Poster Abstracts

1. Title not available online - please see the printed booklet.

Seth Abels (Ball State University), Dr. Emil Khisamutdinov (Ball State University)

Abstract not available online - please check the printed booklet.

2. Investigating the Conformational Changes of DsRNA Upon Binding to TRBP

Roderico Acevedo (The Pennsylvania State University), Declan Evans (The Pennsylvania State University), Katheryn A. Penrod (The Pennsylvania State University), Scott A. Showalter (The Pennsylvania State University)

Abstract not available online - please check the printed booklet.

3. Establishing a link between Translational Recoding and Human Disease

Vivek M. Advani (Dept. of Cell Biology and Molecular Genetics, University of Maryland, College park), Ashton Trey Belew (Dept. of Cell Biology and Molecular Genetics, University of Maryland, College park), Jonathan D. Dinman (Dept. of Cell Biology and Molecular Genetics, University of Maryland, College park)

Abstract:
Gene expression can be controlled at the level of mRNA stability, and prior studies from our laboratory have explained how Programmed -1 Ribosomal Frameshifting (-1 PRF) fits within this paradigm. Computational analyses suggest that 10-15% of eukaryotic mRNAs contain at least one potential -1 PRF signal(1). The overwhelming majority of predicted “genomic” -1 PRF events are predicted to direct translating ribosomes to premature termination codons. We have demonstrated that these can function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway(2). In published work we have explored the biological significance of the connection between -1 PRF and NMD on telomere maintenance in yeast(3). More recently we extended this line of inquiry to human cells, demonstrating that a sequence element in the mRNA encoding Ccr5p harbors a -1 PRF signal which functions as an mRNA destabilizing element through NMD(4). In the current work we are exploring the link between global changes in -1 PRF rates and human health using yeast and human cell-based models of two diseases, X-linked Dyskeratosis Congenita (X-DC) and Spinocerebellar ataxia 26 family (SCA26) as models. Preliminary findings suggest these genetically inherited defects result in translational fidelity defects (i.e. changes in rates of -1 PRF, +1 PRF, and stop codon recognition), with attendant effects on mRNA abundance, gene expression and telomere maintenance. These studies establish a paradigm for understanding the linkage between translational fidelity and human disease.

References:
1. Belew AT et al. PRFdb: a database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals. BMC Genomics [Internet]. 2008 Jan [cited 2014 Oct 15];9(1):339.
2. Belew AT et al. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Res [Internet]. 2011 Apr 1 [cited 2014 Oct 14];39(7):2799–808.
3. Advani VM, Belew AT, Dinman JD. Yeast telomere maintenance is globally controlled by programmed ribosomal frameshifting and the nonsense-mediated mRNA decay pathway. Transl (Austin, Tex) [Internet]. 2013 Apr 1 [cited 2014 Oct 14];1(1):e24418.
4. Belew AT et al. Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway. Nature [Internet]. Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.; 2014 Jul 9 [cited 2014 Jul 9];512(7514):265–9.

Keywords: -1 PRF, translational fidelity, X-DC

4. Title not available online - please see the printed booklet.

George M. Allen (Department of Microbiology, The Ohio State University), Vineeta A. Pradhan (Department of Microbiology, The Ohio State University), Frank J. Grundy (Department of Microbiology, The Ohio State University), Tina M. Henkin (Department of Microbiology, The Ohio State University)

Abstract not available online - please check the printed booklet.

5. Investigating the Role of Translational Recoding in Stress Response

Avan Antia (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Vivek M. Advani (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park)

Abstract not available online - please check the printed booklet.

6. Hepatocyte-Specific Deletion Of A Splicing Regulatory Protein Causes Spontaneous And Severe Nonalcoholic Steatohepatitis In Mice

Waqar Arif (Departments of Biochemistry and Medical Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign), Gandar Dhatar (Department of Biochemistry, University of Illinois at Urbana-Champaign), Qinyu Hao (Department of Cellular and Developmental Biology, University of Illinois at Urbana-Champaign), Jian Ma (Departments of Bioengineering and Cellular and Developmental Biology, University of Illinois at Urbana-Champaign), K V Prasanth (Department of Cellular and Developmental Biology, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Departments of Biochemistry and Medical Biochemistry, College of Medicine, Institute of Genomic Biology, University of Illinois at Urbana-Champaign)

Abstract:
Non-Alcoholic Steatohepatitis (NASH) is emerging as one of the most common liver disease in the American population. It is a metabolic disorder in which fat accumulation within the liver (steatosis) is associated with inflammation, hepatic injury and cirrhosis without significant consumption of alcohol. A recent study examining SNPs in patients with NASH revealed a significant association for pathways involved in mRNA splicing. To further investigate factors involved in NASH pathology, we screened various RNA splicing factors for changes in expression in fatty liver mice models. To our surprise we found a specific downregulation of SRSF1, a highly conserved pre-mRNA splicing factor. This trend was also verified in human fatty liver patient samples which showed a striking downregulation of SRSF1 as well.
To study the function of SRSF1 in liver we created a hepatocyte-specific knockout (HKO) using Cre-loxP technology. Histological and serum analyses of these mice revealed spontaneous and progressive NASH-like liver injury including steatosis, inflammation, and fibrosis. We hypothesized that lack of SRSF1 in hepatocytes results in misregulated splicing and expression of specific transcripts leading to the development of NASH. In order to identify the underlying transcriptome defects, we performed a high-resolution RNA-Seq on livers of five-week old wildtype and SRSF1 HKO mice. Computational analysis of the data revealed hundreds of genes with altered splicing and expression many of which are related to fatty acid metabolism, lipid peroxidation and inflammation. In addition to splicing regulation, SRSF1 has also been shown to regulate the composition of nuclear speckles. Astonishingly, we observe a significant loss of MALAT1 localization to speckles in SRSF1 HKO hepatocytes. Taken together our results demonstrate that SRSF1 is essential for maintaining transcriptome integrity in hepatocytes and that impairment of its activity induces spontaneous NASH pathology in mice.

Keywords: Splice Regulation, SRSF1, Liver Disease

7. Selectivity in substrate binding by the exonuclease Rrp6p

Armend Axhemi (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland OH), Sukanya Srinivasan, Ulf-Peter Guenther, Deepak Sharma (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland OH), Elizabeth V. Wasmuth (Structural Biology Program & Louis V Gerstner Jr. Graduate School of Biomedical Sciences, Sloan Kettering Institute, New York, NY), Christopher D. Lima (Structural Biology Program & Howard Hughes Medical Institute, Sloan Kettering Institute, New York, NY ), Eckhard Jankowsky (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland OH)

Abstract not available online - please check the printed booklet.

8. ATP-dependent G-guadruplex unfolding by Bloom helicase exhibits low processivity

Hamza Balci (Physics Department, Kent State University), Jagat Budhathoki (Physics Department, Kent State University), Edward J. Stafford (Physics Department, Kent State University), Jaya G. Yodh (Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign)

Abstract:
Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5' ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50-70% of reeling attempts under physiological salt and pH conditions. Such unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation.

Keywords: Bloom, G-quadruples, FRET

9. An AUG triplet at the 5’-termini of Escherichia coli’s ptrB mRNA regulates downstream coding sequence expression

Heather J. Beck (Miami University), Ian Fleming (Ohio State University), Gary Janssen (Miami University)

Abstract not available online - please check the printed booklet.

10. Genome-wide analysis of splicing kinetics reveals differential rates of splicing and turnover for mammalian introns

Karan Bedi (Department of Radiation Oncology, University of Michigan), Jayendra Prasad (Department of Radiation Oncology, University of Michigan), Brian Magnuson (Department of Radiation Oncology, University of Michigan), Artur Veloso (Department of Radiation Oncology, University of Michigan), Michelle T Paulsen (Department of Radiation Oncology, University of Michigan), Mats Ljungman (Department of Radiation Oncology, University of Michigan)

Abstract:
Splicing is carried out by spliceosomes and involves splice sites recognition, removal of introns and ligation of exons. Components of the spliceosome interact with the elongating RNA polymerase II (RNAPII) allowing for splicing concurrent with transcription. Here we used BruChase-seq to obtain temporal resolution of splicing kinetics genome-wide and found that the emergence of completely spliced transcripts in the nascent RNA pool was surprisingly slow and variable even within transcripts. Minor class introns were spliced out slower than major class introns and some transcripts, such as from ribosomal protein-coding genes, were rapidly spliced in a spliceosome-independent manner. Slowly spliced introns had an increased density of RNA polymerase II suggesting that transcription elongation through these introns occurred at a slower rate than for other introns. Together these genome-wide studies suggest that splicing and post-splicing turnover occur at different rates for different introns revealing a novel layer of gene regulation.

Keywords: BruChase-Seq, splicing, intron turnover

11. Transcriptome-wide mapping reveals pseudouridylation as a dynamic process in budding yeast mRNA.

Douglas A. Bernstein (Biology, Ball State University ), Schraga Schwartz (Weizmann Institute of Science ), Maxwell R. Mumbach (Biology, Stanford), Gerald Fink (Biology, MIT), Aviv Regev (Biology, MIT)

Abstract:
Pseudouridylation is the most abundant post-transcriptional modification playing critical roles in RNA structure and function in all kingdoms of life. While numerous pseudouridylation sites in noncoding RNAs have been mapped, quantitative assessment of pseudouridine transcriptome-wide has been technically impractical. We have developed a novel pseudouridine sequencing technique that maps and quantitatively measures pseudouridylation transcriptome-wide. Pseudouridine sequencing has identified hundreds of novel pseudouridines in mRNAs. These modifications are dependent upon specific pseudouridine synthases, and occur at consensus motifs. Previous data indicated that pseudouridylation is induced in S. cerevisiae snRNAU2 by stress. We used pseudouridine sequencing to assess pseudouridylation transcriptome wide under a variety of stress conditions. We find pseudouridylation levels change significantly at a number of sites in noncoding RNAs during cold and nutrient stresses. During heat stress however, pseudouridylation is induced at over two hundred sites in mRNA. The majority of these modifications are dependent on the pseudouridine synthase Pus7, and occur at a Pus7 consensus motif. In addition, PUS7 deletion renders S. cerevisiae heat sensitive and Pus7 localization changes in response to heat stress suggesting Pus7 facilitated pseudouridylation is important for heat shock survival, and Pus7 localization plays a role mediating mRNA pseudouridylation during heat stress. In conclusion we have mapped pseudouridine transcriptome-wide, and we find pseudouridylation to be a dynamic process important for yeast stress response.

Keywords: pseudouridine, post-transcriptional modification , Pus7

12. Title not available online - please see the printed booklet.

Divyaa Bhagdikar (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus OH), Frank J. Grundy (Department of Microbiology, The Ohio State University, Columbus OH), Tina M. Henkin (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus OH)

Abstract not available online - please check the printed booklet.

13. Do ribosomal proteins function in multiple stages of 60S subunit assembly?

Stephanie Biedka (Department of Biological Sciences, Carnegie Mellon University), Hailey Brown (Department of Biological Sciences, Carnegie Mellon University), Jelena Jakovljevic (Department of Biological Sciences, Carnegie Mellon University), John Woolford (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Assembly of the structural domains of eukaryotic 60S ribosomal subunits occurs in a hierarchical fashion. Previous studies in which individual yeast large subunit ribosomal proteins (RPLs) were depleted revealed that these mutants fell into either early, middle, or late phenotypic classes, as assayed by precursor ribosomal RNA (pre-rRNA) processing and preribosome composition. While this initial approach was critical in the development of the hierarchical model, its main shortcoming is that many RPLs contact more than one domain of the large subunit, and depletion does not allow us to determine if a given RPL functions in more than one stage of assembly. L7 (uL30) and L20 (eL20) are two such RPLs that contact multiple rRNA domains in the mature large subunit. To begin addressing whether they might play roles in multiple stages of assembly, we are making site-directed mutations in L7 and L20. These RPLs are known to function early in large subunit assembly, but their proximity to 5S rRNA in the mature large subunit suggests that L7 and L20 could also function in later stages of assembly. Our site-directed mutations focus on disrupting the interactions between these RPLs and both 5S and 25S rRNA, allowing us to investigate the importance of the contacts that L7 and L20 make with 5S and 25S rRNA. We are studying the effects of these mutations on growth, pre-rRNA processing, preribosome composition, and RNA structure in order to determine if L7 and L20 might have additional roles later in large subunit assembly in addition to their known early roles.

Keywords: yeast ribosome assembly, preribosomes

15. Specific Early Gene Transcription Termination Signal Binding by Vaccinia Capping Enzyme

Rachel Boldt (Dept. of Biological Sciences, SUNY Buffalo), Paul Gollnick (Dept. of Biological Sciences, SUNY Buffalo)

Abstract:
Vaccinia virus, a member of the poxvirus family, is a double stranded, cytoplasmic DNA virus. Vaccinia genes are expressed in three different temporal classes; early, intermediate, and late. Early gene transcription termination occurs in a site-specific manner and depends on U5NU signal in the nascent RNA. A specific interaction between the viral mRNA capping enzyme and the U5NU sequence is required for proper transcription termination.
The Vaccinia mRNA capping enzyme is a heterodimeric protein composed of a large, 97kDa subunit (D1) and a small, 33kDa subunit (D12). The D1 subunit contains the active sites for capping activities. The N-terminal domain has both guanylyl-transerase and NTPase activities. The C-terminal domain contains the methyl-transferase active site; however dimerization with D12 is required for full enzymatic activity. Both subunits are required for its transcription termination activity.
The N-terminal domain of the D1 capping enzyme subunit has been shown to crosslink to the U5NU termination signal in RNA. Here, we differentiate specific U5NU sequence binding from non-specific RNA 5’end binding associated with mRNA capping. Chimeric oligonucleotides with 10 bases of DNA at the 5’ends followed by 26 bases of RNA were used in binding studies. Capping enzyme was found to have a higher affinity for U5NU containing oligos compared to oligos without the termination signal. Furthermore, chimeric oligos containing the U5NU were able to stimulate transcription termination in vitro. These chimeric oligos will be used in future studies to identify the specific residues within D1 that interact with the U5NU sequence. Both alanine scanning and a crosslinking followed by mass spec approach will be used.

Keywords: Vaccinia, Transcription

16. Effective and long lasting KD of nuclear retained lncRNA in a broad range of mouse tissues achieved with single stranded LNA enhanced antisense oligonucleotides

Gillian Browne, Barbara Gould, Henrik M. Pfundheller, Niels M. Frandsen, Ni (Exiqon, Vedbaek, Denmark), Johnathan Lai, Peter Mouritzen (Exiqon, Vedbaek, Denmark), Bodo Brunner (Sanofi-Aventis Deutschland GmbH), Mike Helms (Sanofi-Aventis Deutschland GmbH), Katharina M. Michalik (R&D Biologics / Nucleic Acid Therap., Frankfurt, Germany, FF Institute for Cardiovascular Regeneration), Reinier A. Boon (R&D Biologics / Nucleic Acid Therap., Frankfurt, Germany, FF Institute for Cardiovascular Regeneration)

Abstract:
One of the most interesting results from the ENCODE NGS project is the observation that a major part of the transcriptome is non-coding. The few lncRNAs that have been studied suggest that they have important and diverse functions involved in epigenetics and regulation of both transcription and translation. There is therefore an urgent need for the development of tools for functional analysis of lncRNAs in cell cultures and animals.

For this purpose Exiqon has focused on single strand LNA enhanced antisense technology that catalyzes RNaseH dependent degradation of target RNA. We have developed an empirically derived design algorithm that provides antisense oligonucleotides (ASOs) that achieve potent target KD with a high hit-rate. To illustrate the power of the design algorithm we show that by screening just 10 ASOs with high design scores we identified three capable of potent KD of the nuclear retained and ubiquitously expressed lncRNA Malat1 in a broad range of tissues upon systemic delivery in mice.

We selected one ASO for extended analysis. 12 different tissue samples were selected 2 days, 5 weeks and 15 weeks after the last ASO administration. We report broad range KD of Malat1 although with some differences between tissue types. Interesting variations in the duration of the KD were observed. This suggests that the long term fate of intracellular ASOs differ significantly between cell types. In conclusion we show that LNA enhanced gapmers are important and powerful tools for the analysis of lncRNA function both in cell cultures as well as animal models which make them particularly promising antisense drugs of the future.

Keywords: lncRNA, gapmers, NGS

17. Implications of Weak GQ Unfolding Activity of RecQL5 Helicase

Jagat B. Budhathoki (Department Of Physics, Kent State University), Hamza Balci (Department Of Physics, Kent State University), Parastoo Maleki (Department Of Physics, Kent State University), William Roy (Department Of Physics, Kent State University), Jaya G. Yodh (Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign), Pavel Janscak (Institute of Molecular Cancer Research, University of Zurich)

Abstract:
RecQL5 is a homologue of E.Coli RecQ helicase and is one of five members of RecQ family of helicases in humans. Even though some other members of this family are associated with different syndromes, such as Bloom (BLM) and Werner (WRN) syndromes which result from deficiencies in respective helicases, RecQL5 has not been directly linked with a specific syndrome. Both Werner and Bloom helicases are considered to be involved in resolving non-canonical DNA structures, such as G-quadruplex (GQ). Deficiencies in this activity are known to result in DNA breaks and genomic instability. Whether RecQL5, which is not directly associated with a syndrome, also possesses GQ unfolding activity is an important question that could provide further insight onto the significance of GQ structures in the syndromes associated with RecQ family of helicases. In our earlier work, we showed that BLM helicase unfolds GQs with and without ATP, which we attributed the latter to be due to binding of BLM to the vicinity of GQ and interacting with it. Our single molecule FRET studies show that the RecQL5 lacks GQ unfolding activity in the absence of ATP for the same DNA substrates used in the BLM study under physiological salt and pH conditions. However, RecQL5- mediated GQ unfolding takes place in the presence of ATP although, at a significantly lower efficiency compared to BLM. Our studies of this activity under different salt conditions and for different GQ constructs demonstrated a strong dependence on GQ stability. Therefore, we conclude that RecQL5 is a less efficient GQ destabilizer but may assist the more efficient helicases, such as BLM and WRN, in this task. Overall, these results are consistent with GQs being significant components of Bloom and Werner syndromes, and might explain why deficiencies in RecQL5 are not directly associated with a similar syndrome.

Keywords: G Quadruplex, RecQ helicase, Unfolding

18. Construction of Nucleic Acid Nano-architectures Based on Flexible tetra-U Linking Module

My N. Bui (Department of Chemistry, Ball State University, Muncie IN 47306), Garrett A. Drinnon (Department of Chemistry, Ball State University, Muncie IN 47306), Emil F. Khisamutdinov (Department of Chemistry, Ball State University, Muncie IN 47306)

Abstract:
The progression in the field of RNA nanotechnology involves the design and synthesis of artificial RNA nanoparticles with wide spectra of applications in pharmacology, medicine, biomaterials, electronics etc. The unique properties of RNA molecules to form non-canonical, local or long- range interactions result in a large library of structural motifs that can be used for RNA nanoparticle construction. Naturally occurring RNA 3D motifs have been used previously in architectonics, which is a computer-aided approach for design artificial RNA nanostructures in a controllable fashion. However, most of the natural RNA modules fold into desired 3D conformations only if certain conditions are satisfied, e.g. pH, presence of mono- and/or divalent ions, and proteins. Here, we report the design and characterization of a variety of RNA nanoarchitectures designed in silico using an artificial tetra U-linking RNA module. This flexible RNA building block can be used to design RNA nanoparticles with a variety of geometrical shapes, utilizing an approach previously reported by Khisamutdinov, et. al. 2015. We found that the RNA nanoarchitectures, including a triangle, square, pentagon and hexagon, possess similar self-assembly properties in vitro, with an individual nanoparticle yield of 65.0 ± 6.0 %. The measured melting points by means of UV-thermal denaturation resulted in similar TM values of 80.0 ± 2.0 °C. We further demonstrated that nanoparticles can be assembled with high efficiency, either from a mixture of DNA and RNA or from DNA strands alone. This study demonstrates the importance of artificial RNA building blocks for the RNA nanotechnology field.

