Poster abstracts

1. A phased-addition strategy enhances the yield of RNAs obtained by in vitro transcription with modified transcriptional initiators

Katie L. Adib (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Ila Marathe (Department of Chemistry and Biochemistry, Center for RNA Biology, Department of Microbiology, The Ohio State University), Stella M. Lai (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Tien-Hao Chen (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Edward Behrman (Department of Chemistry and Biochemistry, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology, Department of Microbiology, The Ohio State University)

Abstract not available online - please check the printed booklet.

2. Translational recoding in human disease: identifying the molecular pathologies underlying X-linked Dyskeratosis Congenita and Spinocerebellar Ataxia 26.

Vivek M Advani (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park MD 20742), Jonathan D. Dinman (Department of Cell Biology and Molecular Genetics, University of Maryland, College Park MD 20742)

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. The overwhelming majority of predicted “genomic” -1 PRF events would direct translating ribosomes to premature termination codons, and we have demonstrated that these can function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway. In published work we have explored the biological significance of the connection between -1 PRF and NMD on telomere maintenance in yeast. More recently we extended this line of inquiry is 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. 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 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.

Keywords: -1 PRF , NMD

3. Thinking outside the S-box: an investigation of an unusual S-box family member

George M. Allen (Department of Microbiolgy, Center for RNA Biology, and The Ohio State University), Vineeta A. Pradhan (Department of Microbiolgy, Center for RNA Biology, and The Ohio State University), Frank J. Grundy (Department of Microbiolgy, Center for RNA Biology, and The Ohio State University), Tina M. Henkin (Department of Microbiolgy, Center for RNA Biology, and The Ohio State University)

Abstract not available online - please check the printed booklet.

4. Dissecting the Mechanisms of Pumilio Mediated Repression

Rene Arvola (Genetics Training Grant Program, Department of Biological Chemistry, University of Michigan), Chase Weidmann (Genetics Training Grant Program, Department of Biological Chemistry, University of Michigan), Aaron Goldstrohm (Department of Biological Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

5. Evolutionary dynamics of RNA binding proteins expressions in mammalian species

Abhijit S. Badve (Department of Bio-Health Informatics, School of Informatics, IUPUI,Indinapolis), Vishal K. Sarsani (Department of Bio-Health Informatics, School of Informatics, IUPUI,Indinapolis), Yaseswini Neelamraju (Department of Bio-Health Informatics, School of Informatics, IUPUI,Indinapolis), Sarath C. Janga (Department of Bio-Health Informatics, School of Informatics, IUPUI,Indinapolis)

Abstract:
RNA binding proteins (RBPs) play important roles in controlling the post-transcriptional fate of RNA molecules, yet their evolutionary dynamics remains largely unknown. As expression profiles of genes encoding for RBPs can yield insights about their evolutionary trajectories on the post-transcriptional regulatory networks across species, we performed a comparative analyses of RBP expression profiles across 8 tissues (brain, cerebellum, heart, lung, liver, lung, skeletal muscle, testis) in 7 mammals (human, chimpanzee, gorilla, orangutan, macaque, rat, mouse). Noticeably, orthologous gene expression profiles suggest a significantly higher expression level for RBPs than their non-RBP gene counterparts - which include other protein-coding and non-coding genes, across all the mammalian tissues studied here. This trend is significant irrespective of the tissue and species being compared, though RBP gene expression distribution patterns were found to be generally diverse in nature. Our analysis also shows that RBPs are expressed at a significantly lower level in human and mouse tissues compared to their expression levels in equivalent tissues in other mammals chimpanzee, orangutan, rat, etc. which are all likely exposed to diverse natural habitats and ecological settings compared to more stable ecological environment humans and mice might have been exposed, thus reducing the need for complex and extensive post-transcriptional control. Further analysis of the similarity of orthologous RBP expression profiles between all pairs of tissue-mammal combinations clearly showed the grouping of RBP expression profiles across tissues in a given mammal, in contrast to the clustering of expression profiles for non-RBPs, which frequently grouped equivalent tissues across diverse mammalian species to group together, suggesting a significant evolution of RBP expression after speciation events. Calculation of species specificity indices for RBPs across various tissues, to identify those that exhibited restricted expression to few mammals, revealed that about 30% of the RBPs are species-specific in at least one tissue studied here.

