Talk abstracts

Friday 01:00-01:15pm: Antagonistic splicing factors hnRNP A1 and SRSF2 compete for binding to snRNA 7SK

Daniel C. Totten (Department of Biological Sciences, University of Pittsburgh), Andrea J. Berman (Department of Biological Sciences, University of Pittsburgh)

Abstract:
Transcription of protein-coding genes in metazoans is tightly regulated through controlled pausing and restarting of RNA polymerase II enforced by P-TEFb, a critical transcription factor1. P-TEFb is regulated through the reversible association with the snRNA 7SK and the 7SK-P-TEFb RNP, a ribonucleoprotein, which includes SRSF22. Upon P-TEFb dissociation from this RNP, 7SK RNA binds to several hnRNPs and forms the 7SK-hnRNP RNP3. To better understand the maintenance of these mutually exclusive RNPs, we investigated the binding of hnRNP A1 and SRSF2 to Stem III of 7SK RNA. We show for the first time that the RNA binding domains of hnRNP A1 melt Stem III and bind to its 3’ half with modest affinity, whereas the single RNA binding motif of SRSF2 is unable to open Stem III to access its binding sites and results in a 10-fold lower affinity for Stem III. Further, hnRNP A1 and SRSF2 efficiently compete and occlude binding by the competitor protein upon saturating conditions; both proteins bind the same RNA molecule when lower concentrations of proteins are used. However, SRSF2 is only efficiently recruited from Stem III to a competing RNA in the presence of UP1, suggesting a model in which hnRNP A1 and SRSF2 help maintain mutually exclusive RNPs through occlusion of the competitor protein, yet work together to aid in the transition between the two 7SK RNPs.

References:
1. Guo, J. and D.H. Price, RNA polymerase II transcription elongation control. Chem Rev, 2013. 113(11): p. 8583-603.
2. Ji, X., et al., SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase. Cell, 2013. 153(4): p. 855-868.
3. Diribarne, G. and O. Bensaude, 7SK RNA, a non-coding RNA regulating P-TEFb, a general transcription factor. RNA Biol, 2009. 6(2): p. 122-8.

Keywords: 7SK RNA, hnRNP A1, SRSF2

Friday 01:15-01:30pm: ESRP2 controls an adult splicing program in hepatocytes to support postnatal liver maturation

Amruta Bhate (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana Champaign), Darren J. Parker (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana Champaign), Thomas W. Bebee (Departments of Medicine and Genetics, Perelman School of Medicine, University of Pennsylvania), Jaegyoon Ahn (Department of Integrative Biology and Physiology, University of California, Los Angeles), Russ P. Carstens (Departments of Medicine and Genetics, Perelman School of Medicine, University of Pennsylvania), Auinash Kalsotra (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana Champaign)

Abstract:
Although major genetic networks controlling early liver specification and morphogenesis are known, the mechanisms responsible for postnatal hepatic maturation are poorly understood. Here we employ global analyses of the mouse liver transcriptome to demonstrate that postnatal remodeling of the liver is accompanied by large-scale transcriptional and post-transcriptional transitions that are cell-type-specific and temporally coordinated. Combining detailed expression analyses with gain- and loss-of-function studies, we identify Epithelial Splicing Regulatory Protein 2 (ESRP2) as a conserved regulatory factor that controls the postnatal switch of approximately 20% of splice isoforms in mouse and human hepatocytes. The normal shift in splicing coincides tightly with dramatic postnatal induction of ESRP2 in hepatocytes such that deletion of Esrp2 in mice results in failure of neonatal-to-adult splicing transitions in a spectrum of genes involved in cell proliferation, differentiation and adhesion. We further demonstrate that forced expression of ESRP2 in immature mouse and human hepatocytes is sufficient to drive a reciprocal shift in splicing. Phenotypic and biochemical characterization of Esrp2 null mice reveals defects in cell proliferation, hepatic zonation abnormalities, and reduction in albumin production. Taken together, these findings define a direct role for ESRP2 in the generation of conserved repertoires of adult splice isoforms that facilitate postnatal liver growth and maturation.

References:
Bhate, A; Parker, D et al. 'ESRP2 Controls an Adult Splicing Program in Hepatocytes to Support Postnatal Liver Maturation' Nature Communications (in press)

Keywords: Alternative splicing, ESRP2, liver maturation

Friday 01:30-01:45pm: The chromatin remodeling complex SWI/SNF is a master regulator of meiotic splicing in S. cerevisiae

Srivats Venkataramanan (Department of Molecular Cell and Developmental Biology, UCLA), Stephen Douglass (Department of Molecular Cell and Developmental Biology, UCLA), Anoop Raj Galivanche (Department of Molecular Cell and Developmental Biology, UCLA), Suman K Pradhan (Department of Biochemistry, UCLA), Tracy L Johnson (Department of Molecular Cell and Developmental Biology, UCLA)

