Srinivasan (Center for RNA Molecular Biology, Case Western Reserve University), Elizabeth Wasmuth (HHMI and Memorial Sloan Kettering Institute), Christopher Lima (HHMI and Memorial Sloan Kettering Institute), Eckhard Jankowsky (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract not available online - please check the printed booklet.
Friday 01:12-01:24pm: CELF1 is necessary for myofibril organization and cardiac morphology during embryonic development
Yotam Blech-Hermoni (Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine), Michael W. Jenkins (Department of Pediatrics, Case Western Reserve University School of Medicine), Oliver Wessely (Department of Cellular and Molecular Medicine, Lerner Research Institute Cleveland Clinic), Andrea N. Ladd (Department of Cellular and Molecular Medicine, Lerner Research Institute Cleveland Clinic)
Abstract:
CUG-BP and Elav-like family member 1 (CELF1) is an RNA-binding protein expressed in a variety of tissues, including the embryonic and adult heart, where it is restricted to the cardiomyocytes (the contractile cells). CELF1 regulates alternative pre-mRNA splicing in the nucleus, as well as transcript degradation and translation in the cytoplasm. While it is predominantly cytoplasmic in other embryonic tissues, its expression in skeletal and cardiac muscle cells is predominantly nuclear, and this pattern is conserved from frog to mouse. Upon CELF1 knockdown in cultured chicken primary embryonic cardiomyocytes, we observed a decrease in cell proliferation as well as profound disorganization of myofibrillar structure. While cells continued to beat, sarcomeres shifted from rod-like striations along thick, long fibrils to globular puncta arranged in thin, often web-like, matrices. These effects are accompanied by changes in the splicing patterns of genes involved in contractility and the regulation of cardiac gene expression. Following knockdown of celf1 in frog (Xenopus laevis) embryos, we observed aberrant cardiac morphology (smaller, thicker, and often misshapen hearts). We are using optical coherence tomography – a non-invasive imaging modality – to evaluate changes in cardiac function and 3D structure in both fixed and live embryos. We have demonstrated that frog celf1 can regulate alternative splicing, upon expression in cultured cells, and this activity can be repressed by co-expression of a nuclear-retained dominant negative CELF protein. We will use this dominant negative protein to dissect the involvement of nuclear versus cytoplasmic functions of celf1 in cardiac structure and function during embryonic development.
Keywords: RNA binding protein, CELF1, heart development
Friday 01:24-01:36pm: Pnrc2 is required for rapid decay of oscillating transcripts via their 3’ UTR
Thomas L. Gallagher (Molecular Genetics, The Ohio State University), Nicolas L. Derr, Kiel T. Tietz (Molecular Genetics, The Ohio State University), Courtney E. French, Jasmine M. McCammon, Michael L. Goldrich (Molecular and Cell Biology, University of California, Berkeley), Steven E. Brenner (Molecular and Cell Biology, University of California, Berkeley), Sharon L. Amacher (Molecular Genetics, The Ohio State University)
Abstract:
The vertebrate segmentation clock controls segment formation by regulating oscillating gene expression. The clock cycles rapidly with a period of 30-120 minutes, depending upon the vertebrate model. At the core of the clock are a family of auto-inhibitory transcriptional repressors encoded by the hairy/Enhancer of split-related (her or Hes) genes. Although many studies have elucidated transcriptional mechanisms important for oscillatory expression, fewer have investigated mechanisms of post-transcriptional processing that promote rapid clearance of Hes/her transcripts. In a zebrafish screen for genes involved in cyclic transcript regulation, we uncovered the tortuga mutation. tortugab644 mutants accumulate cyclic transcripts post-splicing. The gene affected in tortuga mutants is pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in promoting mRNA decay. We hypothesize that Pnrc2 is part of an mRNA decay complex that recognizes elements and/or structural features of developmentally-regulated transcripts. In support of this hypothesis, we find that pnrc2 genetically interacts with upf1, a nonsense-mediated decay (NMD) core component.
In order to define the instability feature(s) recognized by the Pnrc2 decay complex, we have developed an inducible in vivo reporter system to test transcript stability in zebrafish embryos. We show that the 3’ UTR of her1 is sufficient to confer instability to an otherwise stable heterologous transcript in a Pnrc2-dependent manner. Deletion constructs reveal that important features reside in the proximal half of the her1 3’ UTR and studies are underway to define the instability determinants. In parallel, we are comparing the her1 3’ UTR with other potential Prnc2-regulated targets that we identified via RNA-seq of Pnrc2-depleted embryos. Our goal is to define the regulatory logic controlling targeted degradation of oscillating transcripts.
Keywords: mRNA decay, pnrc2, oscillating expression
Friday 01:36-01:48pm: Mechanistic insight into the association of the NMD factor, Upf1p, with mRNA in Saccharomyces cerevisiae
DaJuan L. Whiteside (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)
Abstract:
Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control pathway that identifies and targets aberrant mRNAs for accelerated degradation. The rapid turnover of nonsense-containing mRNAs promotes fidelity in gene expression by preventing the accumulation of truncated polypeptides that could have deleterious effects to the cell. While three core proteins are required for NMD in yeast - namely Upf1p, Upf2p, and Upf3p - Upf1p is the only factor with known enzymatic activity and an ability to bind RNA directly. Key mechanistic events underlying NMD substrate recognition currently remain unclear, including the dependence of Upf1p binding on mRNA translation and how specifically the NMD machinery is recruited to target substrates.
We have developed a biochemical assay involving RNA immunoprecipitation (RIP) coupled with quantitative RT-PCR to evaluate mRNA features and protein requirements for Upf1p binding in the yeast Saccharomyces cerevisiae. Specifically, chromosomally expressed, epitope-tagged Upf1p is immunoprecipitated from yeast cell lysates and associated RNA quantified by real-time PCR. Taqman probes specific to endogenous or plasmid-based mRNA reporters allow for multiplex PCR reactions and the evaluation of multiple RNAs in a single sample.