References:
1. Kechavarzi, B. and S.C. Janga, Dissecting the expression landscape of RNA-binding proteins in human cancers. Genome Biol, 2014. 15(1): p. R14.
2. Necsulea, A., et al., The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature, 2014. 505(7485): p. 635-40.
3. Merkin, J., et al., Evolutionary dynamics of gene and isoform regulation in Mammalian tissues. Science, 2012. 338(6114): p. 1593-9.
4. Brawand, D., et al., The evolution of gene expression levels in mammalian organs. Nature, 2011. 478(7369): p. 343-8.

Keywords: RBP expression, evolution, mammalian species

6. SHARP protein causes the remodeling of SRA1 lncRNA structure

Sumirtha Balaratnam (Kent State University), Joel Caporoso (The University of Akron), Stephanie M. Bilinovich (The University of Akron), Dr. Thomas Leeper (The University of Akron), Dr. Soumitra Basu (Kent State University)

Abstract:
Prostate cancer is one of the two most prevalent forms of cancer in men. The proliferation of prostate cells in the cancer can be directly correlated to transcription of several oncogenes mediated by the nuclear androgen receptor (AR). Androgen receptor stimulation of prostate cancer has been correlated with co-activation from the steroid responsive activator RNA (SRA1). SRA1 is a bifunctional transcript that has recently been found to act not only as a code for protein, but also as a long non-coding RNA (lncRNA). SRA1 has been found to involve in the SRC-1 complex that regulates the transcription of AR. However, the SRA1 assembles in a Ribonucleoprotein (RNP) complex that reduces SRA1 incorporation into SRC-1 complex. SMRT/HDAC1 Associated protein (SHARP) is one protein in the RNP complex that has been previously shown to interact with SRA1 RNA through its four RNA Recognition Motifs (RRMs) and repress the RNA activity. However the specific binding sites of SHARP-RRMs with the SRA1 and their functional roles are still unknown. We investigated the binding of two of the RRM subdomains of SHARP to SRA1 by RNase T1 footprinting. The data show that there was a specific cleavage site at the G52 position of the SRA1-STR7 arm by SHARP-RRM2 which led to the remodeling of the entire SRA1 RNA structure. However, SHARP -RRM1 didn’t show any effect on the SRA1 structure. In addition, the preliminary NMR and Chemical shift perturbation studies indicate that there is a direct interaction between the SHARP and SRA1 RNA. Further studies are required to completely analyze the detailed binding and functional roles of SHARP with SRA1. These studies have potential assist in the development of anti-prostate cancer therapy.

References:
1. Siegel R, Ward E, Brawley O, Jemal A, (2011) The impact of eliminating socioeconomic and racial disparities on premature cancer deaths,Ccancer Journal of Clinicians, 61; 212-36.
2. Whitw JW, (1895), The Results of Double Castration in Hypertrophy of the prostate, Ann Surg, 22; 1-80.
3. Li R, Wheeler T, Dai H, Frolov A, Thompson T, Ayala G,(2004), High level of androgen receptor is associated with aggressive clinicopathologic features and decreased biochemical recurrence-free survival in prostate: cancer patients treated with radical prostatectomy, Am J Surg Pathol, 28; 928-34

Keywords: SRA1 lncRNA, SHARP protein, prostate cancer

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

Erkan Bayir (Department of Biological Sciences, University of Pittsburgh), Paula J. Grabowski (Department of Biological Sciences, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

8. A knockout mouse model to define the roles of the Epithelial splicing regulatory proteins in development and epithelial cell function

Thomas Bebee (Department of Genetics, Perelman School of Medicine, University of Pennsylvania), Katherine Sheridan (Department of Genetics, Perelman School of Medicine, University of Pennsylvania), Benjamin Cieply (Department of Genetics, Perelman School of Medicine, University of Pennsylvania), Juw Won Park (Department of Microbiology, Immunology, & Molecular Genetics, University of California Los Angeles), Yi Xing (Department of Microbiology, Immunology, & Molecular Genetics, University of California Los Angeles), Russ Carstens (Department of Genetics, Perelman School of Medicine, University of Pennsylvania)

Abstract not available online - please check the printed booklet.

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

Divyaa Bhagdikar (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.