References:
Khisamutdinov, E. F., M. N. Bui, D. Jasinski, Z. Zhao, Z. Cui and P. Guo 2015 Simple Method for Constructing RNA Triangle, Square, Pentagon by Tuning Interior RNA 3WJ Angle from 60 degrees to 90 degrees or 108 degrees. Methods Mol Biol 1316:181-193.

Keywords: RNA nanotechnology, Nucleic acid nanoparticles, RNA 3D motif

19. Integrated techniques for the structure determination and RNA binding properties of SHARP

Joel Caporoso (Chemistry, The University of Akron), Caroline Davis (Chemistry, The University of Akron), Stephanie Bilinovich (Pathology, University of North Carolina), Louis Ray, Shaun Christie (Chemistry, The University of Akron), Sumirtha Balaratnam, Soumitra Basu (Chemistry, Kent State University), Thomas Leeper (Chemistry, The University of Akron)

Abstract:
SMRT/HDAC1 Associated Repressor Protein (SHARP) is a multi-domain protein that is involved in multipletranscription regulatory pathways. Four RNA Recognition Motifs (RRMs), that are found near the N-terminus of the protein, are believed to mediate its epigenetic action. Previous data has suggested that SHARP is involved in interacting and regulating the activity of the Steroid Receptor Activator RNA (SRA) through the use of these RRMs. Steroid Receptor Activator RNA (SRA) is a long, noncoding RNA (lncRNA) transcript that also encodes for the biosynthesis of another protein, SRA1p. lncRNAs typically do not participate in translation to make protein, but instead regulate gene expression by interacting with RNA-binding proteins, becoming coactivators for transcription factors, and repressing promoters. SRA, in particular, has been proposed to be involved in multiple complexes that regulate the transcription of nuclear steroid receptors. SHARP has the capability to repress the activity of SRA RNA through complex formation by direct interaction with its RRMs. Chemical shift perturbation studies via 15N heteronuclear single quantum correlation (HSQC) of SHARP and SRA RNA will be presented and indicate that there is a direct interaction between the two species. A preliminary nuclear magnetic resonance (NMR) structural model of a SHARP RRM with the likely binding pocket for the RNA will be discussed. Due to the size restraints of the ribonucleoprotein complexes, other techniques such as Small Angle X-ray Scattering (SAXS) and Paramagnetic Relaxation Enhancement (PRE) will be combined with traditional NMR data to examine the interactions. These and other biochemical data indicate that SHARP RRM 2 may be involved in structural remodeling of the STR7 loop of SRA.

Keywords: SHARP, SRA, NMR

20. Genome wide alternative polyadenylation landscape in the forage legume Red clover (Trifolium pratense L.)

Manohar Chakrabarti (Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546), Randy D. Dinkins (USDA-ARS, Forage-Animal Production Research Unit, Lexington, KY 40546), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546)

Abstract:
Polyadenylation at the 3΄ end of the mRNA is a key step in the mRNA processing with significant bearing on gene expression, mRNA stability, translation ability and others. In recent years evidences are emerging depicting prevalence and role of alternative polyadenylation (APA) on various developmental processes and diseases in humans (Elkon et al., 2013; Shi, 2012). In plants, recent research points towards possible implication of APA on cellular signaling, developmental processes, defense response and oxidative stress tolerance (Chakrabarti and Hunt, 2015; Hunt, 2014). To this end, genome-wide APA analyses of non-model crop species under diverse developmental and physiological conditions are of significant importance. In this study, we have conducted a genome-wide APA analysis of different tissue types of red clover (Trifolium pratense L.), a cool season forage legume, grown widely throughout the northeastern USA.
To this end, poly (A) tag libraries were prepared from leaf, root and flower tissues of red clover using a recently developed protocol (Pati et al., 2015) and were mapped to the red clover genome. The sequence data were used to assemble a list of poly(A) sites, and to study possible occurrences of alternative poly(A) site choice in the three tissues that were sampled. With respect to the latter, evidence supporting the occurrence of tissue-specific APA has been obtained, supporting the hypothesis that tissue-specific APA is an important contributor to regulated gene expression during the development of the plant. Ongoing analyses are focusing on the genomic locations (introns, coding regions, etc.) of PACs displaying tissue-specific APA, and on Gene Ontology (GO) analysis of the genes depicting tissue-specific APA.
This study reveals a genome-wide landscape of APA in a non-model crop species, red clover. Together with previous studies on genome-wide transcriptome analysis of APA in different plants (Wu et al., 2014; Wu et al., 2011), these results will provide comprehensive understanding of the contributions that APA makes to the regulation of gene expression in plants.

References:
1. Chakrabarti, M., and Hunt, A.G. (2015). Biomolecules 5, 1151-1168.
2. Elkon, R., Ugalde, A.P., and Agami, R. (2013). Nat Rev Genet 14, 496-506.
3. Hunt, A.G. (2014). Curr Opin Plant Biol 21, 128-132.
4. Pati, P.K., Ma, L., and Hunt, A.G. (2015). Methods Mol Biol 1255, 159-174.
5. Shi, Y. (2012). RNA 18, 2105-2117.
6. Wu, X., Gaffney, B., Hunt, A.G., and Li, Q.Q. (2014). BMC Genomics 15, 615.
7. Wu, X., Liu, M., Downie, B., Liang, C., Ji, G., Li, Q.Q., and Hunt, A.G. (2011). Proceedings of the National Academy of Sciences, USA 108, 12533-12538.

Keywords: Alternative polyadenylation, red clover, Trifolium

21. Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme

Tien-Hao Chen (Department of Chemistry and Biochemistry and Center for RNA Biology, Ohio State University), Akiko Tanimoto (Department of Chemistry and Biochemistry, Ohio State University), Vicki Wysocki (Department of Chemistry and Biochemistry, Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry and Center for RNA Biology, Ohio State University)

Abstract:
RNase P is an endonuclease essential for maturation of all transfer RNAs (tRNAs). Among all enzymes in nature, RNase P occupies a unique place: it can use either an RNA- or a protein-based active site for catalyzing the cleavage of the 5ꞌ-leader from precursor tRNAs (pre-tRNAs). These distinct RNase P variants appear to be the products of parallel evolution. The well-studied RNA-based RNase P uses a specificity domain to recognize the pre-tRNA substrate and a catalytic domain to perform cleavage. Coincidentally, the newly-discovered proteinaceous RNase P (PRORP) also possesses two RNA-processing domains: a pentatricopeptide repeat (PPR) domain and a metallonuclease (NYN) domain. Here, we combined chemical modification of lysines and mass spectrometry to identify putative substrate-contacting residues on PRORP. Site-directed mutagenesis of these candidate contact sites in PRORP, either individually or in pairs, was used to validate the inferences from our chemical footprinting studies. Binding assays helped delineate lysines in the first PPR motif as important for substrate recognition – PRORP K101A and K101A/K109A led to three- and six-fold increase compared to the wild type in the dissociation constants for pre-tRNA binding. On the other hand, pre-tRNA processing assays identified a lysine in the NYN domain as a contributor to cleavage of the scissile phosphodiester linkage, as the cleavage rates of PRORP K439A, K101A/K439A and K109A/K439A decreased two-fold relative to the wild type. These findings indicate that PRORP utilizes two small regions in the PPR and NYN domains, respectively, to bind and cleave the pre-tRNA. Our results also show that protein- and RNA-based forms of RNase P have distinct modules for substrate recognition and cleavage, an unexpected parallel in their mode of action given their independent evolution.

Keywords: RNA-Protein interaction, Mass Spectrometry, PRORP

22. The DEAD-box helicase Dhh1p couples translation elongation to mRNA decay

Ying-Hsin Chen (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 ), Sophie Martin (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 ), Aditya Radhakrishnan (Program in Molecular Biophysics, Johns Hopkins University School of Medicine), Najwa Al Husaini (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 ), Rachel Green (Program in Molecular Biophysics, Johns Hopkins University School of Medicine), Jeff Coller (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH 44106 )

Abstract:
mRNA decay controls the steady-state levels of mRNAs. In eukaryotic cells, the major pathway of mRNA decay initiated with 3’ deadenylation, followed by 5’ decapping and exonucleolytic digestion in a 5’ to 3’ direction. Our labs recently measured the global decay rate of mRNA and investigated the correlation between mRNA half-lives and codon usage based upon the classical translational efficiency (cTE) scale. We found that mRNA stability correlates well with codon usage. The transcripts with a higher ratio of optimal codons are more stable compared to those composed with non-optimal codons. We also demonstrate that codon optimality impacts ribosome translocation. How is translation elongation coupled to mRNA decay is the subject of our current work.

The DEAD-box helicase Dhh1p is a highly abundant enzyme (>50,000 per cell), far exceeding the levels of all other mRNA decay factors in yeast. Previous results in our lab have shown that endogenous Dhh1p can associate with slowly moving ribosomes. In current study we demonstrate that DHH1 is a sensor of codon optimality- consolidating an mRNA into decay if elongation rate is slow. First, we find that mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a DHH1-dependent manner. We find that these effects on the mRNA decay are sensitive to the concentration of DHH1 in the cell and the number of slowly moving ribosomes on an mRNA. Second, using a tethering system, we find that DHH1 saturates an mRNA with ribosomes when bound. Third, over-expression of DHH1 leads to the accumulation of ribosomes specifically on mRNAs with low codon optimality in ribosome profiling experiments. Finally, biochemical pull-downs show that DHH1 is preferentially associated with mRNAs with suboptimal codon choice.

These findings are consistent with a model that Dhh1p can bind on transcripts with slower translational elongation rate and act to promote mRNA decay.

References:
1. Vladimir Presnyak, N. A.-H., Ying-Hsin Chen, Sophie Martin, Nathan, Morris, N. K., Sara Olson, David Weinberg, Kristian E. Baker, Brenton R. & Graveley, a. J. C. Codon optimality is a major determinant of mRNA stability. Cell (2015).
2. Sweet, T., Kovalak, C. & Coller, J. The DEAD-box protein Dhh1 promotes decapping by slowing ribosome movement. PLoS Biol (2012).

Keywords: DHH1, codon optimality, mRNA decay

23. SHARP RNA recognition motif optimizations, extensions, and mutations for use in 2D and 3D NMR experiments

Shaun Christie (University of Akron), Joel Caporoso, Louis Ray, Dr. Thomas Leeper (University of Akron)

Abstract:
SMRT/HDAC Associated Repressor Protein interacts with the long noncoding RNA, produced by SRA, by binding at the RRMs. Three projects were done to prepare the truncated proteins for use in 2D and 3D NMR experiments. The first focuses on RRM 3 and its optimiza- tion during the purification process. The second focuses on RRM 2-4, which was found to be missing two alpha helices that may be important for protein stability. These helices can also in- teract with RRM 3 as well due to the tight association of RRMs 3 and 4. The two step PCR ex- tension of RRM 2-4 was assumed to work based on sequencing and agarose gels. The final project focused on serine to cysteine mutations of RRM 2 to allow site directed spin labeling for use in NMR PRE experiments. Both mutants developed in this project do not affect the protein structure or the residues surrounding the mutation based on wild type and mutant HSQC spec- tra. The spectra of the spin labeled samples does show broadening some resonances, but may need reductant removed from the buffer sfter the labeling reaction.

Keywords: Protein-RNA interaction, NMR PRE

24. Combined multi-color fluorescence and ultra-high resolution optical tweezers

Cho-Ying Chuang (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan), Miles L Whitmore (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan), Jess L West (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan), Matthew J Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan)

Abstract:
We present a single-molecule instrument that combines ultra-high resolution optical tweezers with multicolor confocal fluorescence microscopy. Timeshared dual optical traps were interlaced and synchronized with three fluorescence excitation lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting detectors (one for each excitation laser). Our new instrument enables the simultaneous measurement of DNA tether extension changes (e.g., from helicase or polymerase motion) and multiple fluorescently labeled observables (e.g., internal protein conformation dynamics via FRET or precise stoichiometry of complexes via multi-colored fluorophore counting). We demonstrated our instrument by measuring the binding and unbinding of fluorophore-labeled single stranded DNA oligonucleotides to a complementary tethered strand of DNA. Further, we combined multi-channel sample chambers with precise computer control of fluorescence measurement and triggered chamber motion to implement an automated ‘molecular assembly line.’ This allowed us to precisely add individual molecules of different types to a single DNA tether while conserving fluorescence photons and reducing photobleaching. In the future, these instrumentation advancements should enable the precise single-molecule assembly and measurement of complex, multi-component molecular machine systems.

References:
M.J. Comstock, Taekjip Ha, and Y.R. Chemla, Nature Methods 8, 335-340 (2011)

Keywords: optical tweezers, fluorescence microscopy, single-molecule

25. Characterizing Interactions Between La-Related Protein 1 and Poly(A) Binding Protein

Gabrielle Ciotti (Department of Biological Sciences, University of Pittsburgh), Roni Lahr (Department of Biological Sciences, University of Pittsburgh), Hiba Al-Ashtal (Department of Biological Sciences, University of Pittsburgh), Andrea Berman, PhD. (Department of Biological Sciences, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

26. HIV-1 subtype-specific differences in tRNALys targeting to viral RNA primer binding site

Roopa Comandur (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University), Erik D. Olson, William A. Cantara, Christopher P. Jones (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University)

Abstract:
Human tRNA^Lys3 serves as the primer for reverse transcription in human immunodeficiency virus type-1 (HIV-1). All three tRNA^Lys isoacceptors are selectively packaged into virions through specific interactions between human lysyl-tRNA synthetase (hLysRS) and the viral Gag protein. tRNA^Lys3 must be released from hLysRS prior to annealing to the complementary primer binding site (PBS) in the 5´-UTR of the HIV-1 genome in order to initiate reverse transcription. This release is partly facilitated by the interaction of hLysRS with a tRNA-like element (TLE) in the HIV-1 genome(1). In the HIV-1 NL4-3 isolate the TLE is located proximal to the PBS and contains a U-rich anticodon-like loop. Small angle X-ray scattering (SAXS) analysis of the NL4-3 PBS-TLE domain has revealed that this region mimics the 3D structure of tRNA(2). The goal of this project is to establish whether the tRNA mimicry and hLysRS binding is conserved across two prototypical HIV-1 subtypes. Here, we study the 5`-UTR region of the MAL isolate, which contains a 23-nt insertion that is known to result in an alternative secondary structure relative to the NL4-3 isolate(3). Fluorescence anisotropy assays used to investigate the binding of hLysRS to MAL-derived genomic RNA constructs revealed high-affinity binding to a 229-nt MAL construct (Kd=47±13 nM). Truncation of this construct at the 3` end yielded a 98-nt construct with ~10-fold reduced binding (Kd=485±133 nM). No binding was observed to smaller stem-loop elements derived from the 98-nt construct and point mutations of this construct did not significantly affect binding. The tertiary structure of both the 229-nt and the 98-nt MAL RNAs was analyzed using SAXS, revealing that these RNAs show remarkable structural similarity to analogous NL4-3-derived RNAs. The results suggest that specific recognition of the HIV-1 PBS-TLE domain by hLysRS is largely based on overall 3D topology rather than sequence-specific recognition.

References:
1. Jones, C. P., Saadatmand, J., Kleiman, L., and Musier-Forsyth, K. (2013) Molecular mimicry of human tRNA^Lys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing, RNA 19, 219-229.
2. Jones, C. P., Cantara, W. A., Olson, E. D., and Musier-Forsyth, K. (2014) Small-angle X-ray scattering-derived structure of the HIV-1 5' UTR reveals 3D tRNA mimicry, Proc Natl Acad Sci USA 111, 3395-3400.
3. Goldschmidt, V., Paillart, J. C., Rigourd, M., Ehresmann, B., Aubertin, A. M., Ehresmann, C., and Marquet, R. (2004) Structural variability of the initiation complex of HIV-1 reverse transcription, J Biol Chem 279, 35923-35931.

Keywords: HIV-1, TLE, MAL

27. Splicing Factor SRSF2 Positively Regulates the Alternative Splicing of MDM2

Daniel F. Comiskey Jr. (Department of Pediatrics, The Ohio State University), Dawn S. Chandler (Department of Pediatrics, The Ohio State University)

Abstract not available online - please check the printed booklet.

28. Rbfox2 plays a major role in regulating alternative splicing in the adult liver

Gandhar Datar (School of Molecular and Cellular Biology, University of Illinois Urbana-Champaign), Waqar Arif (School of Molecular and Cellular Biology, Department of Biochemistry, College of Medicine, University of Illinois Urbana-Champaign), Amruta Bhate (Department of Biochemistry, University of Illinois Urbana-Champaign), Auinash Kalsotra (School of Molecular and Cellular Biology, Department of Biochemistry, College of Medicine, University of Illinois Urbana-Champaign)

Abstract:
Alternative splicing (AS) is a key gene regulatory mechanism that supports normal liver development and function. Aberrant AS has been cited to cause abnormal development and disease pathologies. Splicing factors are an essential component of this type of regulation, increasing or decreasing the expression of certain isoforms by binding to pre-mRNA as part of a spliceosome complex. Rbfox2, a member of the RNA-binding FOX (Rbfox) family of splicing factors is known as a master tissue-specific factor, regulating splicing of many genes including other splicing factors in the brain, muscle, and embryonic stem cells. While the role of Rbfox2 is well-defined in neural and muscle tissues; its function as a splicing regulatory factor in the liver is not understood. We generated a liver-specific knockout (LKO) of Rbfox2 using an albumin-driven CRE recombinase and characterized alternative splicing regulation in Rbfox2-deficient hepatocytes. Splicing assays analyzing the isoforms of over 100 developmentally regulated genes were performed in adult WT and Rbfox2 LKO mice. Several key genes that control hepatic growth showed strong splicing defects including Nf2, an upstream regulator of the Hippo Pathway, and NPRL3, a member of the GATOR1 complex inhibiting the TORC1 Pathway. In addition, histological analysis revealed early onset of fibrosis in LKOs. An integrative analysis of ENCODE generated eCLIP and RNA-seq data from Rbfox2 depleted HepG2 liver cells is being currently analyzed to uncover Rbfox2’s direct gene targets in hepatocytes. Taken together, our results define a critical role for Rbfox2 in regulating hepatocyte-specific splicing and maintaining appropriate liver functioning.

Keywords: Alternative Splicing, Liver, Regulation

29. Trans-Splicing and the Co-Evolution of SNF Proteins with their RNA Targets

Rex Meade Strange (University of Southern Indiana), L. Peyton Russelburg (University of Southern Indiana), Kimberly J. Delaney (University of Southern Indiana)

Abstract:
Although the mechanism of pre-mRNA splicing has been well characterized, the evolution of spliceosomal proteins is poorly understood. The U1A/U2Bʺ/SNF family of RNA binding spliceosomal proteins participates in both the U1 and U2 small interacting nuclear ribonucleoproteins (snRNPs). Both snRNPs play a role in cis-slpicing, the predominant mode of pre-mRNA splicing in many eukaryotic organisms. However, some species are capable of trans-splicing, an alternative mechanism that does not employ the U1 snRNP. In SNF phylogenetic analysis of protostome species, we observed a correlation between trans-splicing species (nematodes and platyhelminths) and increased phylogenetic branch lengths of the U1A/U2Bʺ/SNF protein family. We found that nematodes (~70-80% of pre-mRNAs are trans-spliced) have experienced higher rates of SNF sequence evolution than arthropods (predominantly cis-spliced) at both the nucleotide and amino acid levels. We mapped substitutions to functionally important regions of the SNF protein, specifically to amino acids that are predicted to disrupt protein:RNA and protein:protein interactions. Finally, we investigated SNF’s RNA targets: the U1 and U2 snRNAs. Both exhibit reduced sequence conservation in comparison to that observed in arthropods, suggesting the RNAs have co-evolved with SNF in order to maintain the necessarily high affinity interaction that has been characterized in other species.