Our studies have revealed the following: (i) Upf1p preferentially binds nonsense-containing mRNA, (ii) Upf1p binding to RNA is not dependent on the other NMD factors Upf2p or Upf3p, (iii) translation enhances, but is not absolutely required for Upf1p binding, (iv) Upf1p displays a length-dependent association with nonsense-containing mRNAs and, lastly, (v) secondary structure downstream of a premature termination event can stabilize NMD substrates and modify Upf1p association with the mRNA. Additionally, analysis of previously characterized mutant alleles of Upf1p reveal that neither the ATP binding or hydrolysis activity of Upf1p is required for its binding to RNA. Taken together, our data provide insight into the mechanism of Upf1p association with mRNA and NMD substrate recognition in yeast.
Keywords: mRNA decay
Friday 01:48-02:00pm: Significance of novel RNA-protein interaction in U12-dependent splicing
Jagjit Singh MS (Center of Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH-44115), Kavleen Sikand Ph.D., Tupa Basu Roy M.S, Girish C. Shukla Ph.D.
Abstract:
Recently, a second class of spliceosome that removes a distinct set of introns was revealed. Even though minor class introns constitute only <1 % of the total introns, their host genes are involved in very critical cellular functioning for example cell proliferation, DNA replication and repair. U12 and U6atac snRNAs, the functional analogs of U2 and U6 of major class spliceosome, play a pivotal role in the removal of U12-dependent introns by performing almost similar RNA-RNA interactions with the intron. In addition, U6atac-U12 interaction is known to establish an intermolecular base paired helix I region. Beyond these known functional regions of U6atac and U12 snRNAs, several regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that 3’ stem-loop region of U6atac snRNA serves as U12-dependent spliceosome specific guide element. In this work, we show the functional requirement of an evolutionary redundant substructure of U6atac 3’ stem-loop in minor class in vivo splicing. Our in vitro data show that C-terminal RRM (RNA recognition motif) of p65, U11/U12 18s complex protein, interacts with distal stem-loop substructure of U6atac 3’ stem-loop. Further, we show that the functionally important p65 protein-binding apical stem-loop of U12 snRNA can be replaced by U6atac stem-loop. Using binary splice site mutation suppressor assay, we demonstrate that the exchange of these RNA elements between U12 and U6atac is functional in in vivo splicing. In addition, we found that structure of this distal 3’ stem loop is more important as compared to the sequence for in vivo splicing. We have also shown that 3’ end of U6atac from distant species with different secondary structure and sequence could catalyze both the splicing as well as the interaction with p65 to different extent, indicating its evolutionarily conserved function. These observations support the idea that both the RNA-RNA as well as the RNA-protein interactions occurring in minor class spliceosome are extremely flexible as compared to major class spliceosome.
Keywords: U12-dependent splicing, U6atac snRNA, p65
Friday 02:00-02:12pm: Conformational control of the U2 snRNA by Cus2p
Sandy Tretbar (a University of Wisconsin-Madison, Biochemistry Department, 433 Babcock Dr, Madison, WI, 53706), Aaron A. Hoskins (a University of Wisconsin-Madison, Biochemistry Department, 433 Babcock Dr, Madison, WI, 53706)
Abstract:
Tremendous RNA rearrangements occur during yeast spliceosome assembly and catalysis. These include both intermolecular rearrangements between different snRNAs and/or the pre-mRNA substrate or intramolecular conformational dynamics within an individual snRNA. One example of intermolecular dynamics involves structural transitions within the U2 snRNA. Two mutually exclusive conformations of the U2 snRNA promote either binding to the pre-mRNA branchsite (stem IIa) or the catalytic steps of splicing (stem IIc). Two key players in this process are yeast Cus2p and Prp5p. Prp5p hydrolyzes ATP to promote duplex formation between the U2 snRNA and the pre-mRNA; however, this function is only required in the presence of Cus2p. The detailed biochemical relationship between Cus2p, Prp5p, and U2 snRNA conformation remains unexplored.
Using an in vitro model system, we show that Cus2p binds to the stem II region of the U2 snRNA (U2 core) with a clear preference for binding to the stem IIa conformation. Prp5p appears to remodel the Cus2p/U2 core interaction resulting in formation of a Prp5/U2 core complex. Preliminary single molecule FRET analysis reveals that U2 core RNA is dynamic and that Cus2p stabilizes a particular FRET conformation of the RNA consistent with stem IIa formation. We hypothesize that Cus2p may function as a chaperone to conformationally select the stem IIa form of the U2 snRNA. Cus2p then holds the snRNA in this conformation until arrival of Prp5p in preparation for branchsite duplex formation. Significantly, this demonstrates how two different classes of RNA binding proteins (the RRMs of Cus2p and the DEAD-box of Prp5p) can work collaboratively to regulate and control RNA conformation.
Keywords: U2 snRNA, Cus2p, Prp5p
Friday 02:12-02:24pm: Phosphorylation regulates assembly of cancer-relevant splicing factors SF3b155 and SPF45
Jake A. Kloeber (Center for RNA Biology, University of Rochester Medical Center, Rochester, NY), Sarah Loerch (Center for RNA Biology, University of Rochester Medical Center, Rochester, NY), Clara L. Kielkopf (Center for RNA Biology, University of Rochester Medical Center, Rochester, NY)
Abstract not available online - please check the printed booklet.
Friday 02:24-02:36pm: Codon optimization is a major determinant of mRNA half-life in Saccharomyces cerevisia
Vladimir Presnyak (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Ying-Hsin Chen, Sophie Martin, Najwa Al Husaini, Nicholas Kline (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Nathan Morris (Statistical Science Core in the Center for Clinical Investigation, Case Western Reserve University, Cleveland, OH), Sara Olson, Brenton Graveley (Department of Genetics and Developmental Biology, Institute for Systems Genomics, University of Connecticut Health Center, Farmington, CT), Kristian Baker (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Jeff Coller (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH)
Abstract not available online - please check the printed booklet.
Friday 02:36-02:48pm: NusA-dependent transcription termination in bacteria
Smarajit Mondal, (Dept. of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University), Alexander V. Yakhnin (Dept. of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University), Paul Babitzke (Dept. of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University)
Abstract not available online - please check the printed booklet.