10. Interaction between a G-quadruplex domain and 40s ribosomal subunit is critical for IRES A mediated translation initiation of hVEGF

Debmalya Bhattacharyya (Department of Chemistry and Biochemistry, Kent State University), Paige Diamond (Department of Biochemistry, Cell and Molecular Biology, Drake University), Soumitra Basu (Department of Chemistry and Biochemistry, Kent State University)

Abstract:
Human VEGF (hVEGF) is a mitogenic growth factor involved in both physiological and pathological angiogenesis. The 5′-untranslated region (UTR) of hVEGF mRNA is known to harbor two internal ribosomal entry site (IRESs), which under stress, are independently capable of initiating cap-independent translation. One of the IRESs, IRES A is a 293 nt sequence located towards the end of the 5′-UTR is responsible for cap-independent translation initiation at the canonical AUG start codon. The IRES A harbors a G-quadruplex (GQ) forming domain which is known to modulate cap-independent translation. G-quadruplex structures in the 5′-UTR of mRNA are known to regulate translation in general by inhibiting cap-dependent and augmenting cap-independent IRES mediated translation. The exact mechanism by which the GQ structures modulate the cap-independent translation is still unresolved. Our studies begin to shed light on the possible mechanistic role of GQ structures in cap-independent translation. We show here the direct interaction of a GQ structure in IRES A and it binds to the 40S ribosomal subunit with high affinity. The 40S ribosomal subunit binds to the 5′-proximal end of the IRES A which includes the GQ forming sequence. We also show that 40S subunit preferentially binds to the IRES in presence of the GQ structure with higher affinity. The GQ domain plays the most vital role in steering the IRES activity in a bicistronic plasmid in human cancer cells. The GQ in IRES A is an essential domain for the recruitment of 40S ribosomal subunit, which most likely is a key step for cap-independent initiation of translation.

References:
1. Bugaut, A.; Balasubramanian, S. Nucleic Acids Res 2012, 40, 4727.

2. Morris, M. J.; Negishi, Y.; Pazsint, C.; Schonhoft, J. D.; Basu, S. Journal of the American Chemical Society 2010, 132, 17831.

Keywords: G-quadruplex, IRES, hVEGF

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

Jamie Bingaman (Department of Chemistry and Center for RNA Molecular Biology, Pennsylvania State University), Sixue Zhang (Department of Chemistry, University of Illinois), Sharon Hammes-Schiffer (Department of Chemistry, University of Illinois), Phil Bevilacqua (Department of Chemistry and Center for RNA Molecular Biology, Pennsylvania State University)

Abstract not available online - please check the printed booklet.

12. Identification of mRNA Target of Human PUF Proteins

Jennifer Bohn (Cellular Biotechnology Training Program and Department of Biological Chemistry University of Michigan), Trista Schagat (Promega Corporation and Department of Biological Chemistry, University of Michigan), Jamie Van Etten, Aaron Goldstrohm (Department of Biological Chemistry, University of Michigan), Richard McEachin (Department of Computational Medicine & Bioinformatics, University of Michigan), Ashwini Bhasi (Department of Computational Medicine & Bioinformatics, University of Michigan), Aaron Goldstrohm (Department of Biological Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

13. The C-terminal domain of the vaccinia capping enzyme D12 subunit is important for its role in transcription termination

Rachel Boldt (Department of Biological Sciences, SUNY Buffalo), Dr. Paul Gollnick (Department 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 requires a specific viral form of RNA polymerase. Termination of early gene transcription occurs in a site-specific, signal-dependent manner involving a U5NU signal in the nascent RNA together with several protein factors including, NPHI and the viral mRNA capping enzyme. The viral Capping Enzyme recognizes and binds a U5NU sequence which signals for termination to occur. In turn NPHI releases the terminated transcripts using a forward translocation method.
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.
Here we have made mutations in the C-terminal region of D12 and tested mutant D1 D12 dimers for early gene transcription termination activity. We have found that truncating D12 by as few as 3 amino acids from its C-terminal end inhibits transcription termination. These mutant proteins are fully active in capping activities suggesting that the C-terminal region of D12 is specifically involved in transcription termination function of the capping enzyme.
We have also detected an interaction between the C-terminal end of D12 and an as of yet unidentified viral factor using a Mts-Atf-Biotin label transfer reagent. Further work is being carried out to identify this factor and to determine whether this interaction is important for capping enzymes role in transcription termination.

Keywords: vaccinia , transcription termination

14. BLM Helicase does not require ATP to Unfold G-Quadruplex

Jagat B. Budhathoki (Physics, Kent State University, USA), Sujay Ray (Physics, Kent State University, USA), Vaclav Urban (Institute of Molecular Genetics ASCR, Prague, Czech Republic), Pavel Janscak (Institute of Molecular Cancer ,University of Zurich, Zurich, Switzerland), Jaya G. Yodh (Physics,Center for the Physics of Living Cells,University of Illinois at Urbana-Champaign), Hamza Balci (Physics, Kent State University,USA)

Abstract not available online - please check the printed booklet.