Keywords: Trans-Splicing, RRM, U1ASNF

30. Analysis of the debranching reaction with backbone branched RNA substrates

Sourav Kumar Dey (Department of Chemistry, Carnegie Mellon University.), Linda A. Peteanu (Department of Chemistry, Carnegie Mellon University.), Subha R. Das (Department of Chemistry, Carnegie Mellon University.)

Abstract:
The lariat RNA generated during pre-mRNA splicing includes a branch-point adenosine residue that is linked through the 2'-O position to the 5'-end of the RNA sequence. This 2'-5'-phosphodiester linkage in the lariat RNA backbone is debranched by the lariat debranching enzyme (Dbr1p). Following debranching, some introns can participate in highly important biological processes like snoRNA biogenesis, microRNA pathways etc. In the Das lab we have facile access to 2'-5' phosphodiester linked backbone branched RNA (bbRNA) which are mimics of the lariat introns and that enables us to study the cleavage mechanism of Dbr1p. To examine the kinetics of the debranching reaction, we have synthesized a dual labeled bbRNA with Cy3 and Cy5 dyes that acts as donor and acceptor in a FRET based assay. By following the increase in the donor emission, we have been able to find the kinetics constants of the Dbr1p enzyme. We have also synthesized an analogue of the native substrate where the 2'-5' phosphodiester bond is replaced by a trizole linkage. This 'click-branched' RNA serves as a non-cleavable substrate mimic of Dbr1p. Using the FRET based kinetics we show that this 'click-branched' RNA is a competitive inhibitor of Dbr1p. We examine the dependence of the inhibition activity on the length of the stem and the 2'-5' branch of the 'click-branched' RNA. We are currently using these 'click-branched' RNAs in single molecule FRET studies to understand its structure, dynamics and binding to Dbr1p. An initial design for single molecule FRET studies of the branched RNA and the debranching reaction is also presented.

Keywords: Debranching enzyme , Backbone branched RNA, FRET

31. RNA Substrate Recognition and Specificity of Non-Canonical 3'-5' RNA Polymerases.

Samantha Dodbele (The Ohio State Biochemistry Program), Yicheng Long (The Ohio State Biochemistry Program), Jane Jackman (The Ohio State Biochemistry Program, The Center for RNA Biology at Ohio State)

Abstract not available online - please check the printed booklet.

32. Title not available online - please see the printed booklet.

Catey Dominguez (Center for Childhood Cancer, Research Institute at Nationwide Childrens), Dawn Chandler (Center for Childhood Cancer, Research Institute at Nationwide Childrens)

Abstract not available online - please check the printed booklet.

33. Promoting A Novel Approach to Cellular Gene Expression Alteration

Joseph Dong, Christopher Giromini, Woojin Han, Leanne Young (UMD), Aniekanabasi Ufot, Ki Kim, Rajan Patel, Autusa Pahlavan, Sonya Hatten (UMD), Johnathan Dinman (UMD)

Abstract:
A novel method for delivering small interfering RNA (siRNA) to alter cellular gene expression was recently developed at the NIH. This method uses a modular vehicle consisting of a specific ligand coupled to a Hepatitis B Virus-derived RNA binding domain (HPV-RBD). The system enables researchers to deliver siRNAs to specific cell types through cell-specific receptor/ligand interactions. These interactions trigger cells to internalize the receptor/ligand complex via receptor-mediated endocytosis (RME). When the delivery vehicle is internalized, so is the RNA cargo bound to HPV-RBD. The research objective is to develop and refine this novel small-molecule delivery system. Two novel recombinant delivery proteins are being developed: One with Interleukin-8 fused to the HPV-RBD, the other with Machupo Virus GP1 joined to HPV-RBD. After incubating with specific siRNA cargo, the recombinant proteins will be exposed to CEM (a human T-cell line) or HeLa (epithelial) cell cultures. We predict the IL-8 vehicle will specifically deliver RNAs to T-cells through the IL8 receptors CXCR1 and CXCR2, while the Machupo virus GP1, which targets the ubiquitous transferrin receptor, will deliver RNAs to all cells. qRT-PCR will be used to measure changes in specific mRNA levels in both the CEM and HeLa cells. A major limitation to safe, effective, and targeted delivery of therapeutic RNA to living cells is the harshness of conventional techniques. The gentle nature of this technology has the potential to overcome this limitation and could provide a platform for the expansion of personalized medicine.

Keywords: gene expression, RNA interference, receptor mediated endocytosis

34. Title not available online - please see the printed booklet.

Jey Sabith Ebron (Center for Gene regulation in Health and Disease,Cleveland State University), Girish C. Shukla

Abstract not available online - please check the printed booklet.

35. Title not available online - please see the printed booklet.

Dylan Fortman (Chemistry and Biochemistry; The Ohio State University), William Swinehart (Chemistry and Biochemistry; The Ohio State University), Mara Abad (Chemistry and Biochemistry; The Ohio State University), Jane Jackman (Chemistry and Biochemistry; The Ohio State University)

Abstract not available online - please check the printed booklet.

36. Title not available online - please see the printed booklet.

Jane K. Frandsen (Ohio State Biochemistry Program, Center for RNA Biology, OSU), Anna V. Sherwood (Molecular Cellular and Developmental Biology, Center for RNA Biology, OSU), Alexandar L. Hansen (CCIC NMR Facility, OSU), Frank J. Grundy (Department of Microbiology, Center for RNA Biology, OSU), Mark P. Foster (Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, Center for RNA Biology, OSU), Tina M. Henkin (Department of Microbiology, Ohio State Biochemistry Program, Molecular Cellular and Developmental Biology, Center for RNA Biology, OSU)

Abstract not available online - please check the printed booklet.

37. Exploring the function of the EJC and NTC during zebrafish development

Thomas L. Gallagher (Molecular Genetics, The Ohio State University), Kiel T. Tietz (Molecular Genetics, The Ohio State University), Pooja S. Gangras (Molecular Genetics, The Ohio State University), Natalie C. Deans (Molecular Genetics, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University), Sharon L. Amacher (Molecular Genetics, The Ohio State University)

Abstract:
Components of the exon junction (EJC) and nineteen complexes (NTC) are extraordinarily conserved across developmental phyla. Depletion of these factors in vertebrate systems reveals cell- or tissue-specific defects, despite the presumed ubiquitous role of the NTC for pre-mRNA splicing and the EJC for nuclear export, localization, and/or decay of mRNA. These observations hint at shared and non-overlapping roles for EJC and NTC factors and/or EJC and NTC regulation by cell- or tissue-specific regulators. However, the precise roles of the EJC and NTC in post-transcriptional gene regulation during development and differentiation are largely unknown. To characterize developmental and tissue-specific roles of the EJC and NTC, we have isolated zebrafish mutants of core components of each complex.

Using genome-wide SNP association mapping, we molecularly identified the honu lesion as a nonsense mutation in the splicing factor Cdc5l. Cdc5l is a core component of the NTC and plays an important role in pre-mRNA splicing and mitotic progression in mammalian and yeast cells. In zebrafish, honu mutants do not complete segmentation and accumulate cyclic transcripts that are normally cleared rapidly during segmentation. This accumulation occurs post-splicing since expression of nascent transcripts appears normal in these mutants. High resolution detection of cyclic transcripts in honu mutants reveals a pronounced nuclear accumulation defect, suggesting that mRNA export may be compromised. Since late stage spliceosomal components like the NTC interface with the EJC in mammalian cells, we are now exploring the possibility that EJC mutants share similar defects in mRNA stability and localization with honu mutants. Using CRISPR/Cas9 technology, we have generated null alleles in the core EJC genes magoh, eif4a3, and y14/rbm8a. Phenotypic characterization is underway, in addition to global transcript profiling by RNA-seq of wild-type and mutant embryos. In parallel, we are exploring the potential impact that mutation of EJC and NTC core factors has on nonsense mediated decay (NMD) by quantifying Upf1-regulated NMD candidates in EJC and NTC mutants.

Keywords: EJC, NTC, Cdc5l

38. Unraveling the role of the exon junction complex in zebrafish

Pooja S. Gangras (Molecular Genetics, The Ohio State University), Thomas L. Gallagher (Molecular Genetics, The Ohio State University), Kiel T. Tietz (Molecular Genetics, The Ohio State University), Natalie C. Deans (Molecular Genetics, The Ohio State University), Sharon L. Amacher (Molecular Genetics, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

39. Regulation of expression of RNP-4F splicing assembly factor in Drosophila melanogaster

Sushmita Ghosh (Dept. of Biology, Miami University), Shelby E. Thomas (Dept. of Biology, Miami University), Lindsey M. Abraham (Dept. of Biology, Miami University), Jack C. Vaughn (Dept. of Biology, Miami University)

Abstract:
The Drosophila rnp-4f gene encodes RNP-4F protein which is a splicing assembly factor. Several observations suggest that rnp-4f expression may be regulated by a feedback pathway. Northerns and RT-PCR studies suggest that there is a developmental switch that controls the levels of the two isoforms. Northerns show that the long mRNA isoform level peaks in the mid-embryo stage. Westerns and RT-PCR show that high levels of RNP-4F protein correspond to elevated levels of the short rnp-4f mRNA isoform during very early embryo stages and late stages of fly development. Evolutionary conservation of the rnp-4f stem-loop and developmental regulation of alternative transcript levels suggest functional significance of this 5’-UTR stem-loop structure. RNA electrophoretic mobility shift assay using in vitro transcribed RNA (rnp-4f 5’-UTR 177-nt stem-loop) and whole embryo protein extract from wild-type embryos and dADAR mutant embryo protein combined with qPCR analysis and RNAi studies suggest that dADAR is one of the two proteins that bind to the stem-loop. However, the identity of the other protein remains unknown. A structural study has revealed that there exists a conserved sequence on U6-snRNA to which the RNP-4F chaperone may bind. A stretch of 12 nucleotides within the 5’-UTR 177-nt in the rnp-4f mRNA shares significant sequence similarity with the conserved binding site on U6-snRNA. Another level of similarity is that in both cases the consensus sequence lies within a long stem-loop secondary structure. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the ɸC31 system. The results confirm our previous observation that presence of the regulatory stem-loop enhances RNP-4F protein expression. However, in that study, the enhancement factor protein was not identified. We show here that overexpression of RNP-4F transgene results in additional translation, as indicated by the GFP reporter in the fluorescent images. Based on all our findings we propose a model in which RNP-4F binds to the stem-loop in the rnp-4f mRNA 5’-UTR (long isoform) and regulates its own expression via a feedback pathway.

Keywords: rnp-4f gene, UAS-GAL4 system, expression control

40. High throughput screen for inhibitors of RAN translation

Katelyn M. Green (Cellular and Molecular Biology Graduate Program, University of Michigan), Michael G. Kearse (Department of Neurology, University of Michigan), Peter K. Todd (Cellular and Molecular Biology Graduate Program and Department of Neurology, University of Michigan; Veteran Administration Medical Center, Ann Arbor, MI)

Abstract not available online - please check the printed booklet.

41. Dissecting the requirement of Arabidopsis RanGAP1 subcellular targeting and GAP activity for its cellular and developmental functions

Anna H. N. Griffis (Center for RNA Biology and Department of Molecular Genetics, The Ohio State University), Joanna Boruc (Department of Plant Systems Biology, VIB, and Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium), Thushani Rodrigo-Peiris (Department of Molecular Genetics, The Ohio State University), Xiao Zhou, Bailey Tilford (Department of Molecular Genetics, The Ohio State University), Daniel Van Damme (Department of Plant Systems Biology, VIB, and Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium), Iris Meier (Center for RNA Biology and Department of Molecular Genetics, The Ohio State University)

Abstract:
RanGAP is the GTPase activating protein of Ran, in vertebrates involved in nucleocytoplasmic transport, spindle organization and post-mitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis RanGAP1. A double null mutant of the two Arabidopsis RanGAP paralogs is gametophyte lethal. Here, we have created a series of rangap mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, severity of developmental phenotypes increases but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development requires the GAP activity of RanGAP and is susceptible to reductions in RanGAP protein level, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP form the nuclear envelope.

Keywords: RanGAP, Ran, mitosis

42. Using the ykkCD riboswitch as a caging compound for targeted drug delivery

Evan Jones (Ball State University), Von Grogg (Ball State University), Timea Gerczei (Ball State University )

Abstract:
Riboswitches are highly conserved elements in the mRNA that regulate gene expression by directly binding to a small molecular ligand. Ligand binding triggers an allosteric structural change that regulates expression of downstream genes by attenuating transcription or translation. The focus of this study is the ykkCD riboswitch from Bacillus subtilis. Earlier studies showed that ykkCD appears to specifically recognize tetracycline (tet) antibiotics and upregulates expression of downstream genes by attenuating transcription. The goal of this work is to demonstrate that the ykkCD riboswitch is a good candidate to be used as a caging compound or molecular sensor. To achieve this goal we will show that ykkCD is stable and functional under wide variety of conditions (temperature, pH, salt concentrations) and retains its stability and functionality when attached to nanoparticles. We present fluorescent binding assays and thermal melting studies to show that the ykkCD riboswitch is stable under a variety of conditions that are encountered in mammalian cells.

Keywords: Riboswitch , nano technology, fluorescent binding assays

43. RNA triangle, square and pentagon nanoparticles for potent immunomodulation

Sijin Guo (Department of Chemistry, Nanobiotechnology Center, University of Kentucky), Hui Li (Department of Pharmaceutical Sciences, College of Pharmacy, Markey Cancer Center, Nanobiotechnology Center, University of Kentucky), Daniel L. Jasinski (Department of Pharmaceutical Sciences, College of Pharmacy, Markey Cancer Center, Nanobiotechnology Center, University of Kentucky), Jiao Chen (Center for Research on Environmental Disease, Graduate Center for Toxicology, College of Medicine, University of Kentucky), Jian Fu (Center for Research on Environmental Disease, Graduate Center for Toxicology, College of Medicine, University of Kentucky), Peixuan Guo (Department of Pharmaceutical Sciences, College of Pharmacy, Markey Cancer Center, Nanobiotechnology Center, University of Kentucky)

Abstract:
Modulation of immune response through cytokine induction is a vital process in vaccine development and immunology therapy. Herein, we reported the development of a new generation of potent immunomodulators using RNA nanotechnology. RNA triangle, square and pentagon nanoparticles are successfully self-assembled by stretching the interior angle of thermodynamically stable 3WJ motif of phi29 bacteriophage DNA packaging motor pRNA. When immunological adjuvants were incorporated, their immunomodulation effect for cytokine TNF-alpha and IL-6 induction was greatly enhanced in vitro and in vivo, while RNA polygon controls induced unnoticeable effect. The RNA nanoparticles were delivered to macrophages specifically. The degree of immunostimulation is size and shape dependent as well as the number of the payload per nanoparticles. Stronger immune response was observed as the number of adjuvants per polygon was increased. This finding demonstrates that RNA nanotechnology such as developing RNA nanoparticles based on pRNA has the great potential to develop potent immunomodulators.

References:
[1] Guo,P. (2010) The emerging field of RNA nanotechnology. Nat. Nanotechnol., 5, 833–842.
[2] Khisamutdinov,E.F., Jasinski,D.L. and Guo,P. (2014) RNA as a boiling-resistant anionic polymer material to build robust structures with defined shape and stoichiometry. ACS Nano,8, 4771–4781.
[3] Jasinski,D.L., Khisamutdinov,E.F., Lyubchenko,Y. and Guo,P. (2014) Physicochemically Tunable Poly-Functionalized RNA Square Architecturewith Fluorogenic and Ribozymatic Properties. ACS Nano, doi:10.1021/nn502160s .

Keywords: immunomodulation, RNA polygon, 3WJ

44. Mutation of human WDR4 impairs tRNA m7G46 methylation and causes a distinct form of microcephalic primordial dwarfism

Ranad Shaheen, Rana Alomar (Department of Genetics, King Faisal Specialist Hospital and Research Center), Ghada M.H. Abdel-Salam, Hanan H. Afifi, Samira I. Ismail, Bayoumi A Emam (Clinical Genetics Department, National Research Centre, Cairo, Egypt ), Michael P. Guy (Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry), Mohamed S. Abdel-Hamid (Medical Molecular Genetics Department, National Research Centre, Cairo, Egypt), Eric M. Phizicky (Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry), Fowzan S Alkuraya (Department of Genetics, King Faisal Specialist Hospital and Research Center)

Abstract not available online - please check the printed booklet.

45. Promoting a Novel Approach to Cellular Gene Expression Alteration (PANACEA)

Joseph Dong, Christopher Giromini, Woojin Han, Sonja Hatten, Ki Kim (University of Maryland), Autusa Pahlavan, Rajan Patel, Aniekanabasi Ufot, LeAnne Young (University of Maryland)

Abstract:
A novel method for delivering small interfering RNA (siRNA) to alter cellular gene expression was recently developed at the NIH. This method uses a modular vehicle consisting of a specific ligand coupled to a Hepatitis B Virus-derived RNA binding domain (HPV-RBD). The system enables researchers to deliver siRNAs to specific cell types through cell-specific receptor/ligand interactions. These interactions trigger cells to internalize the receptor/ligand complex via receptor-mediated endocytosis (RME). When thedelivery vehicle is internalized, so is the RNA cargo bound to HPV-RBD. The research objective is to develop and refine this novel small-molecule delivery system.Two novel recombinant delivery proteins are being developed: One with Interleukin-8 fused tothe HPV-RBD, the other with Machupo Virus GP1 joined to HPV-RBD. After incubating with specific siRNA cargo, the recombinant proteins will be exposed to CEM (a human T-cell line) or HeLa (epithelial) cell cultures. We predict the IL-8 vehicle will specifically deliver RNAs to T-cells through the IL8 receptors CXCR1 and CXCR2, while the Machupo virus GP1, which targets the ubiquitous transferrin receptor, will deliver RNAs to all cells. qRT-PCR will be used to measure changes in specific mRNA levels in boththe CEM and HeLa cells.A major limitation to safe, effective, and targeted delivery of therapeutic RNA to living cells is the harshness of conventional techniques. The gentle nature of this technology has the potential to overcome this limitation and could provide a platform for the expansion of personalized medicine

Keywords: siRNA, endocytosis, medicine

46. Drought Stress Changes Poly (A) Site Choice in Arabidopsis thaliana

Guijie Hao (Department of Plant and Soil Sciences, University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky)

Abstract:
mRNAs polyadenylation is an important step in eukaryotic gene expression. Polyadenylation impacts gene expression through determining the coding and regulation potential of the mRNA, especially mRNAs of genes that may be polyandenylated at more than one position. Alternative polyadenylation (APA) has potential effect to gene regulation and function. In Arabidopsis, APA may be linked with stress responses through a polyadenylation complex subunit (CPSF30) that is inhibited by calmodulin and by disulfide remodeling; calcium signaling (through calmodulin) and disulfide remodeling (through the generation of reactive oxygen species) are typically associated with various stress responses in plants, suggestive of a possible connection.
In order to explore this, a study was done to examine the effects that an artificial drought stress (triggered by growth of plants in mannitol) has on genome-wide poly (A) site choice. We treated wild type Arabidopsis with different concentrations of mannitol, for varying lengths of time. RNA was extracted after harvesting the whole seedlings and poly (A) tag libraries were prepared and sequenced on the Illumina platform. The sequence data was analyzed to assess gene expression and alternative polyadenylation. The gene expression analysis demonstrated genes from certain GO categories are up- or down- regulated by the simulated drought stress; these categories include responses to hormone stimulus, secondary metabolism, transmembrane transport, regulation of transcription, and protein phosphorylation. Moreover, the results showed that drought stress caused significant changes in the usage of poly (A) sites that lie within 5’-UTRs; specifically, the poly (A) site profiles for a number of genes were shifted to increased usage of sites within 5’ UTRs, site that would truncate the mRNA.
These results indicate that drought stress alters poly (A) site choice for many genes, and may play negative regulatory roles for genes that possess poly (A) sites that fall within 5’-UTRs. They also suggest that APA plays an important role in the responses of Arabidopsis to drought stress. Future studies will include 3’-RACE confirmation of selected genes, and comparisons of the effects of drought stress with other abiotic and biotic stresses.