Friday 02:48-03:00pm: Numerous C/D box snoRNAs are associated with the spliceosome, form non-canonical ribonuclear protein complexes and regulate alternative splicing
Marina Falaleeva (Biochemistry, University of Kentucky, USA), Amadis Pages (Universitat Pompeu Fabra, Barcelona, Spain), Zaneta Matuszek (Biochemistry, University of Kentucky, USA), Eduardo Eyras (Universitat Pompeu Fabra, Barcelona, Spain), Ruth Sperling (The Hebrew University of Jerusalem, Jerusalem, Israel), Stefan Stamm (Biochemistry, University of Kentucky, USA)
Abstract not available online - please check the printed booklet.
Friday 03:30-03:42pm: Regulation of the RNA-Activated Protein Kinase (PKR) by Highly Structured Direct and Indirect Bacterial Riboswitches
Chelsea Hull (Chemistry and Center for RNA Molecular Biology), Philip C. Bevilacqua (Chemistry and Center for RNA Molecular Biology)
Abstract not available online - please check the printed booklet.
Friday 03:42-03:54pm: Phylogenetic, Structure and thermodynamic insight of HIV-1 Intronic splicing silencer (ISS)
Niyati Jain (Department of Chemistry, Case Western Reserve University), Brittany D. Rife (Department of Pathology, Immunology and Laboratory of Medicine, College of Medicine, and Emerging Pathogens Institute, University of Florida, Gainesville), Christopher E. Morgan (Department of Chemistry,Case Western Reserve University), Blanton S. Tolbert (Department of Chemistry,Case Western Reserve University)
Abstract:
Alternative splicing is a crucial process occurring during the human immunodeficiency virus type 1 (HIV-1) life cycle. This process is primarily regulated by interaction between the various splice sites within the viral RNA genome and trans host partners that regulate the splicing process by activating or repressing splice site usage. The host proteins hnRNP A1 and ASF regulate the splicing pattern in HIV-1 transcript by binding to highly conserved 3’splice site ssA7. Splice site A7 is 175 nucleotide long RNA and composed of 3 stem- loop structures that contain splicing regulatory elements ISS (SL1), ESE3 (SL2) and ESS3 (SL3) that bind to trans host proteins and thus regulate the splicing pattern .The mechanism by which this ssA7 recruits these host proteins and regulate the splicing pattern in HIV-1 is not well characterized. The high- resolution solution structure of ssA7 RNA and its interaction studies with its host factors will give the insight about the mechanism involved. Solving the NMR structure of large RNA like ssA7 is challenging and so we have applied divide and conquer approach. The ESS3 solution structure was recently solved and now we are focusing on Intron splicing silencer (ISS).
The ISS is a 50-nucleotide long stem-loop RNA with several non-canonical elements that may add to the dynamics and function of this molecule. Toward understanding the structure -based mechanism of the ISS function, NMR spectroscopy has been used to probe its solution properties. Here, we present a solution structure of ISS.
Keywords: HIV-1, Intronic splicing silencer, NMR
Friday 03:54-04:06pm: Next-generation tools for RNA enzymology: Determination of rate and equilibrium constants for large populations of RNA substrate variants using high throughput sequencing.
Hsuan-Chun Lin (Department of Biochemistry, School of Medicine, Case Western Reserve University), Courtney Niland (Department of Biochemistry, School of Medicine, Case Western Reserve University), Jing Zhao (Department of Biochemistry, School of Medicine, Case Western Reserve University), David R. Anderson ( Zicklin School of Business, Baruch College, CUNY ), Eckhard Jankowsky (Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University), Michael E. Harris (Department of Biochemistry, School of Medicine, Case Western Reserve University)
Abstract:
Structure-function studies of RNA binding and RNA-processing reactions, in which the effects of specific variations in sequence on specific reaction parameters such as binding kinetics, equilibrium binding affinity and catalytic rate, have provided deep insights into biological function to be gained. Nonetheless, our perspective is severely limited by the relatively small number of sequence variants that can be analyzed. Using RNase P processing of pre-tRNA as an experimental system are developing a set of tools based on high-throughput sequencing and competitive kinetic analysis to accurately and simultaneously determine kinetic and equilibrium binding constants for large RNA substrates. The resulting high-density structure-function data sets are providing unique insights into patterns of molecular recognition and the nature of specificity in RNA-protein interactions. Although powerful, an inherent limitation of competitive multiple turnover kinetics is that product inhibition, inactive substrate populations and multiphasic kinetics can limit precision. Using single turnover reactions which conform more directly to simple exponential kinetics should allow high resolution data sets to be gained for both binding kinetics and effects on catalysis. A similar approach is being developed to determine equilibrium binding constants by analyzing the distribution of sequences in free and bound populations separated by EMSA using simple competitive binding models. Since the high throughput kinetics gives us the entire affinity distribution, the classical methods such as sequence logos and position weight matrix provide restricted views of specificity that ignore information contain the affinity distribution accordingly. We develop a new multivariable regression method which considers the interaction between different positions. In combination these approaches are providing a comprehensive understanding of how substrate sequence and structure affect binding affinity, association kinetics and catalysis.
Keywords: Enzyme Kinetics, RNase P, high-throughput sequencing
Friday 04:06-04:18pm: Entropy As a Key Factor for One Step Formation of Phi29 pRNA 3WJ from Three RNA Fragments
Daniel Binzel (Nanobiotechnology Center, Markey Cancer Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA), Emil Khisamutdinov (Nanobiotechnology Center, Markey Cancer Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA), Peixuan Guo (Nanobiotechnology Center, Markey Cancer Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA)
Abstract:
The applicational use of functional RNA nanoparticles requires the creation of chemically and thermodynamically stable RNA core scaffolds. We have discovered an ultra-stable three-way junction (3WJ) in the bacteriophage phi29 motor packaging RNA (pRNA). This 3WJ can assemble from three short RNA oligos with high efficiency. Thermodynamic studies during assembly of the 3WJ indicated no dimer intermediate formation as the RNAs were only seen as monomer components or assembled three way junction; meaning the 3WJ does not follow the usually seen single assembly pathway of A + B + C ↔ AB + C ↔ ABC. A + B + C leads to the formation of ABC efficiently within a rapid time. Additionally, the 3WJ displayed high thermodynamic stability by exhibiting a melting temperature (TM) of 59.3 ± 1.7 °C with the substitution of 2’-fluoro (2’-F) nucleotides increasing the TM to 69.8 ± 2.0 °C, while the DNA 3WJ displayed a melting temperature of only 48.9 ± 3.2 °C. Furthermore, the Gibbs free energy of formations (ΔG°37) were found to be -36, -28, and -15 kcal/mol for 3WJ2’-F, 3WJRNA, and 3WJDNA, respectively. Surprisingly, the added stability, indicated by the more negative ΔG°37, in the two RNA species of the pRNA-3WJ was attributed to the more favorable change in entropy (ΔS°), compared to DNA instead of the commonly seen more favorable enthalpy change. The unique thermodynamic stability and folding pathway of the pRNA-3WJ makes it an ideal candidate for RNA nanoparticles and nanoscaffolds.