Abstract:
Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae. One of the most striking is the splicing of meiotic transcripts. At the onset of meiosis, Saccharomyces cerevisiae, like other eukaryotes, undergoes a dramatic reprogramming of gene expression. This includes regulated transcription and splicing of a number of meiosis-specific transcripts. Splicing of a subset of these is dependent upon the meiosis-specific splicing activator Mer1. The discovery that spliceosome assembly occurs co-transcriptionally, while RNA polymerase is engaged with the chromatin-template, raises important questions about the mechanisms by which chromatin modification influences splicing, particularly regulated splicing. Here we show a crucial role for the chromatin remodeler Swi/Snf in meiotic regulation of splicing. We find that the complex affects meiotic splicing in multiple ways. First, meiosis-specific Swi/Snf downregulation regulates downregulation of ribosomal protein genes, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs (the RPGs) to Mer1-regulated transcripts. Secondly, expression of Mer1 itself is absolutely dependent on Swi/Snf—Snf2 is poised at the Mer1 promoter, and the timing of Snf2 downregulation allows elegant coordination of these mechanisms. Hence, the Swi/Snf complex directs the regulated splicing of meiotic genes, establishing it as a master regulator of meiotic splicing in Saccharomyces cerevisiae. Moreover, meiosis serves as an elegant example of a poorly understood, but undoubtedly important regulatory mechanism employed by cells, whereby redistribution of limiting gene-regulation machineries to different classes of targets serves as an important adaptive strategy in response to environmental and growth conditions.

Keywords: pre-mRNA splicing , Meiosis, chromatin

Friday 01:45-02:00pm: Unexpected mechanistic features of a multifunctional tRNA methyltransferase

Aiswarya Krishnamohan (Dept. of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Jane E. Jackman (Dept. of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

Friday 02:00-02:15pm: Title not available online - please see the printed booklet.

Zhao J (Department of Biochemistry, CWRU School of Medicine, Cleveland, OH 44109), Lin H-C, Niland CN, Jankowsky E, Harris ME

Abstract not available online - please check the printed booklet.

Friday 02:15-02:30pm: Identification of novel proteins involved in nuclear export of pre-tRNAs in Saccharomyces cerevisiae

Kunal Chatterjee (Department of Molecular Genetics, The Ohio State University), Jingyan Wu (Department of Genetics, Stanford University), Yao Wan (Department of Molecular Genetics, The Ohio State University), Anita Hopper (Department of Molecular Genetics, The Ohio State University)

Abstract:
tRNAs perform essential role of delivering amino acids to the cytoplasmic protein synthesis machinery. To execute this role, eukaryotic tRNAs must be escorted out of the nucleus, their site of synthesis, to the cytoplasm, their site of function. Via primary nuclear export, newly transcribed, end-matured, partially modified tRNAs are shuttled to the cytoplasm. Genetic and biochemical studies have identified a set of RanGTPase binding proteins called β-importins implicated in this tRNA movement. Recent in vivo biochemical data in yeast have shown two such members of β-importin family, Los1 (Exportin-t in vertebrates) and Msn5 (Exportin 5), serve overlapping but distinct functions in tRNA export1. Although Los1 and Msn5 both participate in tRNA nuclear export, they cannot be the only nuclear exporters for tRNAs as los1Δ msn5Δ double mutant cells are viable2. An unbiased screening representing ~90% of the total yeast proteome conducted recently in our laboratory uncovered novel genes involved in tRNA subcellular localization3. Two genes MEX67 and MTR2 when inactivated, cause accumulation of end-matured unspliced tRNAs in the nucleus as determined by FISH and Northern hybridization assays. Moreover, concurrent overexpression of Mex67 and Mtr2 suppresses tRNA export defects associated with los1Δ. Interestingly, the Mex67-Mtr2 complex is the principal yeast nuclear export factor for bulk mRNA4 and also contributes to ribosomal subunit nuclear export5. Hence, it is possible that this complex binds tRNAs as well and may constitute a missing alternate pathway for nuclear export. The total proteome screening also uncovered Crm1 to be involved in tRNA nuclear export. Crm1, which functions in nuclear export of proteins with Leucine rich motifs and pre-ribosomal particles6, may provide yet another pathway for tRNAs to be transported from the nucleus to the cytoplasm. Our studies lead to the hypothesis that tRNAs may highjack both the mRNA and protein nuclear export machineries as alternative pathways for tRNA nuclear export.

References:
1. Huang H.-Y.,Hopper A.K . In vivo biochemical analyses reveal distinct roles of β-importins and eEF1A in tRNA subcellular traffic. Genes Dev. 29:772-783 (2015).
2. Hopper A.K. tRNA post-transcriptional processing, turnover, and subcellular dynamics in the yeast Saccharomyces cerevisiae. Genetics 194:43-67 (2013).
3. Wu J, Bao A, Chatterjee K, Wan Y, Hopper A.K. Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics (in revision).
4. Okamura M, Inose H, Masuda S. RNA Export through the NPC in Eukaryotes. Genes (Basel). 6:124-49 (2015).
5. Faza MB, Chang Y, Occhipinti L, Kemmler S, Panse VG.Role of Mex67-Mtr2 in the Nuclear Export of 40S Pre-Ribosomes. PLoS Genet 8(8) (2012).
6. Tschochner H, Hurt E. Pre-ribosomes on the road from the nucleolus to the cytoplasm. TRENDS in Cell Biology.13: 255–263 (2003).

Keywords: tRNA export, Mex67-Mtr2, Crm1

Friday 02:30-02:45pm: Direct interaction and functional coupling of the DEAD-box RNA helicases, eIF4A and Ded1p, from Saccharomyces cerevisiae

Zhaofeng Gao (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Andrea Putnam, Heath Bowers, Ulf-Peter Guenther, Xuan Ye (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Angela Hilliker (Department of Biology, The University of Richmond, Richmond, Virginia), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH )

Abstract:
The eukaryotic translation initiation factor 4F (eIF4F) consists of the large scaffolding and RNA binding protein eIF4G, the cap-binding protein eIF4E, and the DEAD-box RNA helicase eIF4A. In Saccharomyces cerevisiae, another DEAD-box RNA helicase, Ded1p also interacts with eIF4G. Here, we show that the two DEAD-box helicases are physically and functionally coupled.