Keywords: Alternative polyadenylation, drought, Arabidopsis thaliana

47. Defining the role of MRB10130 in uridine insertion and deletion RNA editing in trypanosoma brucei

Gregory L. Harrison Jr. (Department of Microbiology and Immunology, SUNY Buffalo School of Medicine), Michelle Ammerman (Department of Microbiology and Immunology, SUNY Buffalo School of Medicine), Laurie K. Read (Department of Microbiology and Immunology, SUNY Buffalo School of Medicine)

Abstract:
In Trypanosoma brucei, the majority of the mitochondrially-encoded RNAs require the post-transcriptional addition and deletion of uridines to create translatable open reading frames through an essential process termed Uridine Insertion/Deletion RNA Editing. The enzymes that catalyze RNA editing are contained in the RNA Editing Core Complex. Recent studies have also identified the Mitochondrial RNA Binding Complex (MRB1), comprising a dynamic network of protein-protein and protein-RNA interactions, as an essential component of the editing machinery. Combining a comprehensive yeast two-hybrid (Y2H) screen and in vivo immunoprecipitation and mass spectrometry, we identified an MRB1 core and at least two TbRGG2 subcomplexes in MRB1. Interestingly, one of the proteins in the Y2H screen, MRB10130, interacted with a large number of MRB1 components within both the MRB1 core and TbRGG2 subcomplexes. MRB10130 is predicted to be composed almost entirely of alpha-helical repeats that resemble ARM/HEAT repeats, and such proteins often act as organizers of protein-protein interactions. Thus, we hypothesize that MRB10130 is involved in coordinating interactions within the MRB1 complex. Here, we show that MRB10130 is essential for both growth and RNA editing in T. brucei. We performed glycerol gradient sedimentation analysis of affinity purified MRB10130-PTP and compared that to a similar analysis of whole cell extracts. We show that MRB10130 co-purifies MRB1 core and TbRGG2 subcomplexes, but our data suggest that MRB10130 is more loosely associated with MRB1 than are other proteins, consistent with an organizing function. Furthermore, we use a cell line harboring inducible MRB10130 RNAi and an epitope-tagged MRB1 core component to show an RNA-enhanced MRB10130 involvement in maintaining interactions between the MRB1 core and TbRGG2 subcomplexes. We also show that MRB10130 is able to weakly bind RNA in vitro. We propose a model in which MRB10130 and RNA work synergistically to stabilize interactions between the MRB1 core and TbRGG2 subcomplexes.

Keywords: Trypanosoma brucei, RNA editing

48. diffHunter: A general scalable framework for comparing genome-wide protein occupancy profiles to study protein-RNA interactomes

Seyedsasan Hashemikhabir (Biohealth Informatics, Indiana University ), Vishal Kumar Sarsani (Biohealth Informatics, Indiana University ), Sarath Chandra Janga (Biohealth Informatics, Indiana University )

Abstract not available online - please check the printed booklet.

49. Promoting a Novel Approach to Cellular Gene Expression Alteration

Joseph Dong, Christopher Giromini, Woojin Han, Sonja Hatten, Ki Kim (Cell Biology and Molecular Genetics, University of Maryland), Autusa Pahlavan, Rajan Patel, Aniekanabasi Ufot, LeAnne Young (Cell Biology and Molecular Genetics, University of Maryland), Jonathan Dinman (Cell Biology and Molecular Genetics, University of Maryland)

References:
Biragyn, A., Bodogai, M., Olkhanud, P.B., Denny-Brown, S.R., Puri, N., Ayukawa, K., Kanegasaki, S., Hogaboam, C.M., Wejksza, K., and Lee-Chang, C. (2013). Inhibition of lung metastasis by chemokine CCL17-mediated in vivo silencing of genes in CCR4+ Tregs. J. Immunother. 36, 258-267.

Keywords: Gene Expression, RNA interference, Receptor-mediated Endocytosis

50. Title not available online - please see the printed booklet.

Jennie E. Hazen (the Ohio State University), Yicheng Long (Ohio State Biochemistry Program), Jane E. Jackman (the Ohio State Biochemistry Program, the Center for RNA Biology at Ohio State)

Abstract not available online - please check the printed booklet.

51. The role of LUC7L2 in splicing and bone marrow failure

Courtney E. Hershberger (Cellular and Molecular Medicine, Cleveland Clinic Foundation), Naoko Hosono (Taussig Cancer Institute, Cleveland Clinic Foundation), Hideki Makishima (Taussig Cancer Institute, Cleveland Clinic Foundation), Jarnail Singh (Cellular and Molecular Medicine, Cleveland Clinic Foundation), Jaroslaw P. Maciejewski (Taussig Cancer Institute, Cleveland Clinic Foundation), Richard A. Padgett (Cellular and Molecular Medicine, Cleveland Clinic Foundation)

Abstract:
Myelodysplastic syndromes (MDS) are a group of disorders characterized by bone marrow failure and frequent progression to acute myeloid leukemia (AML). MDS is the most common hematological malignancy in patients over 65, with only 35% surviving three years after diagnosis. Recent studies have shown that over 65% of MDS patients harbor recurrent point mutations in several proteins involved in spliceosomal splicing. One of these is the poorly characterized splicing-related protein, LUC7L2. 14% of MDS patients are deficient in LUC7L2 expression and this low expression is correlated with more rapid disease progression and decreased survival. The function of LUC7L2 is unknown, but its yeast ortholog LUC7 is involved in recruitment of early splicing factors. We hypothesize that LUC7L2 acts as a mammalian splicing factor and deficiency of LUC7L2 results in aberrant splicing of oncogenes and tumor suppressor genes that contribute to the pathogenesis of MDS. To test this hypothesis, the protein binding-partners of LUC7L2 were examined by immunoprecipitation followed by mass spectrometry. This experiment confirmed that LUC7L2 interacts with snRNP-associated proteins and SR proteins. RNA crosslinking and immunoprecipitation followed by high-throughput sequencing was performed to identify the RNA binding sites of LUC7L2. The analysis showed that LUC7L2 binds the snRNAs, most often the early splicing factors U1, U2, and U12. It also crosslinks to at least 300 pre-mRNA transcripts where the binding sites are located in exons near splice junctions consistent with a role in exon definition. LUC7L2-knockdown cells show reduced splicing of a subset of introns. Finally, RNA-Seq transcriptomes of AML patient samples with and without LUC7L2-deficiency were compared and differential splicing of at least 44 transcripts was detected. Therefore, LUC7L2 demonstrates the characteristics of a splicing factor that binds pre-mRNA, influencing splicing, and the transcripts it binds are candidates for further study in the pathogenesis of MDS.

Keywords: LUC7L2, Pre-mRNA Splicing, Myeloid Neoplasm

52. Identifying the regulatory role of chromatin in transcription termination

A. Elizabeth Hildreth (Department of Biological Sciences, University of Pittsburgh), Karen M. Arndt, PhD (Department of Biological Sciences, University of Pittsburgh)

Abstract:
Normal cellular growth and development relies on the precise regulation of gene expression. In eukaryotes, the chromatin template acts as a barrier to the transcription machinery. Chromatin consists of nucleosomes, which contain approximately 147 basepairs of DNA surrounding an octamer of histone proteins H2A, H2B, H3, and H4. To faithfully express the genetic material, a host of transcription factors regulate RNA polymerase II activity during transcription initiation, elongation, and termination. The Nrd1-Nab3-Sen1 (NNS) complex regulates transcription termination of many short noncoding RNAs, some short protein-coding RNAs, and intergenic transcripts in yeast. Despite a few studies showing that transcription-coupled histone modifications are important for proper termination, very little is known about the role of chromatin in this pathway. The goal of my project is to elucidate how chromatin influences transcription termination using the NNS pathway in Saccharomyces cerevisiae as a model. To screen for histone residues required for termination by the NNS pathway, I have made use of a plasmid library encoding histones with all possible alanine substitutions and a well-characterized termination reporter. I have identified 10 residues in H3 and H4 with defects in termination, and have begun functionally characterizing their transcription-related phenotypes. Analysis to this point reveals that these amino acid substitutions in the histones cause widely varying phenotypes, perhaps suggesting that these residues promote NNS-dependent termination in different ways. Future work will elucidate the mechanisms by which these histone residues regulate proper transcription termination.

Keywords: chromatin, transcription, S cerevisiae

53. Identification of mitochondrial DNA-associated trans-acting factors potentially required for mtRNA editing in Physarum polycephalum

Jillian Houtz (Center for RNA Molecular Biology, CWRU), Jonatha Gott (Center for RNA Molecular Biology, CWRU)

Abstract:
Extensive RNA editing is required for proper gene expression in the mitochondria of the acellular slime mold Physarum polycephalum. The term RNA editing describes changes in nucleotide sequence from that encoded in DNA, comprising both nucleobase substitutions or conversions as well as nucleotide insertions and deletions; however, the majority (>94%) of editing events in P. polycephalum mtRNAs comprise single cytosine (C) insertions. While it is known that insertional editing in P. polycephalum is co-transcriptional (i.e., non-templated nucleotides are added at the 3' end of the nascent RNA during transcription), the mechanism of insertional editing remains unclear. Preliminary data from chimeric template experiments implicate a requirement for at least one template-associated trans-acting protein factor. Previous work from our lab has yielded a method for fractionation of mitochondrial lysates which reduces the protein complement of the native mtDNA template by greater than 90%. The resulting partially purified mtDNA-protein complex is a substrate for transcription - as well as mtRNA editing - and thus we refer to it as a mitochondrial Transcription Elongation Complex (mtTEC).
Following digestion of the DNA in mtTECs with restriction endonucleases, we have been able to enrich for protein factors associated with discreet regions of the mitochondrial genome that are both actively transcribed and whose transcripts are also extensively edited. When ligated to a short biotinylated DNA cassette, restriction-digested mtTEC DNA fragments can be affinity selected on streptavidin-coated magnetic beads. We have recently demonstrated that these immobilized fragments retain editing competency and thus any potential editing factors. The proteins associated with these editing-competent mtTEC fragments have been sequenced via mass spectrometry, narrowing down the number of potential candidates for factors required for nucleotide insertion to a manageable number. The subset of these proteins that have probable DNA or RNA binding domains will be the first targeted for silencing by RNAi to determine their role (if any) in co-transcriptional mtRNA editing in P. polycephalum.

Keywords: Physarum polycephalum, mitochondria, RNA editing

54. Probing the physiological functions of prolyl-tRNA synthetase-related trans-editing domains

C. Bradley Howard (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Ziwei Liu (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA), Michael E. Hoover (Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA), Michael A. Freitas (Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA)

Abstract not available online - please check the printed booklet.

55. Role of MALAT1 lncRNA in breast cancer disease progression

Mahdieh Jadaliha (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA), Rotem Karni (Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada The Hebrew University-Hadassah Medical School, Jerusalem, Israel), Partha S. Ray (Carle Foundation Hospital, Urbana, Illinois. Department of Surgery, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA), Kannanganattu V. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA)

Abstract not available online - please check the printed booklet.

56. Solution Structure of the HIV-1 Intronic Splicing Silencer and its Interaction with the UP1 Domain of hnRNP A1

Niyati jain (Department of Chemistry, Case Western Reserve University, Cleveland, Ohio), Christopher E. Morgan (Department of Chemistry, Case Western Reserve University, Cleveland, Ohio), Brittany D. Rife (Department of Pathology, Immunology and Laboratory Medicine University of Florida Gainesville, Florida), Marco Salemi (Department of Pathology, Immunology and Laboratory Medicine University of Florida Gainesville, Florida), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University, Cleveland, Ohio)

Abstract:

Alternative splicing is of the HIV life cycle, yet little is known about the structures that control splice site selection. Splicing from donor site D4 to acceptor site A7 removes the RRE to allow expression of late phase viral proteins. Given the importance of this splicing event to controlling viral replication, the activity of splice site A7 is tightly regulated by a complex network of an intronic splicing silencer (ISS), a bipartite splicing silencer (ESS3a/b) and an exonic splicing enhancer (ESE3). RNA chemo-enzymatic probing studies have shown the isolated splice site A7 locus folds into three RNA stem loops, where the regulatory elements localize to distinct apical loops. The host hnRNP A1 protein binds SL1(ISS) and SL3(ESS3) to effectively repress splice site A7, whereas the ASF protein counteracts hnRNP A1 by binding SL2(ESE3). To gain a better understanding of the RNA structures and protein interactions that regulate A7, we have solved the high-resolution structure of the ISS stem loop. ISS folds into a 53-nt long stem-loop RNA composed of several non-canonical structural features: a UG wobble tract, a stable 2X2 internal loop, a UU bulge and a 5-nt apical loop. As a step towards understanding how hnRNP A1 gets recruited to splice site A7, we have further characterized the interaction of its UP1 domain with ISS. UP1 binds to the apical loop with high affinity and specificity. Collectively, this data provides valuable insights into developing structure based mechanisms of HIV splicing.

Keywords: Alternative splicing , HIV-1 ssA7, hnRNPA1

57. Platination of nucleotides and nucleic acids by cisplatin and its derivatives

Jun Jiang (Department of Chemistry, Wayne State University), Bett Kimutai (Department of Chemistry, Wayne State University), Harjot Mann (Department of Chemistry, Wayne State University), Ahmed El-Moussa (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
Cisplatin and its derivatives have been successfully employed in the treatment of certain types of cancer. Coordination of platinum (II) to the N7 of G residues in DNA is established as the molecular mechanism resulting in cell death. Recent work has shown that RNA molecules are also susceptible towards platination reactions, and certain cisplatin derivatives, such as the amino-acid-linked Oplatin, demonstrate differential site preferences depending on the local RNA folding or nucleotide identities. Therefore, cisplatin and its derivatives are not only important as anticancer drugs, but may also serve as probes of secondary structures within large noncoding RNAs. Nuclear magnetic resonance (NMR) spectroscopy was employed to monitor reactions between NMPs (N = A, G, and C) and Oplatin, and the results were compared with those using the corresponding nucleosides. To determine the RNA product profiles following platination, enzyme digestions of the modified oligonucleotides coupled with mass spectrometry were employed. Together, the results from these two approaches are used to correlate the properties of coordination complexes and their reactivates towards different RNA residues or local structural elements, which is helpful for the design of new RNA-targeting drugs and probes.

Keywords: cisplatin, nucleotide, RNA

58. Increased extracellular osmolarity hits the ribosomal subunits and delays recovery from stress

Raul Jobava (Department of Biochemistry, Case Western Reserve University, Cleveland, OH), Dawid Krokowski (Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH ), Maria Hatzoglou (Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH )

Abstract not available online - please check the printed booklet.

59. Nucleic acid quadruplexes in biotechnological applications

Besik Kankia (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), David Gvarjaladze (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Adam Rabe (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Levan Lomidze (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Nunu Metreveli (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210)

Abstract:
The biotechnological applications of DNA quadruplexes we are investigating are based on the unique properties of the GGGTGGGTGGGTGGG (G3T) oligomer: (i) it is capable of forming a quadruplex with significantly higher stability than the corresponding duplex; (ii) G3T acts as a monomeric unit for a higher degree of tetrahelical architecture; (iii) formation of the quadruplex represents an efficient molecular switch to turn on intrinsically fluorescent nucleotides.
Quadruplex priming amplification (QPA) employs the free energy of G3T as a driving force for endergonic DNA amplification. In addition, intrinsic fluorescence of G3T allows highly specific and sensitive monitoring of product DNA without any external quantification mechanisms. As a result, QPA represents an efficient alternative to PCR with plateau-free, isothermal, simplified and low-cost amplification of nucleic acid signals.
Quadruplex-and-Mg2+ connection (QMC) is based on the tetrahelical architecture of G3T and has two key components: (i) shape complementarity between QMC partners introduced by specific modifications of the quadruplexes and (ii) Mg2+ ions. The on-rate of QMC formation is between 105 - 106 M-1 s-1, while the off-rate is undetectable even at 80 °C. However, QMC dissociates rapidly upon removal of Mg2+ ions (i.e., by EDTA). QMC can create new opportunities in biomedical research by introducing a new class of capture molecules with advantages over the streptavidin-biotin system including reversibility, multiplexing, higher stability and specificity, longer shelf life and low cost.
DNA quadruplexes as structural and recognition elements in nanotechnology. The monomolecular tetrahelical architecture and QMC capabilities of quadruplexes have the potential to revolutionize DNA nanotechnology by introducing fast and error-free self-assembly of extraordinarily stable molecules. The advantages of the tetrahelical structure over DNA duplexes include: (i) monomolecularity of self-assembly, which eliminates errors characteristic of bi-molecular duplex formation; (ii) the ability to be folded/unfolded with little change in ionic strength; (iii) the folded structure is a tenth of the size of the original single strand, which could be used to induce movements in nanomachinery.

Keywords: quadruplexes, NA amplification, NA nanostructures

60. Title not available online - please see the printed booklet.

Alan Kessler (Department of Microbiology and Center of RNA Biology, The Ohio State University), Zdenk Paris (Biology Centre, Institute of Parasitology Czech Republic), Juan Alfonzo (Department of Microbiology, The Ohio State Biochemistry Program, Center of RNA Biology, The Ohio State University )

Abstract not available online - please check the printed booklet.

61. The JAK-STAT pathway is regulated by miRNA mediated Ribosomal Frameshifting.

Yousuf A. Khan (Department of Cell Biology and Molecular Genetics, University of Maryland.), Vivek Advani (Department of Cell Biology and Molecular Genetics, University of Maryland.), Zach Flickinger (Department of Cell Biology and Molecular Genetics, University of Maryland.), Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland.)

Abstract not available online - please check the printed booklet.

62. Design and Synthesis of Nucleic Acid Architectures Based on Flexible tetra-U Linking Module

Emil F. Khisamutdinov (Department of Chemistry, Ball State University, Muncie IN 47306)

Abstract not available online - please check the printed booklet.

63. Promoting a Novel Approach to Cellular gene Expression Alteration

Ki Kim, Rajan Patel, Chris Giromini, LeAnne Young, Woojin Han, Joe Dong (University of Maryland), Sonja Hatten, Autusa Pahlavan, Ani Ufot (University of Maryland)

Keywords: Gene Expression, RNA interference, Receptor mediated endocytosis

64. Reaction kinetics of amino-acid-linked platinum analogues and characterization of platinated nucleosides

Bett Kimutai (Department of Chemistry, Wayne State University), Xun Bao, Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract not available online - please check the printed booklet.

65. Extensive Alternative RNA editing in the NADH dehydrogenase subunit 3 transcript in Trypanosoma brucei

Laura Kirby (Dept. of Microbiology and Molecular Genetics, Michigan State University), Joshua Foster (Dept. of Microbiology and Molecular Genetics, Michigan State University), Yanni Sun (Dept. of Computer Science and Engineering, Michigan State University), Donna Koslowsky (Dept. of Microbiology and Molecular Genetics, Michigan State University)

Abstract:
RNA editing in Trypanosoma brucei is necessary to create functional transcripts for many mitochondrial genes. Specific editing events are directed by small RNA molecules called guide RNAs (gRNAs). In the gRNAs collection identified for the NADH dehydrogenase subunit 3 transcript, a gRNA was identified that could encode an alternative edit. Gene specific primers were used to target the edited mRNA region in question and an abundant alternative edit was identified. This editing event was found to be followed by multiple other alternative editing events that generate a completely unique sequence. Searching the EATRO gRNA transcriptomes with this new sequence identified several highly abundant gRNAs that would encode these edits. These data suggest that different “sets” of gRNAs can be involved in the editing of the same primary transcript, greatly increasing the coding potential of the mitochondrial genome.

Keywords: RNA editing, Trypanosoma brucei, NADH dehydrogenase

66. Gene length as a biological timer for transcriptional regulation following serum stimulation

Killeen Kirkconnell (Radiation Oncology, University of Michigan), Brian Magnuson (Radiation Oncology, University of Michigan), Michelle Paulsen (Radiation Oncology, University of Michigan), Karan Bedi (Radiation Oncology, University of Michigan), Mats Ljungman (Radiation Oncology, University of Michigan)

Abstract not available online - please check the printed booklet.