References:
1. Guo P. The emerging field of RNA nanotechnology. Nature Nanotechnology. 2010; 5(12):833-42.
2. Shu D, Shu Y, Haque F, Abdelmawla S, Guo P. Thermodynamically stable RNA three-way junction as a platform for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology. 2011; 6(10):658-67.
3. Binzel DW, Khisamutdinov EF, Guo P. Entropy-Driven One-Step Formation of Phi29 pRNA 3WJ from Three RNA Fragments. Biochemistry. 2014 Apr 15; 53(14):2221-31.
Keywords: Phi29, Thermodynamics, RNA Nanotechnology
Friday 04:18-04:30pm: DRBD18 is an essential Trypanosome RNA binding protein that modulates RNA abundance and is regulated by arginine methylation
Kaylen Lott (Microbiology and Immunology, SUNY at Buffalo), Shreya Mukhopadhyay (Microbiology and Immunology, SUNY at Buffalo), Jie Yao (Microbiology and Immunology, SUNY at Buffalo), Jun Li, Jun Qu (Pharmaceutical Sciences, SUNY at Buffalo), Yijun Sun (Microbiology and Immunology, SUNY at Buffalo), Laurie Read (Microbiology and Immunology, SUNY at Buffalo)
Abstract:
Unlike most eukaryotes, T. brucei regulates gene expression almost entirely post-transcriptionally, thus RNA binding proteins are key factors in gene regulation. In yeast and humans, RNA binding proteins constitute a significant proportion of proteins that are substrates for arginine methylation, and methylation can dramatically affect their functions. Indeed, our global proteomic screen in T. brucei revealed ~100 arginine methylproteins with known or predicted functions in RNA metabolism. DRBD18 is an RRM (RNA recognition motif)-containing protein that harbors three methylarginines positioned between its two RRMs. DRBD18 repression leads to a severe growth defect in procyclic form T. brucei. RNAseq analysis of DRBD18-repressed cells identified 417 transcripts that decrease in abundance and 568 transcripts that increase 1.8 to 40-fold upon DRBD18 depletion. To begin to understand the role of arginine methylation in DRBD18 function, we performed in vivo cross-linking immunoprecipitation (CLIP) assays in trypanosomes harboring DRBD18 mutated at the identified methylated arginines. Intriguingly, R to F methylmimic DRBD18 exhibits a substantial loss of RNA binding in vivo, while non-methylatable R to K mutants display increased RNA binding. Moreover, in complementation assays, both methylmimic and non-methylatable DRBD18 fail to fully complement growth of the DRBD18 knockdown cell line. Together, these data suggest that DRBD18 RNA binding activity is regulated by arginine methylation, and mis-regulation of its RNA binding can impact parasite growth. When these complemented cells were probed for their ability to restore RNA levels altered by repression of DRBD18, nonmethylatable and methylmimc cells displayed opposite phenotypes on RNA levels. Mass spectrometry reveals that DRBD18 methylmutants bind distinct RNA binding proteins which may influence DRBD18 function. These data are consistent with a model in which methylation modulates DRBD18 affects on protein-RNA and protein-protein interactions.
Keywords: RNA binding protein, arginine methylation, transcriptome
Friday 04:30-04:42pm: Title not available online - please see the printed booklet.
Gayan Mirihana Arachchilage (Department of Chemistry and Biochemistry, Kent State University), Arosha C. Dassanayake (Department of Chemistry and Biochemistry, Kent State University), Soumitra Basu (Department of Chemistry and Biochemistry, Kent State University)
Abstract not available online - please check the printed booklet.
Friday 04:42-04:54pm: Dissecting miRNA function and localization using single molecule approaches
Laurie A. Heinicke (Chemistry, University of Michigan), Sethuramasundaram Pitchiaya (Chemistry, University of Michigan), Elizabeth L. Cameron (Chemistry, University of Michigan), Nils G. Walter (Chemistry, University of Michigan)
Abstract:
MicroRNA (miRNA) genes are transcribed in the nucleus as primary miRNA transcripts (pri-miRNA) and processed via multiple steps to generate mature miRNAs in the cytoplasm. These small non-coding RNA (ncRNAs) associate with components of the RNA-induced silencing complex (RISC) and engage mRNA targets and regulate gene repression via translational inhibition and/or mRNA degradation. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, the intracellular dynamics and assembly of miRNA-associated complexes and the spatiotemporal modulation of miRNA-regulated gene expression remain unclear. To uniquely probe intracellular RNA silencing pathways, our lab has developed a method termed intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) to determine the localization, diffusion constant and assembly state of single miRNA complexes inside living human cells at 30 nm spatial and 100 ms temporal resolution. We have extended the capabilities of iSHiRLoC to simultaneously detect two colors and examined the diffusion and co-localization of microinjected fluorophore-labeled miRNAs and fluorescent protein labeled P-bodies, sub-cellular foci enriched in RNA degrading enzymes. Our data indicate that there are at least two types of temporarily increasing miRNAs that reside in P-bodies for ~1.18 s and >10 s, representing transient and tight association with P-bodies, respectively, wherein the latter likely reflects miRNA-directed mRNA degradation. In addition, we have used iSHiRLoC to probe nuclear functions of miRNAs. We find a significant fraction of microinjected mature let-7-a1 localizes to the nucleus, whereas artificial cxcr4 miRNA localizes to the nucleus at a much lower extent. Inhibition of transcription significantly reduces let-7-a1 nuclear localization, similar to the level of cxcr4 in the absence or presence of transcription inhibitor, suggesting that only let-7-a1 binds RNA targets in the nucleus. We are currently pursuing complementary biochemical approaches, such as subcellular fractionation and Northern blotting, to further validate our iSHiRLoC observations. Together, these studies highlight the versatility of iSHiRLoC by providing biological insight regarding assembly and localization of intracellular miRNAs.