We find that eIF4A suppresses the cold-sensitive ded1 mutant alleles in vivo, and that eIF4A directly binds to Ded1p. Unwinding stimulations are observed between Ded1p and eIF4A, without or with the presence of eIF4G.

We further show that despite the presence of two RNA helicases in the Ded1p-eIF4G/4A complex, Ded1p is the unwinding module. eIF4A and eIF4G exert regulatory functions that depend on the concentrations of Ded1p, eIF4A, eIF4G and ATP. A basic thermodynamic model of the various complexes that form among Ded1p, eIF4G, eIF4A, RNA and ATP indicates that complexes containing eIF4A, eIF4G and Ded1p have the highest thermodynamic stabilities.

Collectively, our findings reveal intricate functional coupling between Ded1p, eIF4A and eIF4G, and demonstrate that eIF4A and Ded1p work simultaneously on eIF4G, and suggests that Ded1p is an integral part of the eIF4F complex.

Keywords: eIF4A, Ded1p, eIF4G

Friday 03:15-03:30pm: Determinants of specific recognition of mischarged Ala-tRNAPro by a bacterial trans-editing domain

Eric M. Danhart (Department of Chemistry and Biochemistry, The Ohio State University), Lexie Kuzmishin, Brianne Sanford, Marina Bakhtina (Department of Chemistry and Biochemistry, The Ohio State University), Marija Kosutic, Ronald L. Micura (2Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to cognate tRNAs. Mistakes in this process lead to errors in protein synthesis that can be deleterious to cells. Prolyl-tRNA synthetase (ProRS) mischarges tRNAPro with Ala; this aberrant product is hydrolyzed by a cis-editing domain (INS) in most bacteria. Bacteria whose ProRS lack the INS domain encode a homologous free-standing trans-editing protein known as ProXp-ala that performs the same function. Enzyme assays show that specific nucleotides in the acceptor stem of tRNAPro are critical for ProXp-ala activity, and a small RNA stem-loop containing these elements, microhelixPro, is a good substrate for Ala deacylation. To define the elements in ProXp-ala that confer acceptor stem specificity, NMR mapping studies were carried out with an uncharged microhelixPro and with a non-hydrolyzable, amide-linked Ala-microhelixPro mimic. We observe similar but significantly stronger chemical shift perturbations (CSPs) in the presence of the charged microhelix, which also displays higher affinity for binding to ProXp-ala. The largest CSPs were mapped to three regions: helix α2 at the top of the active site pocket (aa 27-30), β-strands β2 (aa 43-49) and β6 (aa 128-134) within the active site, and β-strand β4 (aa 80-84). Site-directed mutagenesis and AUC studies are consistent with the critical nature of these residues. Additionally, 15N NMR relaxation experiments revealed that the helix α2 exhibits significant dynamics at the ps-ns timescale. These results allow us to propose a mechanism for recognition of Ala-microhelixPro that involves induced-fit binding, specific protein-RNA contacts, and key contributions from the Ala moiety.

Keywords: tRNA, NMR, RNA-protein interaction

Friday 03:30-03:45pm: High-throughput RNA 3D motif structure prediction and validation

Jian Wu (Department of Molecular Genetics, Center for RNA Biology, Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH 43210 USA), Cuiji Zhou, Ying Wang, James Li, Annie Showers, Dana Drivera (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University, Columbus, OH 43210 USA), Neocles B. Leontis (Department of Chemistry,Bowling Green State University, Bowling Green, OH 43403 USA), Craig L. Zirbel (Department of Mathematics and Statistics, Bowling Green State University, Bowling Green, OH 43403 USA), David M. Bisaro (Department of Molecular Genetics, Center for RNA Biology, Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH 43210 USA), Biao Ding (Department of Molecular Genetics, Center for RNA Biology, Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH 43210 USA. Deceased.)

Abstract not available online - please check the printed booklet.

Friday 03:45-04:00pm: A Riboswitch-Containing sRNA Controls Gene Expression by Sequestration of a Response Reg

Margo P Gebbie (Cell Biology and Molecular Genetics, University of Maryland), Sruti Debroy (Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston), Jonathan Goodson (Cell Biology and Molecular Genetics, University of Maryland), Arati Ramesh (Biochemistry, University of Texas Southwestern Medical Center at Dallas), Danielle Garsin (Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston), Wade Winkler (Cell Biology and Molecular Genetics, University of Maryland)

Abstract:
Adenosyl cobalamine (AdoCbl) is required for the metabolism of ethanolamine (EA) in Enterococcus faecalis, both as an enzyme co-factor and for the induction of the EA utilization (eut) genes. It acts through an AdoCbl-binding riboswitch to induce the eut genes, but the mechanism of control is incompletely understood. Gene expression also requires EA, and this compound activates a two-component system composed of the sensor kinase, EutW, and its cognate response regulator, EutV, by phosphorylation. Active EutV is an antiterminator that binds nascent transcripts by recognizing a dual hairpin substrate and preventing terminator formation. Previously, we found that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual hairpin substrate for EutV. From this, we proposed a model where, in the absence of AdoCbl, EutX sequesters EutV in an inactive complex. In contrast, when AdoCbl is in excess, its binding to the riboswitch prevents EutX/EutV complex formation, freeing EutV to induce gene expression by antitermination. This model assumes that the EutX-EutV interaction is preferred relative to the four different EutV antitermination sites within the eut gene cluster. Therefore, to explore how EutX is able to successfully sequester EutV in vivo, we have purified variants of EutV and EutX and investigated their interactions through various binding assays in vitro. These data suggest that EutX may contain multiple EutV binding sites, a feature that we hypothesize is likely to improve affinity of EutV for EutX, such that it can outcompete the EutV antitermination sites. These data provide important insight into the molecular mechanism of a protein-sequestering sRNA.