67. A heterodimer-based regulatory role for Drosophila truncated dADAR protein isoform function

Fatemeh Kohram (Biology Dept., Miami University), Sushmita Ghosh (Biology Dept., Miami University), Jack C. Vaughn (Biology Dept., Miami University)

Abstract:
The Drosophila rnp-4f gene encodes RNP-4F protein which is a splicing assembly factor. The Drosophila rnp-4f gene codes for two mRNA isoforms (“long” and “short”) which differ by a 177-nt sequence (caused by alternative splicing in the 5’-UTR within intron 0 and exon 2) that forms an evolutionarily conserved stem-loop. Several observations suggest that rnp-4f expression may be regulated by a feedback pathway. Northerns and RT-PCR studies suggest that there is a developmental switch that controls the levels of the two isoforms. Northerns show that the long mRNA isoform level peaks in the mid-embryo stage. Westerns and RT-PCR show that high levels of RNP-4F protein correspond to elevated levels of the short rnp-4f mRNA isoform during very early embryo stages and late stages of fly development. Evolutionary conservation of the rnp-4f stem-loop and developmental regulation of alternative transcript levels suggest functional significance of this 5’-UTR stem-loop structure. RNA electrophoretic mobility shift assay using in vitro transcribed RNA (rnp-4f 5’-UTR 177-nt stem-loop) and whole embryo protein extract from wild-type embryos and dADAR mutant embryo protein combined with qPCR analysis and RNAi studies suggest that dADAR is one of the two proteins that bind to the stem-loop. However, the identity of the other protein remains unknown. A structural study has revealed that there exists a conserved sequence on U6-snRNA to which the RNP-4F chaperone may bind. A stretch of 12 nucleotides within the 5’-UTR 177-nt in the rnp-4f mRNA shares significant sequence similarity with the conserved binding site on U6-snRNA. Another level of similarity is that in both cases the consensus sequence lies within a long stem-loop secondary structure. Here, we describe construction and utilization of several new rnp-4f gene expression study vectors using a GFP reporter in the Φ C31Based on all our findings we propose a model in which RNP-4F binds to the stem-loop in the rnp-4f mRNA 5’-UTR (long isoform) and regulates its own expression via a feedback pathway.

Keywords: dADAR, protein function

68. Being present is a big part of any job, but not the only one: Multiple roles of the trans-acting Nop7-subcomplex in the assembly of yeast ribosomal 60S subunits.

Salini Konikkat (Dept. of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA-15213), John L. Woolford Jr. (Dept. of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA-15213)

Abstract not available online - please check the printed booklet.

69. Potential roles of unconventional G-quadruplexes in rRNA biogenesis

Karen A. Kormuth (Dept. of Biological Sciences, Carnegie Mellon University), Bruce A. Armitage (Dept. of Chemistry, Carnegie Mellon University), John L. Woolford, Jr. (Dept. of Biological Sciences, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

70. Analysis of the bloodstream stage gRNA transcriptome in Trypanosoma brucei.

Laura Kirby (Microbiology and Molecular Genetics, Michigan State University), David Judah (Merial Veterinary Scholars Program, Michigan State University), Yanni Sun (Computer Science and Engineering, Michigan State University ), Scooter Nowak (Entomology, Michigan State University), Donna Koslowsky (Microbiology and Molecular Genetics, Michigan State University)

Abstract:
The mitochondrial genome of Trypanosoma brucei contains many cryptogenes that must be edited following transcription before being translated. This editing is directed sequentially by guide RNAs (gRNAs), which encode the specific insertion and deletion of uridines. We have obtained the gRNA transcriptome from the bloodstream form of the EATRO 164 cell line. Using conventionally accepted fully edited mRNA sequences, ~1 million gRNAs were identified. In contrast, over 3 million reads were identified in our insect stage gRNA transcriptome. The overall ratio of procyclic to bloodstream gRNA reads was 3.5:1. This ratio varies significantly by gene and by populations within genes and, except for the initiating gRNA, no apparent trend relating gRNA abundance and developmental editing pattern was observed. A comparison of related major classes from each transcriptome revealed a median value of ten single nucleotide mutations per gRNA. Mutations were much less likely to occur in the consecutive Watson-Crick anchor region, indicating a strong bias against G:U base pairs in this region.

Keywords: Trypanosoma brucei, RNA editing , transcriptome

71. Title not available online - please see the printed booklet.

Kiel D. Kreuzer (MCDB, Department of Microbiology, Center for RNA Biology, The Ohio State University), Nicholas J. Green (MCDB, Department of Microbiology, The Ohio State University), Frank J. Grundy (Department of Microbiology, Center for RNA Biology, The Ohio State University), Tina M. Henkin (Department of Microbiology, Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

72. Characterization of the interaction between a trans-editing domain and its aminoacyl-tRNA substrate

Alexandra Kuzmishin (Department of Chemistry and Biochemistry, The Ohio State Biochemistry Program, The Center for RNA Biology, The Chemistry-Biology Interface Program, The Ohio State University, Columbus, OH), Eric Danhart (Department of Chemistry and Biochemistry, The Ohio State Biochemistry Program, The Center for RNA Biology, The Chemistry-Biology Interface Program, The Ohio State University, Columbus, OH), Brianne Sanford, Marina Bakhtina (Department of Chemistry and Biochemistry, The Center for RNA Biology, The Ohio State University, Columbus, OH), Ronald Micura (Institute of Organic Chemistry and Center for Molecular Biosciences, Innsbruck CMBI Leopold Franzens University, Innsbruck, Austria), Mark Foster, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Center for RNA Biology, The Ohio State University, Columbus, OH)

Abstract:
Aminoacyl-tRNA synthetases (ARSs) catalyze the esterification of specific amino acids to their cognate tRNA substrates to form aminoacyl-tRNAs. ARSs can also misactivate and mischarge tRNAs with smaller or similarly-sized non-cognate amino acids. Prolyl-tRNA synthetase (ProRS) mischarges Ala and Cys onto tRNA^Pro in vitro. Many synthetases, including most bacterial ProRSs, exhibit proofreading activities to correct errors due to mischarging and thus prevent amino acid misincorporation into proteins. Bacteria that encode ProRS with a functional editing domain (INS) are able to deacylate Ala-tRNA^Pro via post-transfer editing. Some bacteria lack a functional INS domain and instead encode a free-standing INS-homolog, ProXp-ala, that deacylates Ala-tRNA^Pro via post-transfer editing in trans. The tRNA^Pro recognition elements for Caulobacter crescentus ProXp-ala have been identified, although the sites of interaction on ProXp-ala are unknown. NMR chemical shift perturbation studies were used to map regions of interaction between ProXp-ala and an uncharged microhelix derived from the acceptor stem of tRNA^Pro. Binding to a stably mischarged Ala-microhelix^Pro substrate analog was also investigated for the first time. To establish the significance of the identified regions for substrate binding and catalysis, ProXp-ala variants have been generated via site-directed mutagenesis of 27 residues located throughout the protein structure. Analytical ultracentrifugation was used to screen the variants for relative changes in binding affinity to microhelix^Pro and deacylation assays have been used to establish the effect on catalytic activity. Based on these studies, we have generated a comprehensive model for substrate binding and recognition by a trans-editing domain.

Keywords: aminoacyl-tRNA synthetase, ProRS, ProXp-ala

73. Investigating binding kinetics of RNA aptamers via the Open SPR system

Volition La (University of Waterloo )

Abstract not available online - please check the printed booklet.

74. LARP1 interacts directly with 5’UTRs encoding translation machinery

Roni M. Lahr (Department of Biological Sciences, University of Pittsburgh), Hiba A. Al-Ashtal (Department of Biological Sciences, University of Pittsburgh), Andrea J. Berman, Ph.D. (Department of Biological Sciences, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

75. The role of the DEAD-box protein Dbp2 in RNA structure remodeling

Yu-Hsuan Lai (Biochemistry, Purdue University), Sara C. Cloutier (Biochemistry, Purdue University), Elizabeth J. Tran (Biochemistry, Purdue University)

Abstract not available online - please check the printed booklet.

76. Examination of the disordered C-terminal domain of a host regulator of HIV-1 splicing

Jeffrey D. Levengood (Chemistry, Case Western Reserve University), Christopher Morgan (Chemistry, Case Western Reserve University), Blanton S. Tolbert (Chemistry, Case Western Reserve University)

Abstract:
Alternative splicing of the HIV-1 genome is necessary for translation of the complete viral proteome. Host proteins, such as hnRNP A1, are used to regulate splicing at the various donor and acceptor sites along the viral genome. The multi-faceted hnRNP A1 is composed of three domains, two structurally identical RRM domains that form the nucleic acid binding protein UP1, and a glycine rich C-terminal domain that is primarily involved in protein-protein interactions.
A crystal structure of UP1 was solved with a rAGU trinucleotide bound to the protein. This structure revealed that while only RRM1 binds the RNA, RRM2 is responsible for positioning several residues involved in the binding of the substrate. This mode of binding opens the possibility for allosteric regulation of hnRNP A1, presenting a need to study the structural properties of the C-terminus and its potential interactions with RRM2.
A preliminary view of the structure of full length hnRNP A1 has been obtained through SAXS-scored structural modeling. This model revealed the C-terminal domain to be a long, unstructured, disordered polypeptide chain extending away from the UP1 domain. In this model, the C-terminus appears to be independent of UP1 as there are no interactions between the two. The C-terminus is further being studied through the use of NMR with amino acid selective labeling. Mutations to mimic modification of the C-terminus are also being carried out. Finally, the binding of small molecules and peptides to the C-terminus is being examined through the use of both Isothermal Titration Calorimetry (ITC) and NMR.

References:
Levengood, J.D., Rollins, C., Mishler, C. H. J., Johnson, C.A., Miner, G., Rajan, P., Znosko, B.M., and Tolbert, B.S. (2012) Solution Structure of the HIV-1 Exon Splicing Silencer 3. J. Mol. Biol. 415:680-698.

Rollins, C. Levengood, J.D., Rife, B., Salemi, M., and Tolbert, B.S. (2014) Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element. Biochemistry 53(13):2172-84.

Morgan, C.E., Meagher, J.L., Levengood, J.D., Delproposto, J., Rollins, C., Stuckey, J.A., and Tolbert, B.S. (2015) The first crystal structure of the UP1 domain of hnRNP A1 bound to RNA reveals a new look for an old RNA binding protein. J. Mol. Biol.

Keywords: RRM, Splicing, HIV

77. Dual color fluorescent reporters as a tool to study alternative splicing regulation of the Hippo signaling pathway.

Cole Lewis (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Joseph Seimetz (Department of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry and Medical Biochemistry, College of Medicine, Institute of Genomic Biology, University of Illinois, Urbana-Champaign)

Abstract:
The Hippo tumor suppressor pathway is a crucial regulator of cellular proliferation and the maintenance of organ size. We identified a coordinated splicing isoform switch during mouse and human liver development in two crucial genes in the Hippo signaling pathway, NF2 and YAP1. NF2 acts as an upstream regulator while YAP1 is the major downstream effector of the signaling cascade. To define the cis-acting elements and trans-acting factors regulating the NF2 and YAP1 alternative exons, we engineered dual-color fluorescent reporters to monitor their splicing in real time and at single cell resolution. Skipping of the alternative exon within the reporter maintains the reading frame and results in the production of functional dsRED protein, while inclusion results in a frame shift that produces eGFP or BFP. We demonstrate that the fluorescent readouts exhibit strong correlation with the splicing patterns of respective minigenes in a variety of mammalian cell lines. Cotransfection of the reporters with splice site-blocking morpholino oligonucleotides completely abrogates the exon inclusion, demonstrating that the system can be dynamically regulated. Finally, we show that cell confluence has striking effects on the reporter fluorescence output such that rapidly dividing cells at low density produce a mixture of dsRED and eGFP proteins where as contact inhibition due to high cell density results in exclusive production of eGFP. Future efforts will dissect the cell confluence mechanism(s) that dictate the splicing and fluorescence outcome of these reporters.

Keywords: Hippo Pathway, NF2, YAP1

78. Title not available online - please see the printed booklet.

Ranjodh S. Sandhu (Center for Gene Regulation in Health and Disease, Cleveland State University), Unnati Pandya (NYU Langorie Medical Center, New York University), Vishal Nanavaty (Center for Gene Regulation in Health and Disease, Cleveland State University), Bibo Li (Center for Gene Regulation in Health and Disease, Cleveland State University)

Abstract not available online - please check the printed booklet.

79. Global analyses of RNA binding specificity reveal independent contributions of sequence and structure.

Hsuan-Chun Lin (Department of Biochemistry, Case Western Reserve University School of Medicine), Jing Zhao (Department of Biochemistry, Case Western Reserve University School of Medicine), Courtney Niland (Department of Biochemistry, Case Western Reserve University School of Medicine), Brandon Tran (Department of Biochemistry, Case Western Reserve University School of Medicine), Eckhard Jankowsky (Center for RNA Molecular Biology, Case Western Reserve University School of Medicine), Michael E Harris (Department of Biochemistry, Case Western Reserve University School of Medicine)

Abstract not available online - please check the printed booklet.

80. Highthroughput probing of human lncRNA secondary structures

Yizhu Lin (Department of Biological Sciences, Carnegie Mellon University), Gemma May (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Long noncoding RNAs (lncRNAs) comprise a large proportion of the human transcriptome. Over the past decade, lncRNAs have been increasingly recognized as important regulators of multiple gene expression processes, and a growing number have been associated with human development and diseases. Different from mRNAs which are coding for proteins, lncRNA structures and their interactions with proteins are believed to be crucial in lncRNA regulatory functions. To experimentally determine the secondary structures of lncRNAs, a recently developed method, Mod-seq, is applied. In Mod-seq, RNAs are first chemical probed by SHAPE reagent, and then undergo high throughput sequencing to determine their secondary structures. Mod-seq provides substantial improvements in throughput than traditional RNA secondary structure determination methods, allowing quick structural probing of the full length lncRNAs that are thousands nucleotides long. We applied Mod-seq method on two in vitro transcribed human lncRNAs, sno-lncRNA and NEAT1. Sno-lncRNA is found to be deleted in an important human disease, Prader-Willi Syndrome (PWS). Sno-lncRNAs are produced from introns with two imbedded snoRNA genes, which leads to transcription of lncRNAs flanked by snoRNA sequences at both 5’ and 3’ ends without 5’ cap and 3’ poly(A) tails. Our Mod-seq probing showed that the 5’ and 3’ snoRNA sequences in sno-lncRNA have similar secondary structural patterns, and the 4 predicted fox2 protein binding sites share a partial single stranded partial double stranded structure motif that resembles internal loop. NEAT1 is another nuclear lncRNA, it serves as the structural scaffold for paraspeckles. Our Mod-seq probing on NEAT1 is aimed to reveal stable secondary structures and conserved secondary structural motifs across species.

References:
Talkish, J., May, G., Lin, Y., Woolford, J. L. & McManus, C. J. Modseq: highthroughput sequencing for chemical probing of RNA structure. RNA 20, 71320 (2014).

Keywords: lncRNA, RNA secondary structure, high-throughput sequencing

81. hnRNP K regulates HEXIM’s ability to interact with Stem I on 7SK RNA

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

Abstract not available online - please check the printed booklet.

82. Title not available online - please see the printed booklet.

Levan Lomidze (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), David Gvarjaladze (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Nunu Metreveli (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Olia Rcheulishvili (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Besik Kankia (Department of Chemistry and Biochemistry, The Ohio State University, Columbus 43210, USA; Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia)

Abstract not available online - please check the printed booklet.

83. ZFP3, an RNA binding protein involved in Trypanosoma brucei differentiation, is modulated by arginine methylation

Kaylen Lott (Microbiology and Immunology, SUNY at Buffalo), Jun Li (Pharmaceutical Sciences, SUNY at Buffalo), Jun Qu (Pharmaceutical Sciences, SUNY at Buffalo), Laurie Read (Microbiology and Immunology, SUNY at Buffalo)

Abstract not available online - please check the printed booklet.

84. Secondary Structure Probing of 7SK upon HEXIM1 Binding

Le Luo (Department of Chemistry, Case Western Reserve University), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
During transactivation of the HIV-1 proviral genome, P-TEFb is recruited to the nascent RNAP II complex. P-TEFb exists in equilibrium between an inactive and active pool, where the 7SK snRNA along with the cellular protein HEXIM1 sequesters P-TEFb in its inactive state. Previous EMSA studies have demonstrated the specific binding between HEXIM1 and 7SK, however, the mechanism of the HEXIM1-7SK interaction is not clear. A structural profile of 7SK-HEXIM1 complex will demonstrate the specific binding site and binding mechanism, thus, elucidate the molecular mechanism of P-TEFb regulation. In this study, mutagenesis of the 7SK stem loops demonstrated that more than one structural element of this 7SK snRNA are involved in the HEXIM1-7SK binding event; the DMS modification and sequencing profiles will provide the secondary structure of 7SK and the structural changes when binding with HEXIM1.

Keywords: 7SK, HEXIM1, DMS

85. Biochemical and proteomic characterization of alternate exon junction complexes in mammalian cells

Justin Mabin (Molecular Genetics, The Ohio State University), Mengxuan Jia (Chemistry and Biochemistry, The Ohio State University), Lauren Woodward (Molecular Genetics, The Ohio State University), Kate Ehrensberger (Molecular Genetics, The Ohio State University), Vicki Wysocki (Chemistry and Biochemistry, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University)

Abstract:
In mammalian cells, the exon junction complex (EJC) is deposited during pre-mRNA splicing ~24 nt upstream of most exon-exon junctions in a sequence-independent manner. The EJC core is comprised of a stable tetrameric protein complex consisting of eIF4AIII, Y14:Magoh heterodimer, and MLN51. This stable core travels with mRNA to the cytoplasm, and serves as a platform for recruitment of more dynamic peripheral/adapter proteins that direct mRNA export, localization, translation and nonsense-mediated mRNA degradation (NMD). To date, it is unknown if peripheral proteins interact with all EJC cores. In EJCs purified from cultured human cells, we found levels of the so-called core protein, MLN51, and several peripheral proteins to be sub-stoichiometric to those of the core suggesting that they are part of only a subset of EJCs. Spurred by these findings, we have now discovered at least two stable alternate EJCs that are distinguished by the presence of either MLN51 or RNPS1. Biochemical analysis of FLAG-affinity purified MLN51 and RNPS1 protein complexes from HEK293 cells showed that they are unique in composition and size. A comprehensive proteomic analysis confirmed their mutually exclusive nature, and further revealed their distinct compositional differences. We find that RNPS1 is associated with the EJC core (except MLN51), it's previously known partners (ACIN1, SAP18 and PNN), and several SR proteins and splicing factors. On the other hand, MLN51 is mainly associated with the EJC core proteins and is missing almost all of the RNPS1 associated factors. Instead, it purifies the EJC disassembly protein PYM, and some components of the translation and NMD machinery. Our results suggest that EJCs undergo a dynamic remodeling process before or upon MLN51 incorporation. We are now investigating underlying mechanisms that lead to a sequential exchange of peripheral EJC factors and overall EJC maturation.

Keywords: Exon Junction Complex, post-transcriptional gene regulation, Alternate EJCs

86. Synthesis of backbone branched RNA substrates for the investigation of debranching enzyme

Stephanie Mack (Chemistry, Carnegie Mellon University), Subha R. Das (Chemistry, Carnegie Mellon University)

Abstract:
Lariat debranching enzyme (Dbr1p) cleaves the 2'-5'-phosphate linkage in spliced introns. Little is known about the exact mechanism through with Dbr1p functions and how substrate composition affects cleavage. To examine the Dbr1p mechanism, we have synthesized backbone branched RNAs (bbRNAs) that contain a 2'-5'-phoshpate linkage. Here we describe advances to the methods of bbRNA synthesis, particularly in the photodeprotection step. The stem of the bbRNA is synthesized with a 2'-photoprotected residue and the removal of this group is a critical step for branch synthesis. Using the newly optimized method, we obtained different substrates of Dbr1p that include functional group and single atom substitutions. The synthesized substrates are used for a debranching assay analysis of Dbr1p cleavage.