Keywords: miRNA, single molecule , fluorescence microscopy
Friday 04:54-05:06pm: Novel functions of cytochrome C in the cellular response to stress
Raul Jobava (Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Mridusmita Saikia (Department of Pharmacology, Case Western Reserve University, Cleveland, OH ), Andrea Putnam (RNA Center/Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Tao Pan (Department of Biochemistry and Biophysics, University of Chicago, Chicago, IL), Eckhard Jankowsky (RNA Center/Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Maria Hatzoglou (Department of Pharmacology, Case Western Reserve University, Cleveland, OH )
Abstract:
Regulation of cell volume in response to changes of extracellular tonicity is essential for cell survival. Hyperosmotic stress is associated with inflammation, diabetes and other diseases. Severe hyperosmotic stress induces cytochrome C release from the mitochondria, formation of the apoptosome and initiation of apoptosis. Hyperosmotic stress also activates the ribonuclease Angiogenin, which cleaves a small pool of tRNAs in the anticodon loop producing 30-45 nt long half tRNA molecules known as tiRNAs (tRNA-derived stress-induced fragments). The subcellular localization and molecular function of these small RNAs is largely unknown. Here we demonstrate that certain tiRNAs bind cytochrome C in the cytoplasm after it is released from mitochondria. Next generation sequencing revealed that twenty tiRNAs are highly enriched in this cytochrome C ribonucleoprotein complex. Direct binding of cytochrome C to tiRNAs within the cytochrome C ribonucleoprotein complex was confirmed using RNA gel-shift analysis. Our preliminary data suggest, that certain tiRNAs have prosurvival functions during hyperosmotic stress by inhibiting apoptosome formation. Our findings also reveal novel functions of cytochrome C during hyperosmotic stress which can be further explored for therapeutic purposes of protecting healthy cells from apoptosis.
Keywords: tiRNA, cytochrome C, stress
Friday 05:06-05:18pm: Multiple Conformational Sampling Achieved By Alternate Base-Pairing in Loop A of the Hairpin Ribozyme
Patrick Ochieng (Michigan State University), BeiBei Wang (Michigan State University), Michael Feig (Michigan State University), Charles Hoogstraten (Michigan State University)
Abstract not available online - please check the printed booklet.
Friday 05:18-05:30pm: Using Protein mRNA tethering to identify novel splicing factors
Benjamin Cieply (Department of Genetics, Perelman School of Medicine, Univeristy of Pennsylvania), Russ Carstens (Department of Genetics, Perelman School of Medicine, Univeristy of Pennsylvania)
Abstract not available online - please check the printed booklet.
Saturday 08:30-08:42am: Ribosomal proteins influence RNA dynamics during 30S assembly
Sanjaya Abeysirigunawardena (T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore MD ), Hajin Kim (Department of Physics and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL ), Taekjip Ha (Department of Physics and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL ), Sarah Woodson (T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore MD )
Abstract:
Binding of ribosomal proteins with the rRNA has been shown to stabilize productive assembly intermediates that lead to the formation of active ribosomes. Recently our single-molecule FRET experiments showed binding of ribosomal protein S4 to the 5’-domain of 16S rRNA, modulate the dynamics of rRNA during early stages of 30S assembly. Initial highly dynamic S4-rRNA complexes go through a stable non-native intermediate complex before forming the native complex. Furthermore, fluctuations between non-native intermediate complex in which h3 flipped (F), and fully folded native complex with h3 docked under the base of h18 are observed at equilibrium. Ribosomal proteins S17, S16 and S20 bind to the core region of the 5’-domain RNA. These proteins stabilize certain RNA-RNA and RNA-Protein contacts hence can preferentially stabilize different S4-rRNA complexes. In this research S4 titrations in bulk and single molecule ALEX-FRET (Alternative Excitation Foster Resonance Energy Transfer) experiments are used to understand the effects of 5’-domain binding proteins on equilibrium between native and intermediate S4-RNA complexes. Surprisingly, protein S17 stabilizes the non-native intermediate complex, which is in agreement with the hydroxyl radical footprinting experiments. Protein S20 binds farther away from h3 and shows less influence on the equilibrium between native and non-native S4-RNA complexes. Interestingly however, S4 titrations in bulk showed that presence of proteins S20 and S16 together strongly favored the native S4-RNA complex. Neither protein S20 nor S16 showed significant perturbation to the equilibrium when added individually. Our 3-color single molecule ALEX-FRET experiments showed that native S4-rRNA complex becomes is highly preferred in complexes that are bound with protein S16. However, addition of protein S20 to RNA-S4-S16 complex did not increase the preference to native complex. Nevertheless, addition of protein S20 increased the number of stable RNA-S4-S16 complexes. Real-time S16Cy3 binding experiments showed that S16 binds preferably to native S4-RNA complex. Interestingly, binding of S16 protein slows down the motions of helix3 and stabilizes native complex that is required for the addition of more proteins during the ribosomal assembly.
Keywords: RNA-protein interactions, Ribosome assembly, smFRET
Saturday 08:42-08:54am: Title not available online - please see the printed booklet.
Rohan Balakrishnan (Ohio State Biochemistry Program), Kenji Oman (Department of Physics), Ralf Bundschuh (Department of Physics), Kurt Fredrick (Department of Microbiology, The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 08:54-09:06am: Eukaryote-specific extensions of ribosomal proteins are involved in 60S ribosomal subunit assembly
Beril Kumcuoglu (Carnegie Mellon University), Jelena Jakovljevic (Carnegie Mellon University), John L. Woolford (Carnegie Mellon University)
Abstract:
Recently, our lab demonstrated that the domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical manner. Importantly, the function of ribosomal proteins (r-proteins) in pre-rRNA processing correlates with their location in the mature 60S subunit. Depletion phenotypes reveal the earliest defect in the assembly pathway, after which pre-ribosomes are turned over. Therefore, to understand the functions of r-proteins in more detail, we need to construct more specific mutations.