References:
DebRoy, S., Gebbie, M., Ramesh, A., Goodson, J., Cruz, M., van Hoof, A., Winkler, W., and Garsin, D. Science. A Riboswitch-containing sRNA Controls Gene Expression by Sequestration of a Response Regulator. August 2014: 345 (6199), 937-940.

Keywords: Riboswitch, Two Component System, RNA Regulation

Friday 04:00-04:15pm: A mechanistic and structural study of a yet uncharacterized RNA sensor: the ykkCD riboswitch

Beau Champ (Ball State University), Ambar Rana (Ball State University), Nicholas Frecker (Ball State University), Delores James (Ball State University), Timea Gerczei (Ball State University)

Abstract:
Riboswitches are RNA aptamers that are involved in cellular regulatory functions. They fold in a specific 3D shape and often undergo structural transition when bound to the target molecule. This allosteric structural transition typically allows or prevents the downstream translation of a protein. Here, we will present the ykkCD riboswitch, which recognizes the antibiotic tetracycline and regulates expression of a multidrug resistant efflux pump in Bacillus subtilis. We will show that ykkCD directly binds to tetracycline with high affinity and selectivity and upregulates expression of downstream genes by attenuating transcription. To map the conformational change that is triggered by tetracycline binding and leads to upregulation of gene expression we will show perform nucleic acid footprinting studies of the ykkCD aptamer-tetracycline complex.

Keywords: riboswitch, SHAPE, fluorescent binding assays

Friday 04:15-04:30pm: Title not available online - please see the printed booklet.

Bradley P. Klemm (Biological Chemistry, University of Michigan), Kipchumba J. Kaitany, Michael J. Howard (Biological Chemistry, University of Michigan), Adam Z. Thelen (Program in Biomedical Sciences, University of Michigan), Allison J.L. Dewar (Chemistry, University of Michigan), Matthew J. Henley (Program in Chemical Biology, University of Michigan), Carol A. Fierke (Biological Chemistry, Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

Friday 04:30-04:45pm: Structural insights into how the tandem RRMs of hnRNP A1 bind RNA stem loops

Christopher E. Morgan (Department of Chemistry, Case Western Reserve University), Jennifer L. Meagher (Life Sciences Institute, University of Michigan), Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University), Carrie Rollins (Department of Chemistry, Case Western Reserve University), James Delproposto, Jeanne A. Stuckey (Life Sciences Institute, University of Michigan), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
hnRNP A1 is an RNA binding protein involved in many biological functions. We present the first high-resolution crystal structure (1.92 Å) of the two-RRM binding domain of hnRNP A1, UP1, bound to a 5’-AGU-3’ RNA trinucleotide, representing sequence elements of several target stem loops. The structure shows that hnRNP A1 interacts specifically with the AG dinucleotide sequence through a nucleobase pocket formed between RRM1 and the inter-RRM linker where the RNA does not bind to RRM2. Using Isothermal Titration Calorimetry and Molecular Dynamics simulations, we show that two conserved salt bridge interactions at the inter-RRM interface (R75:D155 and R88:D157) stabilize the linker in a position to bind RNA with high affinity. We investigated the structural basis of UP1 binding HIV ESS3 by determining a SAXS-scored structural model of the complex, showing that UP1 binds to the apical loop of ESS3 using RRM1 and the inter-RRM linker only. Kinetic studies of the structural model revealed that mutations within the apical loop of ESS3 reduce binding affinities by slowing down the rate of association. The resulting data provides unique insights into how the tandem RRMs of hnRNP A1 cooperate to bind RNA stem loops.

Keywords: hnRNP A1, ESS3, HIV

Friday 04:45-05:00pm: Structural analysis of the Ccq1-Tpz1-Poz1 telomere subcomplex in fission yeast

Harry Scott (Department of Pharmacology, Case Western University)

Abstract:
Telomeres are nucleoprotein complexes that cap and protect the ends of all linear chromosomes. Telomeres primarily function to maintain genome stability by preventing end-to-end fusions and deleterious induction of DNA damage response pathways. To achieve this, telomere DNA is recognized and bound by a specialized, protein complex called shelterin. The shelterin complex in Schizosaccharomyces pombe assembles from a set of six proteins (Taz1, Rap1, Poz1, Ccq1, Pot1 and Tpz1) that are orthologs of the human shelterin complex. A shelterin sub-complex, comprised of Ccq1-Poz1-Tpz1 (CTP) has been demonstrated to have regulatory roles in telomere extension by bridging regions involved in binding of double- and single-stranded regions of telomere DNA. When this bridge is intact, the telomeres reside in a non-extendable state. Conversely, when the CTP interactions are destabilized, the telomeres adopt a telomerase-extendable state. The current lack of structural information regarding telomerase regulation by shelterin proteins contributes to the elusive nature of telomere homeostasis. We have used electron microscopy to solve the structure of the CTP complex. Genetically designed tags and truncations were used localize individual domains and the termini of the three proteins within the CTP complex. These data provide the first clues regarding the overall architecture of a shelterin complex.

Keywords: telomere, structure, shelterin

Saturday 08:30-08:45am: Alternative translation initiation site usage under environmental stress conditions.