Keywords: Debranching Enzyme

87. A study of aminoglycosides binding to 16S rRNA constructs

Prabuddha Madubashitha (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
The development of resistance in pathogenic bacteria and adverse health effects are among the major challenges of using antibiotics. In developing better antibiotics, the knowledge of the binding of a particular compound to its target at the molecular level is very important. The objective of our study is to gain a better understanding of the binding affinity, binding site and drug-induced structural changes of the bacterial ribosome with potential antibiotics using a combination of biochemical and biophysical studies. Initially the binding of classical and modified aminoglycosides (AGs) to helix 44 (h44) of the aminoacyl-tRNA site (A site) of E. coli ribosomes was studied by using chemical probing. For the biophysical studies, the interactions between AGs and 27mer h44 constructs from E. coli, wild-type human mitochondrial, and a human mitochondrial mutant were studied by using nuclear magnetic resonance spectroscopy. Preliminary data suggest a difference between binding of AGs to wild-type human mitochondrial and human mitochondrial mutant h44 constructs, which can be used to explain the observed AG-related ototoxicity. In addition, a fluorescence-based assay is being employed to further study these interactions. These results will lead to a better understanding of drug-ribosome interactions, including drug side effects, at the molecular level.

Keywords: Ribosome, Aminoglycosides, Ototoxicity

88. Dynamics of G-quadruplex and Telomestatin Interactions at the Single Molecule Level

Parastoo Maleki (Department of Physics, Kent State University, Kent, OH, USA), K. Nagasawa (Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan), H.Balci (Department of Physics, Kent State University, Kent, OH, USA)

Abstract not available online - please check the printed booklet.

89. RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1

Mohammad Adnan Siddiqui (Department of Biological Sciences, University of Toledo), Krishnamurthy Malathi (Department of Biological Sciences, University of Toledo), Sushovita Mukherjee (Department of Biological Sciences, University of Toledo), Praveen Manivannan (Department of Biological Sciences, University of Toledo)

Abstract:
Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded
RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic
protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral
infections and cancer.

Keywords: RNase L, Autophagy, apoptosis

90. Regulation of Eukaryotic Translational Initiation by Programmed -1 Ribosomal Frameshifting.

Arvin Massoudi (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Isabella Swafford (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Jessica Stimely (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Theodore Nikolaitchik (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Adam Kellerman (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park), Savannah Speir, Vivek Advani, Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park)

Abstract not available online - please check the printed booklet.

91. Title not available online - please see the printed booklet.

Ashanti Matlock (Dept. of Chemistry and Biochemistry; Ohio State University), Jane Jackman (Dept. of Chemistry and Biochemistry; Ohio State University)

Abstract not available online - please check the printed booklet.

92. Characterization of two essential components of the trypanosome mitochondrial RNA-binding complex 1, MRB1

Natalie M. McAdams (SUNY Buffalo), Laurie K. Read (SUNY Buffalo)

Abstract:
Kinetoplastid parasites, including Trypanosoma brucei, T. cruzi, and Leishmania spp., are the causative agents of African sleeping sickness, Chagas’ disease, and leishmaniasis in humans, respectively, leading to hundreds of thousands of deaths per year worldwide. Mitochondrial mRNAs in kinetoplastid parasites require extensive remodeling by a unique process termed uridine insertion/deletion RNA editing to create translatable open reading frames. The RNA editing catalytic enzymes are present in a stable multiprotein structure termed the RNA editing core complex (RECC) or 20S editosome. In addition to RECC, a multiprotein complex called the mitochondrial RNA-binding complex 1 (MRB1) in T. brucei is required for efficient RNA editing. We reported the presence of an MRB1 core that contains at least 7 proteins and is essential for RNA editing initiation. We have also identified subcomplexes containing the TbRGG2 RNA binding/annealing protein; known proteins of the TbRGG2 subcomplexes are essential for 3’ to 5’ progression of editing on pan-edited RNAs. Here, we present ongoing studies of two potential TbRGG2 subcomplex proteins, MRB800 and PhyH. RNAi-mediated knockdown of MRB800 or PhyH in insect stage T. brucei leads to a severe growth defect. However, the effects on mRNA levels are different between the two proteins. Depletion of PhyH results in a decrease in only extensively edited transcripts, while removal of MRB800 causes a decrease in all edited transcripts. Both proteins affect the progression of RNA editing within the ATPase 6 transcripts but appear to affect progression at different places along the editing process. In addition, we have begun to characterize the protein-protein interactions exhibited between MRB800, PhyH, and other components of the TbRGG2 subcomplex. These studies provide another piece in the puzzle of our ongoing characterization of the essential and multifunctional RNA editing complex, MRB1.

Keywords: RNA editing , trypanosomes

93. Title not available online - please see the printed booklet.

Katherine M. McKenney (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.

94. Title not available online - please see the printed booklet.

Kyle J. Messina (Department of Chemistry, Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, and Center for RNA Molecular Biology, Pennsylvania State University)

Abstract not available online - please check the printed booklet.

95. Title not available online - please see the printed booklet.

Gayan Mirihana Arachchilage (Department of Chemistry and Biochemistry, Kent State University), Joshua Reid (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.

96. Inhibitory effects of H69-targeting peptides on protein translation in bacteria

Nisansala Muthunayake (Chemistry, Wayne State University), Christine S. Chow (Chemistry, Wayne State University)

Abstract:
Identifying novel drug targets within the bacterial ribosome is an important approach to overcome the well-known problem of antibiotic resistance. The specific region of the ribosome under investigation in this study is helix 69 (H69). Considering the variety of functions of H69 in protein biosynthesis, as well as differences between the bacterial and human H69 sequences, this RNA is an attractive antibacterial drug target. In a previous study, short peptides that specifically bind to H69 were isolated by using phage-display libraries. The main objective of the current study was to investigate the inhibitory effects of these H69-targeting peptides on protein synthesis in bacteria. In order to determine their in vivo activity, minimum inhibitory experiments were carried out with E. coli MRE 600 and lpxA (lipid membrane mutant) strains. Our preliminary results showed inhibition of protein translation in the presence of peptides alone and in combination with other small molecules; however, it was clear that the peptides have cell penetration problems. Therefore, H69-targeting peptides were expressed in vivo as GFP-fusion proteins and their activities were monitored through cellular fluorescence levels. Expression of two different peptides was shown to have an inhibitory effect on bacterial cell growth. These findings will be helpful for future antimicrobial drug development.

Keywords: Ribosome, Helix 69, Peptides

97. Title not available online - please see the printed booklet.

Monali NandyMazumdar (Microbiology, The Ohio State University), Irina Artsimovitch (Microbiology, The Ohio State University)

Abstract not available online - please check the printed booklet.

98. High-Throughput Enzymology Reveals Shared Molecular Recognition of Precursor tRNAs by the RNA and Protein Subunits of RNase P

Courtney N. Niland (Department of Biochemistry, Case Western Reserve University), Jing Zhao (Department of Biochemistry, Case Western Reserve University), Hsuan-Chun Lin (Department of Biochemistry, Case Western Reserve University), David R. Anderson (School of Business, CUNY Baruch College), Eckhard Jankowsky (Department of Biochemistry, Case Western Reserve University; Center for RNA Molecular Biology, Case Western Reserve University), Michael E. Harris (Department of Biochemistry, Case Western Reserve University)

Abstract:
The specificity of ribonucleoproteins is essential to their functions in biology. That function requires the ability to recognize cognate substrates from non-cognate binding sites in the cell. Ribonuclease P, RNase P, is a multi-substrate ribonucleoprotein enzyme that removes the 5’ leader from all pre-tRNAs despite variation in sequence and structure. Previous work demonstrated that the protein and RNA subunits of RNase P intimately contact nucleotides 5’ to the pre-tRNA cleavage site, the 5’ leader, but these positions vary considerably in the E. coli genome. The specificity of the enzyme for different 5’ leader sequences is poorly understood. E. coli RNase P was used to process pre-tRNA substrate pools randomized in their 5’ leader sequences in both the protein and RNA subunit binding sites (N(-1) to N(-6)). Using High-Throughput Sequencing Kinetics, HTS-Kin, to analyze this population we comprehensively determined the rate constants for processing of all possible 5’ leader sequences in the RNase P binding site. The resulting affinity distribution provides a complete description of enzyme specificity and reveals the full context dependence of mutations on processing rate. Importantly, these data sets reveal a strong influence of sequence identity in the binding site of the RNA subunit on sequence specificity and energetic contribution of contacts in the protein subunit binding site. Analyses of individual sequence variants confirmed the accuracy of the high throughput data and are being used to investigate the mechanistic basis for this strong energetic coupling between RNA-protein and RNA-RNA interactions. Surprisingly, this data also revealed pairing of the 5’ leader with the 3’ACCA as a positive determinant despite known enzyme contacts in this region. Further insight into the mechanistic basis for these effects will reveal how RNase P achieves specificity and provide deeper insight into molecular recognition by multi-substrate ribonucleoprotein enzymes.

Keywords: Enzymology, Ribonucleoprotein, RNase P

99. Title not available online - please see the printed booklet.

Sneha Nishtala (Department of Biohealth Informatics, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana 46202), Yaseswini Neelamraju (Department of Biohealth Informatics, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana 46202), Sarath Chandra Janga (Department of Biohealth Informatics, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana 46202)

Abstract not available online - please check the printed booklet.

100. Polyadenylation as a Multifaceted Translational Regulator in Human Pathogens

Hannah Nourie (Department of Biological Sciences, Carnegie Mellon University), Alex Kilbo (Department of Biological Sciences, Carnegie Mellon University), Kausik Chakrabarti (Department of Chemistry, CNAST, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

101. AR Precursor mRNA Splicing: Understanding the Cause and Consequence of Drug Resistance in Prostate Cancer

Narmada Pandey Sapkota (BGES, Cleveland State University), Jey Sabith Ebron (BGES, Cleveland State University), Girish C Shukla, Ph.D. (BGES, Cleveland State University)

Abstract:
Prostate cancer is the most prevalent cancer in American male and only second to skin cancer. Progression of prostate cancer mainly depends on expression and signaling of androgen receptor (AR). AR is a ligand-dependent transcription factor of steroid receptor family, which is required for normal growth and development. It plays a pivotal role in development of prostate cancer. Conventional treatment for the prostate cancer mainly involves androgen deprivation, surgical or medical castration. Nearly 20% of prostate cancer either bypass or sensitize the AR signaling pathway leading to an advanced form of disease, castration resistance prostate cancer (CRPC). Among many molecular mechanism involving AR functions in development of CRPC, expression of novel alternative spliced isoform appears to play an important role. Various, alternatively spliced, AR isoforms have been identified in prostate cancer, however molecular mechanism contributing to the expression of these novel tumors-specific isoforms and their function remains undefined. In this study, we are investigating AR precursor mRNA splicing in an attempt to understand the development of drug resistance, presumably mediated by novel AR mRNA alternatively spliced isoforms in prostate cancer. We are planning to study the effect of AR mutations on AR pre-mRNA splicing using a novel AR minigene reporter construct. Our experiment aims to reveal the consequences of naturally occurring mutations of AR in prostate cancer patients, who have developed resistance to next generation of therapeutics including Enzalutamide (Xtendi) and Abiraterone Acetate (Zytiga). We will discuss the preliminary data obtained from initial AR minigene splicing characterization experiment. Understanding the basic molecular mechanism of alternative splicing of AR in prostate cancer will help better management of prostate cancer.

Keywords: AR Splicing, AR precursor mRNA splicing

102. Title not available online - please see the printed booklet.

Krishna Patel (Department of Chemistry & Biochemistry,The Ohio State University), Paul Yourik (Department of Chemistry & Biochemistry,The Ohio State University), Jane E Jackman (Department of Chemistry & Biochemistry,The Ohio State University)

Abstract not available online - please check the printed booklet.

103. Assaying the Splicing Activity of Novel Human Disease Variants of U4atac snRNA

Maitri K. Patel (Cleveland State University), Rosemary C.Dietrich (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic), Richard A. Padgett (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic)

Abstract:
In eukaryotes, pre-messenger RNA (pre-mRNA) splicing is an essential process in gene expression. Splicing is carried out by a dynamic multi-megadalton RNA-protein complex known as the spliceosome. Sequential transesterification reactions catalyzed by the spliceosome convert pre-mRNA to mRNA by removing the intervening sequences (introns) and joining the coding sequences (exons) together. Small nuclear RNAs (snRNAs) are essential splicing factors. Biallelic mutations of the human RNU4ATAC gene, which codes for U4atac snRNA, have been identified in patients diagnosed with microcephalic osteodysplastic primordial dwarfism type I (MOPD I). MOPD I is an autosomal recessive disorder characterized by extreme intrauterine growth retardation, multiple organ abnormalities, and typically early death. The mutations that have been studied biochemically reduce U4atac snRNA function and impair minor class (U12-dependent) intron splicing. Four novel patient mutations, 37 G>A, 46 G>A, 48 G>A and 118 T>C, have recently been discovered. To evaluate the functional effects of these newly discovered mutations on U12-dependent splicing, we incorporated each of these mutations into a modified human RNU4ATAC gene construct by site-directed mutagenesis. Following verification of the mutations by DNA sequencing, we prepared DNA for use in an in vivo splicing assay that is based on genetic suppression. These mutations are expected to affect the binding of proteins to U4atac snRNA that are important in formation of the catalytically active form of the spliceosome. We do not yet know how the consequent defective U12-dependent splicing affects gene expression and yields the MOPD I disease pathologies, but this study allows us to better understand the mechanistic basis of MOPD I and will serve as an important foundation for further studies and possible therapeutic intervention in the future.

References:
H. He et al., Science 332, 238. (2011)
F. Jafarifar et al., RNA 20(7), 1078. (2014)
E. Kilic et al., Am J Med Genet Part A 167A, 919.(2014)
R. A. Padgett, Trends in Genetics 28(4), 147. (2012)

Keywords: RNA-splicing

104. Modified oligonucleotide detection by MS/MS spectral matching

Mellie June Paulines (Rieveschl Laboratories for Mass Spectrometry Department of Chemistry, University of Cincinnati ), Patrick A. Limbach (Rieveschl Laboratories for Mass Spectrometry Department of Chemistry, University of Cincinnati )

Abstract not available online - please check the printed booklet.

105. Interactions between hnRNP H and G-tracts (DNA/RNA) reveal the specific recognition for GGG-rich nucleic acids

Srinivasa R. Penumutchu ( Department of Chemistry, Case Western Reserve University, Cleveland. ), Jennifer Meagher (Life Sciences Institute, University of Michigan), Theresa Ramelot ( Department of Chemistry and Biochemistry Miami University, Oxford Ohio), Sebla Kutluay ( Department of Molecular Microbiology, School of medicine, Washington University), Paul Bieniasz ( Department of Molecular Microbiology, School of medicine, Washington University), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University, Cleveland. )

Abstract not available online - please check the printed booklet.

106. Pyrollo-C as a Potential Analog to Cytosine for Structural Studies

Claire A. Porterfield (Allegheny College), Martin Serra (Allegheny College)

Abstract not available online - please check the printed booklet.

107. Initial characterization of a regulatory antisense transcript in Toxoplasma gondii

Abby S. Primack (Biological Sciences, University of Pittsburgh), Emily F. Klonicki (Biological Sciences, University of Pittsburgh), Jon P. Boyle (Biological Sciences, University of Pittsburgh)

Abstract:
The obligate, intracellular parasite Toxoplasma gondii infects 30% of the world’s population, is lethal to immunocompromised humans, and causes birth defects and abortion. After host cell invasion, the parasite forms a vacuole and associates with host mitochondria. Host-mitochondrial association (HMA+) is not present in all T. gondii strains and this has allowed us to use forward genetics to identify the responsible gene. The gene, MAF1, is highly expressed in HMA+ strains while the protein product is undetectable in HMA- negative strains. Although all HMA ± strains produce MAF1 transcript, HMA- strains produce a significantly smaller amount of transcript and a larger amount of antisense transcript as compared to HMA+ strains. We hypothesize that a product encoded in the antisense region suppresses MAF1 expression through transcriptional interference. To begin to test this hypothesis, we are characterizing the sense and antisense promoter activities of MAF1. Putative promoter sequences were transfected into T. gondii and promoter strength was quantified using a reporter assay. Through comprehensive sequencing, our results suggest that there are multiple antisense promoter paralogs within each strain. We also demonstrate that the antisense promoter is stronger than its sense counterpart, indicating that we have isolated a highly active antisense promoter region. Future work aims to further characterize MAF1 antisense promoter activity, and ultimately determine the impact of the antisense transcript on MAF1 expression and HMA.

References:
Pernas, L, Y. Adomako-Ankomah, A. J. Shastri, S. E. Ewald, M. Treeck, J. P. Boyle, J. C. Boothroyd. 2014. Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS Biology 12(4): e1001845

Keywords: antisense transcript, pathogen, promoter

108. A transcription factor aids in noncoding RNA-templated replication by DNA-dependent RNA polymerase II

Jie Qu (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA), Ying Wang and Jian Wu (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA), Shaoyi ji and Yi Li (College of Life Sciences, Peking University, Beijing, China), Andrew Wallace, Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA), David M. Bisaro (Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA), Biao Ding (Deceased, Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA)

Abstract:
Some DNA-dependent RNA polymerases (DdRP) possess RNA-dependent RNA polymerase activity, suggesting their evolution from RdRPs that first arose to transcribe RNA templates. This relic RdRP activity is required for transcription of certain noncoding RNAs that regulate gene expression, and for replication of plant viroids and human hepatitis delta virus. Here, we used Potato spindle tuber viroid (PSTVd) as a model to uncover cellular factor(s) that regulate RNA-templated replication. RNA polymerase II (Pol II)-catalyzed replication of PSTVd in the nucleoplasm employs a rolling-circle mechanism that involves the generation of (+)- and (-)-strand RNAs. We report here that the 7-zinc finger (ZF) transcription factor IIIA (NbTFIIIA-7ZF) from Nicotiana benthamiana, a shorter variant of the DNA template-specific 9-ZF TFIIIA (NbTFIIIA-9ZF), interacts with Pol II in vivo and is essential for PSTVd replication. Both forms of NbTFIIIA interact with the (+)-strand, but only NbTFIIIA-7ZF interacts with PSTVd (-)-strand in vitro and in vivo. Antisense-mediated suppression of NbTFIIIA decreased PSTVd replication, but only overexpression of NbTFIIIA-7ZF, and not NbTFIIIA-9ZF, enhanced replication. Consistent with known sites of PSTVd replication, NbTFIIIA-7ZF localizes to the nucleoplasm and the nucleolus, whereas NbTFIIIA-9ZF is confined to the nucleolus. Further, footprinting assays revealed that NbTFIIIA-7ZF binds to a region of PSTVd critical for initiating PSTVd transcription. Our results identify NbTFIIIA-7ZF as a dedicated cellular transcription factor in DdRP-catalyzed RNA-templated replication, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broad role in cellular processes.
This work was supported by NSF grant IOS-1354636 to B. D. and D. M. B.
This abstract is dedicated to the memory of Dr. Biao Ding

Keywords: Viroid, Transcription factor, noncoding RNA

109. Chromatin Regulation of Pericentric Non-coding RNA in S. cerevisiae affects chromosome stability

Apoorva Ravishankar (Biology, West Virginia University), Julia A. Gallo (Biology, West Virginia University), Jen Gallagher (Biology, West Virginia University)

Abstract not available online - please check the printed booklet.