One of our goals is to distinguish the functions of flexible eukaryote-specific extensions of r-proteins from these of their globular domains. Interestingly, eukaryotic elements are clustered at the solvent-exposed side of the 60S subunit while the peptidyl-transferase center is devoid of these additions. We suggest that these eukaryotic elements have a role in stabilization of pre-ribosomes by providing additional protein-protein and protein-rRNA interactions. To test this hypothesis, we systematically truncated and introduced missense mutations to the eukaryotic extensions of four r-proteins (L7, L8, L25, L35). Our data indicate that the extensions of these four proteins are essential for viability. Truncation mutations of L25 and L35 resulted in the same pre-rRNA processing defects as depletions of each protein. In contrast, a partial truncation mutation of L8, along with a missense mutation of L7 resulted in different pre-rRNA processing defects than their depletions. These results led us to propose a model where the N-terminal extension of L8 is involved in a later step of ribosome assembly than its globular domain, potentially rearranging the 5’ end of 25S and the 3’ end of 5.8S rRNAs in domain V.
Our findings indicate that the flexible eukaryotic extensions of ribosomal proteins are involved in 60S subunit maturation. Additionally, our approach provides insights into the evolution of protein-protein and protein-rRNA interactions in ribosomes from bacteria to eukaryotes.
References:
Gamalinda, M., Ohmayer, U., Jakovljevic, J., Kumcuoglu, B., Woolford, J., Mbom, B., Lin, L., et al. (2014). A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains. Genes & development, 28(2), 198–210. doi:10.1101/gad.228825.113
Keywords: pre-rRNA processing, ribosome assembly, ribosomal proteins
Saturday 09:06-09:06am: Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and NMD.
Ashton T Belew (University of Maryland), Arturas Meskauskas (University of Maryland), Sharmishtha Musalgaonkar (University of Maryland), Vivek M Advani (University of Maryland), Jonathan D Dinman (University of Maryland)
Abstract:
Programmed -1 ribosomal frameshift (-1 PRF) signals redirect translating ribosomes to slip back one base on mRNAs. While well characterized in viruses, how this may regulate cellular gene expression is not understood. Here, we describe a -1 PRF signal in the human mRNA encoding CCR5. CCR5 encodes a cytokine receptor which is also utilized by HIV-1 as a co-receptor for entry into CD4+ T-cells. CCR5 mRNA-mediated -1 PRF is directed by a structurally dynamic mRNA pseudoknot. Sequence-specific stimulation of CCR5-mediated -1 PRF is promoted by at least two miRNAs. Mapping the mRNA/miRNA interaction suggests that formation of a triplex RNA structure stimulates -1 PRF by stabilizing the frameshift-promoting mRNA pseudoknot. A -1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay (NMD) pathway. At least one additional mRNA decay pathway is also implicated. The native CCR5 mRNA is shown to be a substrate for NMD, and siRNA knockdown of NMD results in increased expression of CCR5p. Conversely, stimulation of -1 PRF by specific miRNAs results in decreases steady-state abundance of the CCR5 mRNA, and decreased CCR5p expression. Functional -1 PRF signals that appear to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells.
Keywords: Frameshifting, Pseudoknot, miRNA
Saturday 09:18-09:30am: EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation
Sara Elgamal (Microbiology, The Ohio State University), Assaf Katz (Microbiology, The Ohio State University), Steven J. Hersch (Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada), David Newsom, Peter White (Center for Microbial Pathogenesis, The Research Institute at Nationwide Childrens Hospital), William W. Navarre (Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada), Michael Ibba (Microbiology, The Ohio State University)
Abstract:
Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the ∆efp strain, with up to a 45-fold increase in ribosome density observed at the pausing site. The unexpectedly low fraction of PPX motifs that produce a pause in translation led us to investigate the possible role of sequences upstream of PPX. Our data indicate that EF-P dependent pauses are strongly affected by sequences upstream of the PPX pattern. We found that residues as far as 3 codons upstream of the ribosomal peptidyl-tRNA site had a dramatic effect on whether or not a particular PPX motif triggered a ribosomal pause, while internal Shine Dalgarno sequences upstream of the motif had no effect on EF-P dependent translation efficiency. Increased ribosome occupancy at particular stall sites did not reliably correlate with a decrease in total protein levels, suggesting that in many cases other factors compensate for the potentially deleterious effects of stalling on protein synthesis. These findings indicate that the ability of a given PPX motif to initiate an EF-P-alleviated stall is strongly influenced by its local context, and that other indirect post-transcriptional effects determine the influence of such stalls on protein levels within the cell.
References:
Elgamal S, Katz A, Hersch SJ, Newsom D, White P, Navarre WW and Ibba, M. (2014) EF-P dependent pauses integrate proximal and distal signals during translation. PLoS Genetics. In Press.
Keywords: Ribosome profiling, translation, pausing, elongation factor P, proline
Saturday 09:30-09:42am: Base pair recognition by the Thg1 family 3'-5' polymerases
Krishna J Patel (Chemistry and Biochemistry,The Ohio State University), Paul Yourik (Chemistry and Biochemistry,The Ohio State University), Jane E Jackman (Chemistry and Biochemistry,The Ohio State University)
Abstract not available online - please check the printed booklet.