Pieter Spealman (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Cells respond to changes in the environment through coordinated changes in gene expression. Recent work suggests the cellular response to stress involves a shift ribosome initiation preference from AUG translation initiation sites (TISs) to non-AUG, alternative TISs (1). The use of alternative TISs could have wide-ranging effects on gene expression through the generation of novel N-terminal extension protein isoforms. These isoforms qualitatively differ from the canonical type by the inclusion of additional peptides at the N-terminus (2). Examples of N-terminal isoforms have been shown to change protein functions by affecting transcription factor binding targets, subcellular location of enzymes, and altering protein degradation rates.
Ribosome profiling is a deep-sequencing method that reveals the location of translating ribosomes on mRNA (3) and has been used to identify dozens of putative N-terminal extensions in Saccharomyces cerevisiae (4), mouse, and human (5). While this work suggests that these isoforms could play an important role in the stress response, only a small number have been shown to be functional. Here, we use conservation as a proxy for function and compare isoform expression in two species of budding yeast under multiple environmental conditions using ribosome profiling (6). We identify 131 N-terminal isoforms between the two species, 124 of which are condition specific, and 116 of which had not been previously described. Using mass spectrometry data we identified 51 genes with N-terminal isoforms present at the level of the proteome (7). While we find evidence of conservation and purifying selection acting upon N-terminal extensions only 6 genes are observed to have isoforms in both species, suggesting that function may be conserved while condition specificity has diverged. To further evaluate condition specific expression of N-terminal extensions we are constructing a fluorescent reporter assay.

References:
1. Botao L. et al. Wiley Interdiscip Rev. RNA (2014)
2. Michel, A. M. et al. Wiley Interdiscip Rev RNA 4, 473–90 (2013).
3. Ingolia, N. T. et al. Science 324, 218–23 (2009)
4. Gerashchenko, M. V. et al. Proc. Natl. Acad. Sci. U.S.A. 109, 17394–9 (2012).
5. Van Damme, P. et al. Mol. Cell Proteomics 13,1245–61 (2014).
6. Holcik, M. et al. Nat. Rev. Mol. Cell Biol. 6, 318–27 (2005).
7. Iesmantavicius, V. et al. Mol. Cell Proteomics 13, 1979–92 (2014).

Keywords: Translation Regulation, Gene Expression, Stress Response

Saturday 08:45-09:00am: Translation initiation is controlled by RNA folding kinetics via a ribosome drafting mechanism

Amin Espah Borujeni (Chemical Engineering, Penn State University), Howard M. Salis (Chemical Engineering and Biological Engineering, Penn State University)

Abstract:
RNA folding plays an important role in controlling protein synthesis as well as other cellular processes. Existing models have focused on how RNA folding energetics control translation initiation rate under equilibrium conditions, but have largely ignored the effects of RNA folding kinetics. We introduce a new mechanism, called Ribosome Drafting, that explains how the ribosome’s binding rate and the mRNA’s folding kinetics collectively control its translation initiation rate. During cycles of translation, Ribosome Drafting takes place whenever fast-binding ribosomes bind to mRNAs with slow-folding RNA structures, maintaining the mRNA in non-equilibrium states, with an acceleration of protein synthesis. By designing RNA structures with disparate folding kinetics, but equivalent folding energetics, we show that Ribosome Drafting increases protein synthesis rates by over 1000-fold. Overall, we characterized protein synthesis rates from 30 mRNA variants with rationally designed ribosome binding rates, folding kinetics, and folding energetics to validate a non-equilibrium Markov model of translation and to confirm the necessary conditions for Ribosome Drafting. Our computational design, non-equilibrium modeling, and systematic experiments demonstrate a versatile approach for studying how RNA folding kinetics control cellular processes.

Keywords: Biophysical Model, Ribosome Drafting, RNA Folding Kinetics

Saturday 09:00-09:15am: ATP hydrolysis by Upf1 promotes efficient translation termination on substrates of the nonsense-mediated mRNA decay pathway in yeast

Lucas D. Serdar (Center for RNA Molecular Biology, Case Western Reserve University), Dajuan Whiteside (Center for RNA Molecular Biology, Case Western Reserve University), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University)

Abstract not available online - please check the printed booklet.

Saturday 09:15-09:30am: The life and times of transcribed RNA as told through global nascent RNA sequencing

Brian Magnuson (School of Public Health Environmental Health Sciences, University of Michigan, Ann Arbor, MI), Artur Veloso (Novartis Institutes of Biomedical Sciences, Cambridge, MA), Michelle Paulsen (Department of Radiation Oncology, University of Michigan, Ann Arbor, MI), Frederick Derheimer (Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN ), Zahid Bonday (Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN), Mats Ljungman (Department of Radiation Oncology, University of Michigan, Ann Arbor, MI)