110. Ribosome Profiling reveals a conditional role for upstream open reading frames in neuronal differentiation.

Caitlin M. Rodriguez (Department of Neurology, University of Michigan), Sang Y. Chun (Department of Computational Medicine and Bioinformatics, University of Michigan), Ryan E. Mills (Department of Computational Medicine and Bioinformatics, University of Michigan), Peter K. Todd (Department of Neurology, University of Michigan)

Abstract:
Ribosome profiling is a next-generation sequencing method that provides a transcriptome-wide snapshot of ribosome occupancy¹. This technique demonstrates that fully assembled ribosomes frequently occupy the 5’ leader sequence outside of traditional open reading frames, suggesting a potential role for upstream opening reading frame (uORFs) in cellular functions². To explore the potential role of uORFs in neuronal differentiation, we performed ribosome profiling in both undifferentiated and retinoic acid differentiated SH-SY5Y neuroblastoma cells. Using a novel spectral coherence-based classification algorithm to identify regions of active translation (SPECtre), we have identified a series of high confidence uORFs in SH-SY5Y cells, many of which exhibit initiation at non-AUG codons. Interestingly a subset of these transcripts exhibit a strong shift in uORF usage with retinoic acid differentiation as shown by increased conditional ribosome occupancy in the absence of concomitant changes in RNA abundance. 59% of these shifting transcripts demonstrate a negative correlation between uORF and main ORF occupancy with only 5% showing a positive relationship, corroborating the understanding that uORFs typically act to negatively regulate downstream transition³. Taken together, our findings suggest that uORF usage is a common feature in neuronal-like cells and that the use of uORFs is altered upon neuronal differentiation, suggesting a functional role for these processes in neuronal development.

References:
1. Ingolia, NT, et al.,Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 324, 218-23 (2009).
2. Ingolia, NT, et al.,Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes. Cell. 147, 789-802 (2011).
3. Sonenberg, N and Hinnebusch, AG, Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets, 136, 731-45 (2009).

Keywords: ribosome profiling, upstream open reading frame, translation

111. Reverse transcription driven uncoating of mature HIV capsid

Ioulia Rouzina (Biochemistry, OSU), Robijn Bruinsma (Physics, UCLA)

Abstract:
Until recently it was a commonly accepted notion that reverse transcription (RTion) in retroviruses, including HIV, takes place within the cytoplasm of the infected cell after uncoating of the mature capsid. However, accumulating evidence suggests that the RTion process happens largely within the mature capsid core, which protects the viral genome from host factors and maintains high local concentrations of the essential viral proteins within the RTion complex. In this work, we consider the problem of mature HIV capsid uncoating driven by polymerization of double stranded (ds) pro-viral DNA during RTion. The millimolar concentrations of nucleocapsid protein (NC) contained within the capsid drives aggregation of both single-stranded (ss) gRNA and dsDNA, provided the capsid is intact. Flexible gRNA is aggregated by NC into a ribonucleoprotein complex occupying only a small fraction of the capsid volume. While the self-volume of the full-length pro-viral DNA (~104 bp) is the same as of its diploid gRNA genome, the dsDNA is very rigid, and gets condensed by NC into a large toroidal globule. We estimate that the weak dsDNA self-attraction induced by NC can lead to the size of the dsDNA globule similar to the size of the capsid. We predict very low value of mature capsid stability parameter for which it can be uncoated by pro-viral DNA condensed by NC. We describe the phase diagram that relates the volume of double stranded pro-viral DNA synthesized within the capsid, the strength of its NC-induced self-attraction, and the stability of the capsid at the point of uncoating. We discuss the current in vivo evidence for the relationship between the RTion and mature capsid uncoating in HIV.

Keywords: HIV, reverse transcription, mature capsid

112. Understanding the complications of working with G-quadruplex systems in the single molecule FRET assay

William A. Roy, Jr. (Department of Physics, Kent State University), Parastoo Maleki (Department of Physics, Kent State University), Jagat B. Budhathoki (Department of Physics, Kent State University), Hamza Balci (Department of Physics, Kent State University)

Abstract:
We present our studies on the methodology aspect of applying single molecule Förster Resonance Energy Transfer (smFRET) to study G-quadruplex (GQ) systems. These non-canonical nucleic acid structures have proved to be challenging systems to work with at the single molecule level due to their unusual sensitivity to environmental variables and the preparation protocol. Unless thoroughly understood, these sensitivities may lead to misinterpretation of the data and in inconsistent outcomes in measurements. The annealing and storage conditions, the location of fluorophores on the DNA construct with respect to GQ, and the ionic conditions of the experiment are some of the factors that are of critical importance for the outcome of measurements. Some of these factors are well understood and methods to resolve them have been developed in bulk assays, while others are unique for single molecule assays. We will summarize the knowledge and best practices learned from bulk or single molecule measurements that will help address these issues and result in more reliable smFRET data on GQ-involving systems.

Keywords: g-quadruplex, single molecule, FRET

113. Time Flies: Evolutionary Changes in Drosophila SNF RNA Binding Proteins

L. Peyton Russelburg (Biology Department, University of Southern Indiana), Kim Delaney (Biology Department, University of Southern Indiana)

Abstract:
The complex machinery of the spliceosome consists of both RNA and proteins to facilitate a key step in mRNA processing: RNA splicing. The U1A/U2Bʹʹ/SNF protein family participates in both the U1 and U2 snRNPs of the spliceosome. This protein family is well studied because of its unique, high affinity RNA binding interactions. Most metazoans have a single protein, SNF, which binds RNA stemloops in both the U1 and U2 snRNPs; however a gene duplication event in a predecessor of jawed vertebrates gave rise to paralogous family members which rapidly specialized. This resulted in U1A, which segregates to the U1 snRNP, and U2Bʹʹ, which segregates to the U2 snRNP.
Reconstruction of ancestral SNF proteins has given insight into the mechanisms of modern U1A affinity and specificity. Previous work has informed the evolutionary development of the vertebrate proteins; we now turn our interest to the evolutionary pathway of Drosophila SNF. Two early SNF proteins have been selected for initial investigation. One is the last common ancestor of the genus Drosophila (LCA-D) and the other the last common ancestor of fruit flies (LCA-F). After phylogenetic analysis of U1A/U2Bʹʹ/SNF protostome lineages, we used ancestral reconstruction to resurrect the sequences of LCA-D and LCA-F. We aim to characterize the behavior and binding mechanisms of these two ancestral SNF proteins. Understanding of the evolutionary development of SNF’s affinity and specificity for its two unique RNA targets will lead to further insight into the divergence and specialization of U1A and U2Bʹʹ.

Keywords: RRM, U1ASNF, Ancestral Reconstruction

114. Title not available online - please see the printed booklet.

Brianne Sanford (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio, 43205), Chelsea Brown, Hemant Bid, Thomas Bebee, Daniel Comiskey Jr. (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio, 43205), Frank Rigo (Isis Pharmaceuticals, Carlsbad, California, 92010), Peter Houghton (Greehey Childrens Cancer Research Institute, University of Texas Health Science Center, San Antonio, Texas, 78229), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Department of Pediatrics, The Ohio State University, Columbus, Ohio, 43210)

Abstract not available online - please check the printed booklet.

115. Epigenetic regulation of pathogenesis in parasitic protozoa

Samantha Sanford (Graduate School of Public Health, Infectious Disease and Microbiology, University of Pittsburgh), Yossi Weizmann (Department of Chemistry, University of Chicago), Andrea Berman (Department of Biological Sciences, University of Pittsburgh), Kausik Chakrabarti (Department of Chemistry, CNAST, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

116. Translational Control of the Caulobacter Cell Cycle

Jared Schrader (Biological Sciences, Wayne State University), Gene-Wei Li (Cellular and Molecular Pharmacology, UCSF), Bo Zhou (Develepmental Biology, Stanford University), Keren Lasker (Developmental Biology, Stanford University), Jonathan Weissman (Cellular and Molecular Pharmacology, UCSF), Lucy Shapiro (Developmental Biology, Stanford University)

Abstract:
Cellular differentiation is an essential process by which cells carrying identical genes develop into specialized cell types with distinct functions. An important goal in understanding cellular differentiation is to determine how the genetic information encoded in the genome is expressed properly in time and space to ensure the correct cell fate. The bacterium Caulobacter crescentus has proven to be an excellent model organism for studying cellular differentiation processes that occur as a function of the cell cycle. In Caulobacter each cell division is asymmetric, yielding a daughter with a different cell fate. This process requires rapid and specific changes in gene expression during the cell cycle that are controlled at many levels, including transcriptional regulation, transient DNA methylation, differential proteolysis, and protein phospho-signaling. However, relatively little is known about the cell cycle control of mRNA translation.

To understand the role of translational control in the Caulobacter cell cycle we employed ribosome profiling to monitor genome-wide changes in translation throughout the cell cycle. In agreement with data in E. coli and B. subtilis, ribosomes were observed to pause at internal Shine-Dalgarno (SD) sites within the coding sequence. Despite an identical anti-SD sequence of the ribosomal RNA across virtually all bacteria, only half of bacterial genes contain SDsites preceding start codons, including some genomes such as Caulobacter with very poor usage of this sequence. Therefore, the role of the SD is pausing may be the conserved function of this sequence and not translation initiation. While most cell cycle-regulated mRNAs are predominantly controlled at the transcription level, approximately 47% of cell cycle-regulated mRNAs are additionally regulated at the level of translation, including 56 mRNAs whose cell cycle-regulated expression is exclusively regulated at the level of translation. Genes undergoing cell cycle-regulated translational control include many critical genes involved in polar morphogenesis and cell cycle progression. Overall, our data shows that translational control provides an additional layer of regulation of the Caulobacter cell cycle and that this control is largely used to optimally tune the timing of gene expression.

Keywords: Translational Control, Ribosomes, bacteria

117. Effects of ionizing radiation on the RNA of fungi

Lauren Schultz (Department of Chemistry, University of Cincinnati)

Abstract:
Exposure to ionizing radiation in radiotrophic fungi has been shown to cause changes in the regulation of various genes, including some known to be involved in RNA modification. Here we are investigating how ionizing radiation may impact RNA modification levels using the radiotrophic fungi Cladosporium shaperospermum. Initial experiments involve identifying total RNA modification profiles of C. sphaerospermum by LC-MS/MS. C. sphaerospermum samples, both irradiated at varying dosages with ⁶⁰Co and non-irradiated, are analyzed using the same procedure. Our preliminary findings reveal a change in RNA modification profiles for samples exposed to ionizing radiation as compared to unexposed controls. These differences require further studies to determine any role they may play in the organism’s radiotrophism.

References:
[1] Robertson KL, Mostaghim A, Cuomo CA, Soto CM, Lebedev N, et al. Adaption of the Black Yeast Wangiella dermatitidis to Ionizing Radiation: Molecular and Cellular Mechanisms. PloS ONE, 2012. 7(11): e48674.

Keywords: Radiation, Fungi, Mass spectrometry

118. Developmental reprogramming of the transcriptome is an essential component of liver regeneration

Joseph Seimetz (UIUC Department of Biochemistry), Amruta Bhate (UIUC Department of Biochemistry), Edrees H Rashan (UIUC Department of Biochemistry), Waqar Arif (UIUC Department of Biochemistry), Sayeepriyadarshini Anakk (UIUC Department of Molecular and Integrative Physiology), Auinash Kalsotra (UIUC Department of Biochemistry)

Abstract:
Tissue regeneration is a vital mechanism in mature organisms to recover from injury. The mammalian liver—a major metabolic organ in the body—has the unique capacity to undergo robust regeneration in response to damage. Interestingly, this response can be proliferative, hypertrophic, or a combination of both. However, the molecular mechanisms that drive this terminally differentiated tissue into a proliferative and hypertrophic state remain elusive. To determine the underlying mechanisms, we performed a high resolution RNA-seq on toxin-induced, regenerating mouse livers. We identified dramatic changes at both the transcriptional and post-transcriptional levels that cluster to specific cellular processes. Surprisingly, we determined a reciprocal set of mRNA abundance and splice isoform transitions that are normally active during postnatal liver development. We further verified several hundred of these isoform transitions by PCR within individual genes to verify their directionality, temporal dynamics, and cell-type specificity. A computational framework to correlate the regeneration-associated transitions and various cis elements identified a number of RNA-binding proteins that are significantly associated with these splicing alternations. Within this data we identified a subset of developmentally regulated RNA-binding proteins and splicing factors that are markedly induced in the regenerating livers. This suggests that after sustaining damage, these factors are instrumental in activating an embryonic-associated splicing program to support the regenerative process. In summary, our results demonstrate that after sustaining damage, an early postnatal, splicing regulatory network is redeployed in the adult mammalian liver to facilitate regeneration.

Keywords: alternative splicing, development, RNA-binding proteins

119. Title not available online - please see the printed booklet.

Hyosuk Seo (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract not available online - please check the printed booklet.

120. Development of a fluorescence assay for monitoring ligand binding to regulatory RNA thermometers

Jacob Sieg (Department of Chemistry & Biochemistry Ohio University, Athens Ohio), Chunxi Zeng (Department of Chemistry & Biochemistry, Molecular & Cellular Biology Program Ohio University, Athens Ohio), Erin R. Murphy (Department of Chemistry & Biochemistry, Molecular & Cellular Biology Program, Department of Biomedical Sciences Ohio University, Athens Ohio), Jennifer V. Hines (Department of Chemistry & Biochemistry, Molecular & Cellular Biology Program Ohio University, Athens Ohio)

Abstract:
The ompA RNA thermometer regulates translation of virulence essential ompA proteins in Gram-negative bacteria via temperature dependent seclusion of the Shine-Dalgarno sequence. In this study, a molecular beacon style approach was used to develop a moderate throughput assay that screens for ligands that bind the ompA RNA thermometer. Eight aminoglycoside antibiotics known to bind other RNAs were screened using this assay to test its sensitivity. Then aminoglycoside binding was characterized by follow-up EC50 determinations and thermodenaturation experiments. All aminoglycosides in this study were shown to bind the ompA thermometer. Binding was cooperative indicating likely binding at multiple sites. Furthermore, binding was primarily related to aminoglycoside charge, indicating that binding is driven by electrostatics. This information can be used to inform future drug discovery studies of RNA thermometers.

Keywords: thermometer, fluorescence, aminoglycoside

121. High Throughput Sequencing of Partially Edited RNAs Provides Insight into Trypanosome RNA Editing Progression and the Role of the TbRGG2 Subcomplex

Rachel Simpson (Department of Microbiology and Immunology, University at Buffalo School of Medicine), Andrew Bruno (Center for Computational Research, University at Buffalo School of Medicine), Jonathan Bard (Next-gen Sequencing Core, University at Buffalo School of Medicine), Yijun Sun (Department of Microbiology and Immunology, University at Buffalo School of Medicine), Michael Buck (Department of Biochemistry, University at Buffalo School of Medicine), Laurie Read (Department of Microbiology and Immunology, University at Buffalo School of Medicine)

Abstract not available online - please check the printed booklet.

122. Uncovering the role of PSIP1/LEDGF RNA binding protein in breast cancer progression

Deepak K. Singh, Omid Gholamalamdari, Abid Khan (Cell and Developmental Biology, UIUC), Zhang Yang, Shuomeng Guang, Jian Ma (Department of Bioengineering, UIUC), Xiao L. Li, Ashish Lal (Center for Cancer Research National Cancer Institute, Bethesda, MD 20892), Sarah Holton, Rohit Bhargava (Beckman Institute for Advanced Science and Technology, UIUC), Supriya G. Prasanth, Kannanganattu V. Prasanth (Cell and Developmental Biology, UIUC)

Abstract not available online - please check the printed booklet.

123. RNA-Protein interactions in the minor spliceosome

Jagjit Singh M. S. (Center of Gene Regulation in Health and Disease. Cleveland State University, Cleveland, OH-44115), Kavleen Sikand Ph.D., Girish C. Shukla Ph.D.

Abstract not available online - please check the printed booklet.

124. Regulation of androgen receptor function and tumor suppressive role of a microRNA in prostate cancer

Savita Singh (Center for Gene Regulation in Health and Disease, CSU Ohio), Jey S Ebron (Center for Gene Regulation in Health and Disease, CSU Ohio)

Abstract not available online - please check the printed booklet.

125. A semi-rational approach to probe the importance of RNase U2 binding site amino acids

Beulah Solivio (University of Cincinnati Department of Chemistry), Balasubrahmanyam Addepalli (University of Cincinnati Department of Chemistry), Patrick A. Limbach (University of Cincinnati Department of Chemistry)

Abstract not available online - please check the printed booklet.

126. Structure and constraints imposed on the network of miRNA mediated regulation of RNA-binding proteins in the human genome

Rajneesh Srivastava (Biohealth Informatics, IUPUI), Manjunath (Biohealth Informatics, IUPUI), Sarath Chandra Janga (Biohealth Informatics, IUPUI)

Abstract:
MicroRNAs (miRs) and RNA-binding proteins (RBPs) mediate post transcriptional regulation with uncharacterized communication among themselves on a global scale, thus amplifying a new level of complexity of gene expression and regulation. In this study, we aimed to investigate the miR control over RBPs with respect to non-RBPs, at transcript level and its impact at protein level.
We predicted miR targeted transcripts globally using TargetScan and miRanda and calculated the proportion of target transcripts (separately for RBPs and non-RBPs) controlled by each miRs. Such global miR control were analysed for their impact on (a) targeted transcripts’ expression [Human body map RNA sequence data, quantified by SAILFISH] pattern across 16 tissue type (b) RBPs’ transcript half life [HEK293] and (c) targets’ protein abundance (Human Protein Atlas) pattern across 9 tissue type, using equal binning approach with respect to degree of miR regulation.
We observed that miR regulation over proportion of targeted RBP transcripts and targeted Non RBPs transcripts exihibit varying distribution (p-value < 2.2e-16) and also affirms scale free distribution of miRs over available RBP transcripts with more than 50% of unique RBP transcripts targeted by 0.3% extensively regulating miRs including miR-4739, miR-4728-5p, miR-608, miR-149-3p and ~52% of RBP transcripts targeted by 28% weakly regulating miRs. miRs follows a consistent miRs controlling pattern over RBP (further supported by half lives) as well as non-RBP at transcript level in all tissue types, further justify its involvement in degrading/destabilization. However miRs have no significant influence over RBP protein level when compared to Non-RBPs.
Our study suggests that miRs maintains an equilibrium in between RBP transcript availability and corresponding RBP protein levels through complex uncharacterized buffering mechanism or by checking the frequent translation event of RBP transcript thus allowing “protein synthesis as per need” in cells.

References:
Castello, A.; Horos, R.; Strein, C.; Fischer, B. et al. System-wide identification of rna-binding proteins by interactome capture. Nature protocols 2013, 8, 491-500.

Janga, S.C.; Vallabhaneni, S. Micrornas as post-transcriptional machines and their interplay with cellular networks. Advances in experimental medicine and biology 2011, 722, 59-74.

Hashemikhabir S, Neelamraju Y, Janga SC. Database of RNA binding protein expression and disease dynamics (READ DB). Database (Oxford). 2015, doi: 10.1093/database/bav072.

Neelamraju, Y.; Hashemikhabir, S.; Janga, S.C. The human rbpome: From genes and proteins to human disease. Journal of proteomics 2015.

Kechavarzi, B.; Janga, S.C. Dissecting the expression landscape of rna-binding proteins in human cancers. Genome biology 2014, 15, R14.

Keywords: microRNA, RNA binding proteins, post-transcriptional network

127. Investigation of mRNA signals in E. coli aroL that lead to ribosome binding and start site selection

Sarah R. Steimer (Miami University Department of Microbiology), Racheal Devine, Gary R. Janssen

Abstract not available online - please check the printed booklet.