Saturday 09:42-09:54am: Cap homeostasis is independent of poly(A) tail length
Daniel L. Kiss (Center for RNA Biology, Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210), Kenji Oman (Center for RNA Biology, Department of Physics, The Ohio State University, Columbus, Ohio 43210), Julie A. Dougherty (Center for RNA Biology, Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210), Chandrama Mukherjee (Center for RNA Biology, Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210), Ralf Bundschuh (Center for RNA Biology, Department of Physics, Department of Chemistry and Biochemistry, and Division of Hematology, The Ohio State University, C), Daniel R. Schoenberg (Center for RNA Biology, Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210)
Abstract:
Cap homeostasis is a cyclical process of decapping and recapping that maintains the cap on a subset of the cytoplasmic transcriptome. Interfering with cytoplasmic capping results in the redistribution of target transcripts from polysomes to non-translating mRNPs, where they accumulate in an uncapped but nonetheless stable form. It is generally thought that decapping is preceded by shortening of the poly(A) tail to a length that can no longer support translation. Therefore recapped target transcripts would either have to undergo cytoplasmic polyadenylation or retain a reasonably long poly(A) tail if they are to return to the translating pool. We find that in cells that are inhibited for cytoplasmic capping there is no change in the overall distribution of poly(A) lengths or in the elution profile of oligo(dT)-bound cytoplasmic capping targets. Consistent with this observation there is little difference in poly(A) tail length of uncapped target transcripts recovered from non-translating mRNPs and capped forms of the same mRNAs on translating polysomes. Finally, an in silico analysis of cytoplasmic capping targets found significant correlations with genes encoding transcripts having uridylated or multiply modified 3’-ends, and with genes having multiple 3’-UTRs generated by alternative cleavage and polyadenylation.
Keywords: cytoplasmic capping, poly(A), cap
Saturday 09:54-10:06am: Title not available online - please see the printed booklet.
Katherine M. Anderson (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA), Ian M.C. Fleming (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA), Kirk W. Gaston (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, OH 45221, USA ), Pat A. Limbach (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati, Cincinnati, OH 45221, USA ), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA), Juan D. Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University, Columbus, 43210 Ohio, USA)
Abstract not available online - please check the printed booklet.
Saturday 10:06-10:18am: Ribosome release from non-stop mRNAs is essential
Heather A. Feaga (Department of Biochemistry and Molecular Microbiology, Pennsylvania State University), Kenneth C. Keiler (Department of Biochemistry and Molecular Microbiology, Pennsylvania State University)
Abstract:
Messenger RNAs frequently lack a stop codon due to RNase activity or aberrant transcription. In bacteria, when the ribosome initiates transcription on one of these non-stop RNAs, it cannot be released by classical termination factors. All bacteria have an RNA-based solution to this challenge- tmRNA- a molecule that has the activity of both a tRNA and an mRNA. tmRNA enters the A-site of the stalled ribosome and accepts the nascent polypeptide. The ribosome then resumes translation on the reading frame encoded by the mRNA-like portion of tmRNA. It is estimated that about 2-4% of all translation events end in tmRNA mediated release. Surprisingly, some species of bacteria, such as Caulobacter crescentus, can survive when tmRNA is deleted. Using a synthetic-lethal screen, we identified a back up system for tmRNA. ArfBCc can hydrolyze peptidly-tRNA translated from non-stop mRNA in vitro. Homologs are present in the majority of bacteria phyla, but are absent in species where tmRNA deletion has proved lethal. These data suggest that release of non-stop ribosomes may be essential in all bacteria.
Keywords: tmRNA, ArfB, ribosome release
Saturday 10:18-10:30am: Potential for novel regulation via AUG triplets at the 5’ termini of canonical leadered mRNAs of Escherichia coli
Heather Beck (Miami University), Ian Fleming (Ohio State University), Gary Janssen (Miami University)
Abstract not available online - please check the printed booklet.
Saturday 02:00-02:12pm: Helical Defects in MicroRNA Influence Protein Binding by TAR RNA Binding Protein
Roderico Acevedo (The Pennsylvania State University), Nichole Orench-Rivera (The Pennsylvania State University), Kaycee A. Quarles (The Pennsylvania State University), Scott A. Showalter (The Pennsylvania State University)
Abstract not available online - please check the printed booklet.
Saturday 02:12-02:24pm: HIV-1 alters host cellular gene expression in target cell specific manner through microRNA regulation
Debjani Guha (IDM, University of Pittsburgh), Allison Mancini (IDM, University of Pittsburgh), Jessica Sparks (IDM, University of Pittsburgh), Velpandi Ayyavoo (IDM, University of Pittsburgh)
Abstract not available online - please check the printed booklet.
Saturday 02:24-02:36pm: Title not available online - please see the printed booklet.
Priscilla Wong (CNAST, Department of Chemistry, Carnegie Mellon University), Kausik Chakrabarti (CNAST, Department of Chemistry, Carnegie Mellon University)
Abstract not available online - please check the printed booklet.
Saturday 02:36-02:48pm: Global analysis of RNA-protein interaction site and RNA secondary structure dynamics in response to the plant hormone abscisic acid
Matthew R. Willmann (Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA), Mengge Shan (Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA, and Genomics and Computational Biology Graduate Program, University of Pennsylvania, Philadelphia, PA 19104, USA), Fan Li (Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA, and Genomics and Computational Biology Graduate Program, University of Pennsylvania, Philadelphia, PA 19104, USA), Alsu Ibragimova (Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA), Sarah Foster (Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA), Brian D. Gregory (Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA, and Genomics and Computational Biology Graduate Program, University of Pennsylvania, Philadelphia, PA 19104, USA)
Abstract:
RNA-protein interactions are critical effectors throughout the life cycle of RNA molecules. In fact, transcription, pre-mRNA processing (splicing and polyadenylation), translation, subcellular localization, stability, and decay of RNAs are all driven by interactions with RNA-binding proteins (RBPs). Despite this fact, research into the global effect of RBPs on gene expression in plant transcriptomes has been lacking. Our lab has pioneered an RNase-mediated protein footprint sequencing method called protein interaction profile sequencing (PIP-seq) that allows for the global identification of RNA-protein interaction sites (protein-protected sites (PPSs)) and RNA secondary structure in eukaryotic transcriptomes. Here, we use this technique to simultaneously study the dynamics of these features in the moss Physcomitrella patens in response to the plant hormone abscisic acid (ABA). From this analysis, we identify hundreds of RBP interaction sites whose binding affinity changes within 30 and/or 60 minutes of ABA treatment. Several RNA sequence motifs are enriched among the differentially bound PPSs, suggesting that ABA is able to elicit some of its effects by changing the binding of one or more RBPs that regulate multiple mRNAs. Increased binding of at least one of these motifs by the cognate RBP seems to increase the stability of the RNA targets. In addition to changes in RNA-protein interactions and gene expression in response to ABA, we uncover the immediate early changes in RNA secondary structure, which correspond mostly to the formation of highly structured elements. Current research focuses on identifying the RBPs binding to the enriched sequence motifs, and studying the effect of ABA-induced differential binding by RBPs and RNA secondary structure on translation.