Abstract:
RNA is born, matures, and dies through a number of processes – initiation, elongation, termination, processing, engagement, degradation – all of which can be regulated and ultimately define RNA fate. Global analysis using RNA-seq identifies changes in transcript abundance, splicing patterns, and other characteristics, but only from the steady-state pool of RNA, which is a mixture of young, old, and predominantly stable RNA. To address this, we developed two complementary methods: Bru-seq and BruChase-seq. Bru-seq is the brief metabolic labeling of RNA in live cells with bromouridine followed by immunoprecipitation and sequencing. BruChase-seq incorporates a chase period after RNA labeling in a paired sample. Given the initial synthesis state of RNA, we can follow the fate of RNA as it ages and, importantly, infer whether genes are regulated pre- or post-transcriptionally. The protein arginine methyltransferase 5 (PRMT5), an enzyme that modifies histones, transcription factors, splicing factors, and signaling proteins, has the potential to regulate RNA at multiple levels. For this reason, Bru- and BruChase-seq are well-suited to study the multi-faceted PRMT5. We have found, through knockdown and small molecule inhibition in human melanoma cells, that PRMT5 indeed regulates genes at the levels of transcription, splicing, and stability.
While Bru-seq is capable of capturing primary transcription units of genes that are unstable or highly processed (e.g. miRNA), some RNA species, such as enhancer RNA, are often missed. We found that irradiating cells prior to labeling with UVC light enhanced signal near transcription start sites, putative enhancers, and PROMPTs. UV light inhibits elongation but not initiation and therefore polymerases continue to synthesize RNA near initiation sites. We found that in TNF-treated normal human fibroblasts, BruUV-seq signal near gene TSSs is responsive to stimuli and correlates to changes in nascent (Bru-seq) signal. Furthermore, changes in putative active enhancer elements change with nearby transcribing genes and thus a potential application of BruUV-seq is to identify enhancer dynamics within an experiment. Together, Bru-seq and its derivatives provide a complementary and comprehensive picture of RNA regulation at multiple levels throughout its lifespan.

Keywords: gene regulation, rna sequencing, methyltransferase

Saturday 09:30-09:45am: The naturally-occurring non-stop mRNAs from Arabidopsis display relatively low stability

Laura de Lorenzo (Dept. of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546-0312, USA. ), Arthur G. Hunt (Dept. of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546-0312, USA. )

Abstract:
Non-stop decay is an mRNA surveillance pathway that ensure the rapid degradation of mRNA that lack a translational termination codon (1, 2). In plants, yeast, and mammals, polyadenylation sites have been shown to occur within protein-coding regions (3, 4). Such mRNAs would lack a translation termination codon and thus would constitute a class of naturally-occurring non-stop mRNAs. The significance of such mRNAs is not clear, and it is not known as they are, as a group, less functional than their full-length counterparts. Here, we used high-throughput sequencing to test the hypothesis that naturally-occurring non-stop mRNAs in Arabidopsis are less stable than the bulk of the mRNA population. Analysis of mRNAs with 3’ ends in different regions revealed that mRNAs that are polyadenylated within coding regions (non-stop mRNAs) and also within introns had lower relative stabilities than other mRNA isoforms. Interestingly, non-stop mRNAs were enriched in GA-repeat sequence motifs surrounding the poly(A) sites, and the set of genes that encode with non-stop mRNAs was enriched for those associated with regulatory functions. The GA-repeat motifs were found to be conserved in two Brassicaceae species and over-represented in non-stop mRNAs from Medicago truncatula. Our results suggest non-stop mRNAs constitute a mode of negative regulation in Arabidopsis, and that this mode of regulation is conserved in other plant species.

References:
1. Frischmeyer PA, van Hoof A, O'Donnell K, Guerrerio AL, Parker R and Dietz HC. 2002. An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 295: 2258-2261.
2. van Hoof A, Frischmeyer PA, Dietz HC and Parker R. 2002. Exosome-mediated recognition and degradation of mRNAs lacking a termination codon. Science, 295: 2262–2264.
3. Yoon OK and Brem RB. 2010. Noncanonical transcript forms in yeast and their regulation during environmental stress. RNA, 16: 1256-1267.
4. Wu W, Liu M, Downie B, Liang C, Ji G, Li QQ and Hunt AG. 2011. Genome-wide landscape of polyadenylation in Arabidopsis provides evidences for extensive alternative polyadenylation. PNAS, 108: 12533-12538.

Keywords: PAT-seq, non-stop mRNA, Alternative polyadenylation

Saturday 09:45-10:00am: ADARs Antagonize Deadenylase-dependent Destabilization of a Nuclear-retained RNA

Aparna Anantharaman (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Vidisha Tripathi (National Center for Cell Science, Pune, India), Kannanganattu V. Prasanth, Abid Khan, Supriya G. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Je-Hyun Yoon, Myriam Gorospe (Laboratory of Genetics, National Institute of Aging-Intramural Research program, NIH), Johan Ohlson, Helene Wahlstedt, Marie Ohman, Shuomeng Guang, Jian Ma (Department of Molecular Biosciences, the WennerGren Institute, Stockholm University, Sweden, Department of Bioengineering, Carl R. Woese Institute for Genomic Biology, UIUC), Jochen C. Hartner, Shinichi Nakagawa, Tetsuro Hirose, Michael F. Jantsch (TaconicArtemis GmbH, Germany, RNA Biology Laboratory, RIKEN, JAPAN, Institute of Genetic Medicine, Hokkaido University, Japan, Department of Chromosome Biology, Max F. Perutz Laboratories, Austria)

Abstract:
Adenosine deaminases acting on RNA (ADAR) catalyze the editing of adenosine to inosine (A-to-I) within RNA sequences. Most A-to-I editing occurs in noncoding repeat sequences, including introns and UTRs (un-translated regions). The functional significance of this deamination is poorly understood. Here, we demonstrate that ADAR association with the 3’UTR of nuclear-retained Ctn RNA (Cat2 transcribed nuclear RNA) promotes the stability of Ctn RNA. In the absence of ADAR, the cellular abundance and half-life of Ctn RNA are significantly reduced. Unedited Ctn RNA has a shortened poly(A) tail and shows enhanced interaction with PARN [poly(A)-specific ribonuclease] deadenylase. Furthermore, the RNA binding protein HuR/ELAVL1 recruits PARN to unedited Ctn RNA. Our results show that ADAR promotes the stability of Ctn RNA by antagonizing its degradation by PARN and HuR. In addition to Ctn RNA, global transcriptomic analysis revealed that other mRNAs including nuclear and long noncoding RNAs are also stabilized by ADAR. Taken together, our findings suggest a regulatory paradigm wherein ADARs enhance target mRNA stability, by limiting the interaction of RNA-destabilizing proteins with their cognate substrates.