128. alternative polyadenylation in Sarcocystis neurona

Ashley Stevens (Plant and Soil Sciences, University of Kentucky), Dr. Arthur Hunt (Plant and Soil Sciences, University of Kentucky), Dr. Daniel Howe (Veterinary Sciences, University of Kentucky)

Abstract:
This project studied the occurrence of alternative polyadenylation during the growth of Sarcocystis neurona. S. neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis. Among the stages of growth of the parasite are the merozoite and schizont stages. The merozoite is the extracellular parasite that has been lysed from the host cell once the development has completed. The schizont is the intracellular development stage, where it grows and acquires nutrients, eventually producing 64 haploid merozoites. While some gene expression analysis has been conducted, there is no knowledge on polyadenylation in S. neurona. Moroever, there have been no genome-wide studies of poly(A) site choice for any member of the Apicomplexans phylum. Thus, it is not clear if alternative polyadenylation contributes to changes in gene expression in these organisms, and any findings will add to current knowledge. This study addresses the hypothesis that, indeed, alternative polyadenylation does contribute to regulated gene expression in S. neurona.
Poly(A) tags (1) were made to conduct a genome-wide study of poly(A) site choice The tag libraries were sequenced on a MiSeq instrument, returning 11.9 million reads. The data was analyzed using programs including CLC Genomics Workbench and Bedtools, as well as others designed specifically for APA analysis to define sites and assess alternative polyadenylation. The results suggest that S. neurona possesses a distinctive polyadenylation signal that is reminiscent of that seen in higher plants. The results also show multiple instances of APA. The results from this study confirm our hypothesis that there will be changes in poly(A) site choice during the developmental stages of S. neurona.

References:
1. Ma L, Pati PK, Liu M, Li QQ, Hunt AG. High throughput characterizations of poly(A) site choice in plants. Methods. 2014;67(1):74-83.

Keywords: Polyadenylation, Sarcocystis neurona, Apicomplexan

129. Regulation of Eukaryotic Translational Initiation by Programmed -1 Ribosomal Frameshifting.

Arvin Massoudi (Department of Cell Biology and Molecular Genetics), Isabella Swafford (Department of Cell Biology and Molecular Genetics), Jessica Stimely (Department of Cell Biology and Molecular Genetics), Theodore Nikolaitchik (Department of Cell Biology and Molecular Genetics), Adam Kellerman (Department of Cell Biology and Molecular Genetics), Savannah Speir, Vivek Advani, Jonathan Dinman (Department of Cell Biology and Molecular Genetics)

Abstract not available online - please check the printed booklet.

130. Regulation of Eukaryotic Translational Initiation by Programmed -1 Ribosomal Frameshifting.

Abstract not available online - please check the printed booklet.

131. Title not available online - please see the printed booklet.

David E. Symer (Human Cancer Genetics Program, Dept. of MVIMG, Ohio State Univ. Comp. Cancer Center), Dandan He, Jingfeng Li, Katherine A. Yates, Keiko Akagi (Dept. of MVIMG, Ohio State Univ. Comp. Cancer Center), Christopher J Hlynialuk, Zhengqiu Zhou, (Dept. of MVIMG, Ohio State Univ. Comp. Cancer Center), Huiling He, Albert de la Chapelle, Brad N. Bolon (Dept. of MVIMG and Dept. of Veterinary Medicine, Ohio State Univ. Comp. Cancer Center), Richard A. Padgett (Dept. of Cellular and Molecular Medicine, Cleveland Clinic)

Abstract not available online - please check the printed booklet.

132. Suppression screen for the identification of protein interactors of the trp RNA-binding attenuation protein (TRAP)

Courtney E. Szyjka (Department of Biological Sciences, University at Buffalo), Natalie McAdams (Department of Microbiology and Immunology, University at Buffalo), Paul Gollnick (Department of Biological Sciences, University at Buffalo)

Abstract:
Transcriptional regulation of the tryptophan (trp) biosynthetic operon in Bacillus subtilis is controlled by the trp RNA-binding attenuator protein (TRAP). Regulation of this operon was initially described as involving two competing RNA structures present in the leader region upstream of the first gene in the operon. Formation of these structures, designated as the anti-terminator and terminator, is mutually exclusive due to shared bases. In the presence of excess tryptophan, TRAP binds to 11 (G/U)AG repeats in the trp leader region RNA and prevents anti-terminator formation, allowing formation of the terminator and thus the trp genes are not transcribed. In limiting tryptophan conditions, TRAP does not bind and the trp genes are transcribed and translated to produce the tryptophan biosynthesis enzymes. Recent work has shown that, in vivo, the terminator structure isn’t required for transcription attenuation in the presence of tryptophan, indicating that TRAP may have a more direct role in transcription attenuation. Through a genetic selection, a TRAP mutant (E60K) was isolated that is capable of binding RNA and tryptophan at wild-type (WT) levels, but is deficient in transcription attenuation. We have designed a suppression screen to find protein interactors of TRAP that are necessary for transcription attenuation at the trp operon. By controlling the expression of a toxic protein (MazF) with the trp leader region, we seek to identify Bacillus subtilis proteins that will enhance transcription attenuation of the E60K TRAP mutant in vivo.

Keywords: Transcription, Gene regulation, Termination

133. Title not available online - please see the printed booklet.

Supuni Thalalla Gamage (Chemistry department, Wayne State University), Gayani Dedduwa-Mudalige (Chemistry department, Wayne State University), Christine S. Chow (Chemistry department, Wayne State University)

Abstract not available online - please check the printed booklet.

134. Examining 3’UTR interactions that promote rapid clearance of oscillating transcripts

Kiel T. Tietz (Molecular Genetics, The Ohio State University, Columbus), Thomas L. Gallagher, Nicolas L. Derr, Deepika Sharma, Zachary T. Morrow (Molecular Genetics, The Ohio State University, Columbus), Jasmine M. McCammon, Michael L. Goldrich (Molecular and Cell Biology, University of California, Berkeley, CA), Sharon L. Amacher (Molecular Genetics, The Ohio State University, Columbus)

Abstract:
Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of embryonic segments, or somites. This molecular oscillator generates cyclic gene expression in the tissue that will generate somites, called the presomitic mesoderm (PSM), and has the same periodicity as somite formation. Core molecular components of the segmentation clock include the hairy/Enhancer of split-related (her/Hes) family of transcriptional repressors, but additional transcripts such as ras homolog gene family member V (rhov) also cycle. Maintenance of clock oscillations requires that transcriptional activation and repression, RNA turnover, translation, and protein degradation are all very rapid as the clock cycles and a new somite pair forms every 30 minutes in the zebrafish embryo. We isolated a segmentation clock mutant, tortuga^b644, in which cyclic transcript oscillations are abnormal; mutant embryos express elevated levels of cyclic mRNAs such as her1 and rhov in the PSM. We have demonstrated that loss of Proline-rich nuclear receptor coactivator protein Pnrc2 is responsible for cyclic transcript accumulation in tortuga^b644 mutants. In human cell culture systems, Pnrc2 is implicated in mRNA decay by promoting decapping through interactions with Dcp1a and Upf1. Data from our lab support a similar decay function in zebrafish since antisense knockdown of pnrc2 and upf1 at low doses display a level of her1 transcript accumulation similar to tortuga, a phenotype not seen with either low dose pnrc2 or upf1 knockdown alone. We are also interested in cis-acting elements required for the rapid decay of oscillating transcripts. Our preliminary data support the hypothesis that the 3’UTR of cyclic mRNAs is important for Pnrc2-mediated decay since the her1 3’UTR confers instability to otherwise stable transcripts in vivo, in a Pnrc2-dependent manner. We are currently examining specific regions of the her1 3’UTR that are sufficient to induce rapid transcript decay to further define mechanisms responsible for the rapid turnover of oscillating transcripts.

Keywords: Pnrc2, her1, cyclic transcript

135. High Resolution RNA Solution Structure of the Stem Loop II Domain of the Enterovirus 71 Internal Ribosome Entry Site

Michele Tolbert (Department of Chemistry, Case Western Reserve University), Christopher E. Morgan (Department of Chemistry, Case Western Reserve University), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
Enterovirus 71 (EV71) is the major etiological agent involved in hand, foot and mouth disease. Infection is often acute, and may result in severe neurological and/or cardiovascular complications that can lead to severe morbidity and death. There currently are no vaccines or antivirals to help prevent infection or spread of disease, thus underscoring an urgent need to better understand the molecular mechanisms of this serious threat to public health. EV71 is a positive sense, single stranded RNA virus belonging to the Picorniavirdae family. Similar to other picornaviruses, EV71 utilizes a type I Internal Ribosome Entry Site (IRES) to promote viral translation. Type I IRES elements utilize structured domains to recruit host factors, which facilitate ribosomal loading via poorly understood mechanisms. Previous work from our group has shown that interactions between host hnRNP A1 and Stem Loop II (SLII) of the EV71 IRES is necessary for viral translation and replication, thus highlighting SLII as a critical region in the EV71 genome. To better understand the molecular determinants involved in SLII:hnRNP A1 interactions, a high resolution solution structure of the free SLII domain was solved using an integrated NMR and SAXS based approach. Herein, I will discuss the structural and thermodynamic implications involved in hnRNP A1 recognition of SLII.

Keywords: IRES, NMR, EV71

136. Identfication of cap methyltransferase (RNMT) as a component of the cytoplasmic capping complex

Jackson B. Trotman (Biological Chemistry and Pharmacology, The Ohio State University), Chandrama Mukherjee (Biological Chemistry and Pharmacology, The Ohio State University), Daniel R. Schoenberg (Biological Chemistry and Pharmacology, The Ohio State University)

Abstract not available online - please check the printed booklet.

137. Ribose Dynamics of the Leadzyme

Neil A. White (Department of Biochemistry and Molecular Biology, Michigan State University), Minako Sumita (Department of Biochemistry and Molecular Biology, Michigan State University), Charles G. Hoogstraten (Department of Biochemistry and Molecular Biology, Michigan State University)

Abstract:
The lead-dependent ribozyme or leadzyme is among the smallest of the known catalytic RNAs. Lead-dependent cleavage occurs between C6 and G7 within a six-nucleotide asymmetric internal loop. In structures of the leadzyme determined by NMR spectroscopy and X-ray crystallography the scissile phosphate is not positioned for in-line nucleophilic attack. This necessitates a conformational rearrangement of the active site to be consistent with the proposed transition state. We have previously probed the ribose structure and dynamics of the guanosine residues in the asymmetric loop using LNA (locked nucleic acid). We now report a study of the relationship between conformational dynamics and catalytic function at the cleavage-site residue C6. We have substituted C6 with a 2’-hydroxyl-bicyclo [3.1.0] hexane nucleotide, which is restricted to the C3’-endo ribose conformation but, unlike LNA, maintains the nucleophilic 2’-hydroxyl, and found a drastic attenuation of self-cleavage activity. In parallel, we have undertaken NMR spin relaxation experiments to examine ribose repuckering events at the C6 position using our previously-reported site-specific C-13 isotopic labeling scheme. These studies in totality will yield improved understanding of the relationship between RNA backbone dynamics and cleavage mechanism in the leadzyme.

Keywords: leadzyme, conformational dynamics, RNA catalysis

138. Title not available online - please see the printed booklet.

Rebecca N Williams-Wagner (Department of Microbiology and Center for RNA Biology, The Ohio State University ), Frank J Grundy (Department of Microbiology and Center for RNA Biology, The Ohio State University), Tina M Henkin (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

139. Identification of inhibitors of protein-only RNase P from a high throughput screen

Nancy Wu (Program in Chemical Biology, University of Michigan), Carol A. Fierke (Departments of Chemistry and Biochemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

140. 3’ to 5’ exonuclease activity of Pop2p

Xuan Ye (Department of Biochemistry, Case Western Reserve University), Eckhard Jankowsky (Department of Biochemistry, Case Western Reserve University)

Abstract not available online - please check the printed booklet.

141. Epistasis analysis of 16S rRNA ram mutations helps define the conformational dynamics of the ribosome that influence decoding

Lanqing Ying (Department of Microbiology and Center for RNA Biology, The Ohio State University), Kurt Fredrick (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

142. Promoting A Novel Approach to Cellular gene Expression Alteration (PANACEA)

References:
Biragyn, A., Bodogai, M., Olkhanud, P.B., Denny-Brown, S.R., Puri, N., Ayukawa, K., Kanegasaki, S., Hogaboam, C.M., Wejksza, K., and Lee-Chang, C.(2013). Inhibition of lung metastasis by chemokine CCL17-mediated in vivo silencing of genes in CCR4+ Tregs. J. Immunother. 36, 258-267.

Keywords: Gene silencing, RNA interference, Receptor Mediated Endocytosis (RME)

143. Identifying the Role of Muscleblind-Like Protein 1 in Myotonic Dystrophy Type 1 Pathogenesis and Postnatal Liver Development

Kevin Yum (Department of Biochemistry, University of Illinois at Urbana-Champaign), Amruta Bhate (Department of Biochemistry, University of Illinois at Urbana-Champaign), Anthony Chau (Department of Biochemistry, University of Illinois at Urbana-Champaign), Sandip Chorghade (Department of Biochemistry, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, University of Illinois at Urbana-Champaign)

Abstract:
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a microsatellite repeat expansion in the 3’-untranslated region of the dystrophia myotonica-protein kinase gene. In DM1, the repeat-bearing transcripts promote misregulated alternative splicing (AS) in cardiac and skeletal muscle by sequestering the muscleblind-like (MBNL) proteins in the nucleoplasm. Here we demonstrate that MBNL1 knockout (KO) liver closely resembles the aberrant AS patterns previously found in DM1 striated muscle. MBNL1-dependent splicing defects were determined using RT-PCR analysis and unique adult-to-embryonic splicing changes were discovered in MBNL1 KO liver. Additionally, histological analysis of MBNL1 KO liver showed disrupted hepatic architecture and ballooning degeneration of hepatocytes. These results indicate that MBNL1 is required for maintenance of adult liver splicing patterns and loss of MBNL1 function leads to defects that are reminiscent of DM1 pathology. To further validate our hypothesis that loss of MBNL1 function is responsible for splicing misregulation in DM1 liver, recombinant FLAG-tagged MBNL1 adenoviruses were constructed to overexpress MBNL1 in a liver-specific DM1 mouse model. Our preliminary data suggest that loss of MBNL1 function impairs postnatal AS transition of DM1 liver, prompting further evaluation of various tissues other than skeletal and cardiac muscle.

Keywords: myotonic dystrophy, muscleblind-like protein, alternative splicing

144. Tobramycin Variants with Enhanced Ribosome-Targeting Activity

Marina Y. Fosso1 (Department of Pharmaceutical Sciences College of Pharmacy, University of Kentucky), Hongkun Zhu1 (Department of Microbiology Center for RNA Biology, Ohio State University), Keith D. Green (Department of Pharmaceutical Sciences College of Pharmacy, University of Kentucky), Sylvie Garneau-Tsodikova (Department of Pharmaceutical Sciences College of Pharmacy, University of Kentucky), Kurt Fredrick (Department of Microbiology Center for RNA Biology, Ohio State University)

Abstract:
With the increased evolution of aminoglycoside (AG)-resistant bacterial strains, the need to develop AGs with 1) enhanced antimicrobial activity, 2) the ability to evade resistance mechanisms, and 3) the capability of targeting the ribosome with higher efficiency is more and more pressing. The chemical derivatization of the naturally occurring tobramycin (TOB) by attachment of 37 different thioether groups at the 6''-position led to the identification of generally poorer substrates of TOB-targeting AG-modifying enzymes (AMEs). Thirteen of these displayed better antibacterial activity than the parent TOB while retaining ribosome-targeting specificity. Analysis of these compounds in vitro shed light on the mechanism by which they act and revealed three with clearly enhanced ribosome-targeting activity.

References:
Fosso MY, Zhu H, Green KD, Garneau-Tsodikova S, Fredrick K. ChemBioChem 2015, 16, 1565 – 1570.

Keywords: Aminoglycosides, Ribosome, Translocation

145. Analysis of novel 5’-UTR polyadenylation sites in Arabidopsis thaliana

Yingdong Zhu (Biology Department, Miami University), Cheng Guo (Biology Department, Miami University), Chun Liang (Biology Department, Miami University), Qingshun Q. Li (Biology Department, Miami University), Jack C. Vaughn (Biology Department, Miami University)

Abstract:
Precursor mRNA processing including polyadenylation serves as an important basis for posttranscriptional regulation. The poly(A) site is generally located several tens or hundreds of nucleotides downstream of the mRNA termination codon. However, many eukaryotic genes have more than one poly(A) site, which results in a set of mRNA isoforms with different length. The usage of one poly(A) site over another is mainly owing to the relative strength as well as the availability of the site, which is known as alternative polyadenylation (APA). For many years, polyadenylation was only observed in the 3’-UTR and lack of poly(A) signal sequence conservation made its exact position difficult to predict. The first evidence that the 5’-UTR of some mRNAs could encode peptides and potentially control translation of the downstream ORF came from studies on upstream open reading frames (uORFs). The importance of uORF was recently highlighted in an analysis of the uORF among angiosperms, which showed that ~1% of them are evolutionarily conserved among related species. Arabidopsis thaliana is an important model plant for analyzing the transcriptomics, and remarkably, ~30% of its protein coding mRNAs possess a 5’-UTR with one or more uORF. Recently, we profiled the transcriptomes of roots, flowers and leaves in Arabidopsis (wild-type Columbia), and our bioinformatics analysis predicted that a portion of poly(A) sites are located in the 5’-UTR in addition to the conventional 3’-UTR. Verification of a predicted poly(A) site located in the 5’-UTR should meet three conditions: first, it cannot come from the 3’-UTR of another adjacent gene or neighboring regions of the DNA sequence; second, it should contain a poly(A) tail; third, it can maintain a certain expression level. Considering that polyadenylation in the 5’-UTR can produce novel short peptides, and perhaps reduce or eliminate the original transcripts, we hypothesize that it may be related to an unknown regulation mechanism of the corresponding genes. To confirm our predictions from large scale and computational analysis, we are investigating several predicted uORFs for presence of an AUG start codon, a conventional stop codon, and also using 3’-RACE for evidence of a poly(A) tail.

Keywords: alternative polyadenylation, upstream open reading frame, Arabidopsis thaliana

146. Disparate Functions of a Riboswitch at High and Low Cyclic di-GMP in Vibrio cholerae

Benjamin R. Pursley (Department of Microbiology and Molecular Genetics, Michigan State University), Christopher M. Waters (Department of Microbiology and Molecular Genetics, Michigan State University)

Abstract:
Cyclic di-guanosine-monophosphate (c-di-GMP) is a bacterial second messenger signaling molecule that plays a central role in many diverse phenotypes including biofilm formation, motility, cell cycle regulation, and virulence gene expression. In Vibrio cholerae, c-di-GMP mediates the lifestyle transition from a free-living aquatic organism to a human pathogen, although the genetic regulatory changes that underlie this transition are not yet well understood. The Vc2 riboswitch, located upstream of tfoY, a Vibrionaceae-specific gene of unknown function, has remained one of the functionally uncharacterized genetic inputs for c-di-GMP regulation in V. cholerae. We studied the function of Vc2 in vivo at high and low c-di-GMP states and have determined that the Vc2 riboswitch has two separate and distinct functions. At low c-di-GMP, the Vc2 riboswitch is critical for the induction of V. cholerae motility by inducing tfoY expression. Mutations in Vc2 that disrupt c-di-GMP binding also abrogate tfoY induction and reduce V. cholerae motility. Conversely, although tfoY expression is also induced at high c-di-GMP levels, the Vc2 riboswitch does not substantively contribute to downstream gene regulation. Rather, tfoY is transcriptionally upregulated at multiple riboswitch-independent promoters by a c-di-GMP-binding transcriptional activator. The Vc2 riboswitch instead serves to regulate the abundance of an upstream small RNA. This riboswitch aptamer domain is located at the immediate 3'-end of this sRNA and the stability of the transcript is directly correlated to the intracellular concentration of c-di-GMP. We are currently working to understand the mechanism of Vc2-mediated tfoY induction at low c-di-GMP, and we are identifying the regulatory targets of this Vc2 riboswitch sRNA to elucidate its role in global c-di-GMP regulation of V. cholerae.

Keywords: Riboswitch, cyclic-di-GMP, Vibrio cholerae