Keywords: RNA binding proteins, PIP-seq
Saturday 02:48-03:00pm: Comprehensive annotation of Physcomitrella patens small RNA loci reveals 23nt heterochromatic siRNAs dependent on a minimal Dicer-Like gene
Ceyda Coruh (Department of Biology, Penn State University), Sung Hyun Cho (Department of Biochemistry and Molecular Biology, Penn State University), Saima Shahid (Department of Biology, Penn State University), Qikun Liu (Department of Biology, Penn State University), Andrzej Wierzbicki (Department of Molecular, Cellular, and Developmental Biology, University of Michigan), Michael J. Axtell (Department of Biology, Penn State University)
Abstract not available online - please check the printed booklet.
Saturday 03:00-03:12pm: High-throughput probing of human lncRNA secondary structure by Mod-seq
Yizhu Lin (Department of Biological Sciences, Carnegie Mellon University), Gemma E. May (Department of Biological Sciences, Carnegie Mellon University), C. Joel McManus (Department of Biological Sciences, Carnegie Mellon University)
Abstract:
Over the past decade, long noncoding RNAs (lncRNAs) have been increasingly recognized as important regulators of multiple gene expression processes. A growing number have been associated with human development and diseases. One recently discovered lncRNA family, sno-lncRNA, are transcribed from a region responsible for two human neurological diseases, Prader-Willi and Angelman Syndromes (Yin et al., 2012). sno-lncRNAs are so named for their genomic architecture – they contain box C/D or box H/ACA snoRNAs at both their 5’ and 3’ ends. Yin and colleagues have shown that the Rbfox splicing factor Fox2 binds to sno-lncRNAs and that sno-lncRNA expression changes the alternative splicing of Fox2 targets. Because of their unique architecture and potential role as Fox2-binding sponges, understanding the biological function of sno-lncRNAs requires characterization of their structure.
To experimentally determine the secondary structures of sno-lncRNAs, we applied a recently developed method, Mod-seq (Talkish et al., 2014). Mod-seq allowed the rapid identification of SHAPE (1M7) modifications on the full length (1,178 nt) sno-lncRNA2. By calibrating our Mod-seq results with those produce by SHAPE probing of the P4-P5 domain of the tetrahymena group I intron, we calculated SHAPE-scores and used RNAstructure to produce models of sno-lncRNA2 secondary structure. The SHAPE constrained secondary structure of sno-lncRNA2 shows only ~36% similarity with the structure predicted from its sequence alone, underscoring the importance of using chemical modification data for RNA structure modeling. We find that Fox2 binding sites are typically located near internal loops in sno-lncRNA2, suggesting a structural context for the interaction with this splicing modulator. We are currently expanding this approach towards the analysis of other lncRNA, including additional members of the sno-lncRNA family.
References:
Yin, Q.-F., Yang, L., Zhang, Y., Xiang, J.-F., Wu, Y.-W., Carmichael, G. G., & Chen, L.-L. (2012). Long noncoding RNAs with snoRNA ends. Molecular Cell, 48(2), 219–230. doi:10.1016/j.molcel.2012.07.033.
Talkish, J., May, G., Lin, Y., Woolford, J. L., & McManus, C. J. (2014). Mod-seq: high-throughput sequencing for chemical probing of RNA structure. RNA (New York, NY). doi:10.1261/rna.042218.113
Keywords:
Saturday 03:15-03:35pm: The LARP1-motif contains structurally conserved but sequence diverged repeats
Roni M. Lahr (University of Pittsburgh, Department of Biological Sciences. Pittsburgh, PA 15260), Seshat M. Mack (University of Pittsburgh, Department of Biological Sciences. Pittsburgh, PA 15260), Annie Heroux (Brookhaven National Laboratory, Upton, NY 11973), Sarah P. Blagden (Imperial College London, Hammersmith Campus, London, W12 0NN, UK), Andrea J. Berman (University of Pittsburgh, Department of Biological Sciences. Pittsburgh, PA 15260)
Abstract not available online - please check the printed booklet.
Saturday 03:35-03:55pm: Uncovering the RNA binding protein machinery associated with age and gender during liver development
Praneet Chaturvedi (Bio-Health Informatics, School of Informatics & Computing, Indiana University- Purdue University, Indianapolis), Yaseswini Neelamraju (Bio-Health Informatics, School of Informatics & Computing, Indiana University- Purdue University, Indianapolis), Waqar Arif (Departments of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Departments of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Sarath Chandra Janga (Bio-Health Informatics, School of Informatics & Computing, Indiana University- Purdue University, Indianapolis)
Abstract:
Alterations in the expression of genes are known to be associated with changing age and gender although the mechanisms mediating such a change remain unclear. In the present study, we perform an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Majority (~60%) of the age-associated RBPs were found to be increasing in their expression levels with age. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations state that RBPs are important developmental regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. Thus, the compendium of RBPs and this study will not only help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes but also provide a foundation for identifying the regulators contributing to the splicing eQTLs increasingly being discovered in association studies.
References:
1) Innocenti, F., Cooper, G.M., Stanaway, I.B., Gamazon, E.R., Smith, J.D., Mirkov, S., Ramirez, J., Liu, W., Lin, Y.S., Moloney, C. et al. (2011) Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue. PLoS genetics, 7, e1002078.
2) Schroder, A., Klein, K., Winter, S., Schwab, M., Bonin, M., Zell, A. and Zanger, U.M. (2013) Genomics of ADME gene expression: mapping expression quantitative trait loci relevant for absorption, distribution, metabolism and excretion of drugs in human liver. The pharmacogenomics journal, 13, 12-20.
Keywords: Liver Development, Genome-wide Association, Regulatory Proteins