Keywords: ADAR, Ctn RNA, mRNA stability

Saturday 10:00-10:15am: Alternative exon junction complexes differentially mark mRNAs targeted for Nonsense Mediated mRNA Decay

Lauren Woodward (Molecular Genetics The Ohio State University), Justin Mabin (The Ohio State University), Guramrit Singh (Molecular Genetics The Ohio State University)

Abstract:
The spliceosome deposits the exon-junction complex (EJC) ~24nt upstream of exon-exon junctions during pre-mRNA splicing. The EJC is an integral component of spliced RNPs and regulates gene expression at several steps during the lifecycle of mRNA: pre-mRNA splicing and mRNA export from the nucleus to mRNA localization, translation and nonsense-mediated mRNA decay (NMD) in the cytoplasm. The EJC core—composed of the eIF4AIII, Y14, Magoh and MLN51—serves as a binding platform, recruiting a myriad of factors involved in these processes. Intriguingly, proteomic analysis of EJCs purified from HEK293 cells showed that MLN51 and other peripheral EJC proteins are sub-stoichiometric to the EJC core, indicating that they may not be present in all EJCs. We have now established the existence of at least two distinct EJCs in human and mouse cells: MLN51- and RNPS1-containing EJCs. Interestingly, the two complexes differentially associate with the NMD machinery, which detects and degrades mRNAs containing premature stop codons. RNPS1-EJCs are Upf2-rich, whereas MLN51-EJCs are Upf3b-rich. Our findings are in agreement with previous work suggesting distinct branches of EJC-dependent NMD; RNPS1 is Upf2-dependent but Upf3b-independent to trigger NMD, whereas MLN51 is Upf2-independent. Our transcriptome-wide identification of MLN51- and RNPS1-EJC occupancy sites via RIPiT-Seq has revealed that certain endogenous NMD targets are exclusively bound to MLN51-EJCs. These mRNAs are likely to undergo MLN51 (and Upf3b) - dependent but RNPS1 (and Upf2)-independent NMD, predictions that we are currently testing. We are also determining if NMD targets exist that exclusively undergo RNPS1-dependent NMD. Taken together, our findings suggest an exciting possibility that distinct EJCs sort mRNAs into distinct populations, which may be subject to different spatial and temporal regulation by discrete branches of NMD.

Keywords: EJC, alternate EJC, NMD

Saturday 10:45-11:00am: Relocalization of Human Lysyl-tRNA Synthetase following HIV-1 Infection: Implications for tRNA Primer Recruitment

Alice Duchon (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Nathan Titkemeier (Department of Chemistry and Biochemistry, Center for RNA Biology, Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Corine St. Gelais (Center for RNA Biology, Center for Retroviral Research, and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Li Wu (Center for RNA Biology, Center for Retroviral Research, and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, 2Center for RNA Biology, 3Center for Retroviral Research, The Ohio State University, Columbus, OH 43210)

Abstract:
All retroviruses use specific host cell tRNAs to prime reverse transcription of their retroviral RNA genomes into DNA. The primer for reverse transcription in HIV-1, human tRNALys, is selectively incorporated into virions during viral assembly. We previously reported that human lysyl-tRNA synthetase (LysRS) is specifically packaged into HIV-1 leading to the enrichment of tRNALys in virions. Cytoplasmic LysRS is normally localized to a dynamic mammalian multisynthetase complex (MSC). In addition to their well-known physiological function in translation, many tRNA synthetases are mobilized from the MSC to function in a wide variety of non-translational pathways. While some aspects of tRNA primer packaging into HIV-1 particles are now understood, the mechanism by which the LysRS/tRNA complex is diverted from its normal function in translation and recruited into viral particles is unclear. Using immunofluorescence and confocal microscopy we find that LysRS localization is dramatically altered upon HIV-1 infection. In uninfected cells, the majority of LysRS is located in the MSC, as expected, whereas following HIV-1 infection, LysRS is phosphorylated, released from the MSC, and traffics to the nucleus. Treatment with reverse transcription inhibitors does not block nuclear localization, suggesting that early stages of infection trigger LysRS release from the MSC. In contrast, treatment with a MAP kinase inhibitor known to block phosphorylation of LysRS does prevent nuclear localization of LysRS in HIV-infected cells. The implications of nuclear localization of LysRS for HIV-1 infectivity are being investigated.

Keywords: HiV, tRNA, LysRS

Saturday 11:00-11:15am: Characterizing the role of DDX41, a DEAD-box RNA helicase, in blood cancer and the spliceosome

James M. Hiznay (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic), Chantana Polprasert, Hideki Makishima, Xiaorong Gu, Yogen Saunthararajah (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Carsten Mller-Tidow (Department of Hematology and Oncology, University of Halle, Halle, Germany), Eckhard Jankowsky (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Jaroslaw P. Maciejewski (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic), Richard A. Padgett (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic)

Abstract not available online - please check the printed booklet.

Saturday 11:15-11:30am: Mechanisms of unconventional translation initiation at CGG repeats in the Fragile X mRNA, FMR1.

Michael G. Kearse (Dept. of Neurology, Dept. of Biological Chemistry, University of Michigan), Katelyn M. Green, Amy Krans, Caitlin M. Rodriquez, Alexander E. Linsalata (Dept. of Neurology, University of Michigan), Aaron C. Goldstrohm (Dept. of Biological Chemistry, University of Michigan), Peter K. Todd (Dept. of Neurology, University of Michigan; Veterans Affairs Medical Center, Ann Arbor, MI)

Abstract not available online - please check the printed booklet.

Saturday 11:30-11:45am: Enhanced MicroRNA Detection Assays Based on Tyramide Signal Amplification and GammaPNA Probes.

Munira Fouz (Department of Chemistry, Carnegie Mellon University), Lauren Xu (Department of Chemistry, Carnegie Mellon University), Danith Ly (Department of Chemistry, Carnegie Mellon University), Veronica Hinman (Department of biological sciences, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University), Bruce Armitage (Department of Chemistry, Carnegie Mellon University)

Abstract:
The ability to detect spatial and temporal microRNA (miRNA) expression and distribution at different stages of growth cycles are essential for understanding the biological roles of miRNA and miRNA-associated gene regulatory networks. The success of detection methods such as in situ hybridization depends on the stability of the double helix formed by the target and the probe, especially for the detection of shorter RNA sequences like miRNA. Peptide nucleic acids (PNAs) installed with a relatively small, hydrophilic entity at the γ-position of the backbone, i.e. γPNAs, make high affinity probes. Miniprobes, which are short γPNAs designed by splitting a long probe sequence, can recognize a target sequence by hybridizing adjacent to each other with high cooperative affinity. These miniprobes also show high sequence specificity with significant destabilization of duplexes for non-target sequences bearing single mismatch nucleotides. Here we develop enhanced fluorescent detection tools combining the special features of gammaPNA mini-probes and Tyramide Signal Amplification (TSA) technology for detection and quantification of miRNA. The miniprobe design allows incorporation of functional groups for enhanced amplification, improving signal-to-noise ratio. We currently employ these tools to understand the regulatory mechanisms of cell specification during early development of sea star larva; a useful model system to study tissue regeneration. In addition, the approach can be readily generalized to any RNA target.

Keywords: Gamma-modified Peptide Nucleic Acids (PNA), Regenerative biology, Tyramide Signal Amplification (TSA)

Saturday 11:45-12:00pm: RNA as a unique polymer to build functional nanostructures for material and biomedical applications

Hui Li (Nanobiotechnology Center, Markey Cancer Center, and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536), Peixuan Guo (Nanobiotechnology Center, Markey Cancer Center, and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536)

Abstract:
RNA is a "polynucleic acid", but its polymeric nature in material and technological applications is often overlooked. However, similar to other biopolymer such as DNA, protein, and polysaccharide, RNA not only plays a critic role in the biological functioning of both prokaryotes and eukaryotes, but also has intriguing characteristics at the nanometer scale. Recently, we reported the finding that RNA can serve as a unique polymer for the construction of boiling-resistant nanostructures with applications in medicine, immunology, cancer therapeutics, material sciences, single molecule optics, electronics and computer devices. Our platform is based on the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor. The pRNA is a versatile molecule for constructing multivalent 3D architectures with diverse sizes and shapes via loop-loop interactions, palindrome sequences, and three-way junction motif modulating. These chemically-modified RNA nanoparticles are thermodynamically stable, resistant to RNase degradation, and can harbor different functionalities, such as siRNA, ribozymes and aptamers. Upon systemic injection, these RNA nanoparticles targeted a variety of cancer xenografts and metastases in vivo with little accumulation in healthy vital organs and tissues. RNA nanoparticles tested in vivo revealed that they did not induce cytokines, interferon, antibody, and toxicity; and retained favorable pharmacokinetics and biodistribution profiles.

References:
1. Li H, Lee T; Dziubla T; Pi F; Guo S; Xu J; Li C; Haque F; Liang XJ; Guo P. RNA as a Stable Polymer to Build Controllable and Defined Nanostructures for Material and Biomedical Applications. Nano Today. 2015. Accepted.
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, 6:658-67.
3. Guo P. The emerging field of RNA nanotechnology. Nature Nanotechnology. 2010, 5:833-42.
4. Guo P, Zhang C, Chen C, Garver K, Trottier M and Chen C. Inter-RNA Interaction of phage phi29 pRNA to Form a Hexameric Complex for DNA Transportation. Mol. Cell. 1998, 2:149-155.

Keywords: RNA Nanotechnology, Bacteriophage Phi29 pRNA, RNA Nanoparticles

Saturday 12:15-12:35pm: Mechanism of PWI Motif Binding to Nucleic Acids

Harry Chanzu (Dept. of Chemistry, Western Michigan University), Elizabeth I. Lopez (Dept. of Chemistry, Western Michigan University), Denise Flores (Dept. of Chemistry, Western Michigan University), Blair R. Szymczyna (Dept. of Chemistry, Western Michigan University)

Abstract not available online - please check the printed booklet.

Saturday 12:35-12:55pm: Silencing of a telomeric gene by a noncoding RNA derived from its own 3’-UTR

Jayakrishnan Nandakumar (Molecular Cellular and Developmental Biology, University of Michigan), Kamlesh Bisht (Molecular Cellular and Developmental Biology, University of Michigan)

Abstract not available online - please check the printed booklet.