15. Discovery-based Ribonuclease (RNase) mapping with mass exclusion list locating tRNA modifications

Xiaoyu Cao (Chemistry Department, Univerisity of Cincinnati), Patrick A. Limbach (Chemistry Department, Univerisity of Cincinnati)

Abstract not available online - please check the printed booklet.

16. Structure determination and RNA-binding properties of SHARP

Joel A. Caporoso (Department of Chemistry and Biochemistry, The University of Akron), Stephanie M. Bilinovich (Department of Chemistry and Biochemistry, The University of Akron), Thomas C. Leeper (Department of Chemistry and Biochemistry, The University of Akron)

Abstract:
SMRT/HDAC1 Associated Repressor Protein (SHARP) is a multi-domain protein that is involved in multiple transcription regulatory pathways. Of particular interest are the four RNA Recognition Motifs (RRMs) that are found near the N-terminus of the protein. Previous data has suggested that SHARP is involved in interacting and regulating the action of the Steroid Receptor Activator RNA (SRA) through the use of these RRMs. Steroid Receptor Activator RNA (SRA) is a RNA transcript that has recently been found to act not only as code for protein biosynthesis, but also as a long, non-coding RNA (lncRNA). lncRNAs 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, with its four RNA recognition motifs (RRMs), has been shown to interact and form complexes with SRA RNA that are capable of repressing the RNA's activity. 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. This structure and other biophysical data on the SRA STR7 arm indicate that SHARP RRM 2 may be involved in RNA structure remodeling.

Keywords: NMR, SHARP, SRA

17. SHAPE analysis of the rpoH thermosensor from Escherichia coli

Casey Cempre (Denison University Department of Chemistry and Biochemistry), Kelsey Ulanowicz (Denison University Department of Chemistry and Biochemistry), Rachel Mitton-Fry (Denison University Department of Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

18. Exploring role of alternative polyadenylation during tall fescue-endophyte symbiosis under drought stress using genome wide approaches.

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 is a key step during mRNA processing. Although, poly (A) tails are predominantly added at the 3ʹ UTRs of mRNAs, however they can also be added at other parts of transcriptional units, like protein coding regions, introns and 5ʹ UTRs. Such instances of alternative polyadenylation can impact gene expression, mRNA stability and translation ability, etc. There are evidences of alternative polyadenylation being implicated in different developmental processes and human diseases (1-3). Additionally, there are indication of links between polyadenylation and oxidative stress signaling in plants (4, 5).
The current project delves into the role of alternative polyadenylation in the tall fescue-endophyte symbiotic system. Tall fescue is a cool season perennial forage grass, which forms a symbiotic association with endophytic fungi, Epichloe sp. Fescue plants provide shelter, nutrition and mode of dissemination to the fungi, while the fungi produce toxic alkaloids to deter herbivores and also confer resistance to various stresses. Endophyte infected (E+) and free (E-) tall fescue clone pairs were subjected to drought stress for two days, tissue samples in triplicates were collected from leaves and pseudostems, followed by RNA extraction, poly (A) tag library preparation and sequencing in Illumina platform (6). Global gene expression analysis using the poly (A) tags showed over representation of certain GO categories, namely, secondary metabolism, programmed cell death, reproductive processes, amine metabolism and post translational protein modification, etc. between E+ and E- plants under stress, but not between E+ and E- control plants. Comparison of global poly (A) profiles of E+ and E- plants under drought and control conditions indicates that in presence of endophyte there is some difference in poly (A) profile between drought stressed and control plants. Deep sequencing of the samples is underway to obtain more read coverage to substantiate preliminary findings. Additionally, confirmation of candidate genes displaying altered poly (A) profile between E+ and E- plants under drought is being carried out using 3ʹ RACE.

References:
1. Liu et al., Science, 2009. 327 (5961): 94-97.
2. Simpson et al., Cell, 2003. 113 (6): 777–787.
3. Mayr et al., Cell. 2009. 138 (4): 673-684.
4. Zhang et al., PLoS One. 2008. 3 (6): e2410.
5. Delaney et al., Plant Physiology. 2006. 140 (4): 1507-1521.
6. Ma et al., Methods.2014. 67 (1): 74-83.

Keywords: Alternative polyadenylation, Tall fescue, Drought

19. Using SHAPE chemistry to determine the structure of the ykkCD RNA riboswitch

Beau Champ (Chemistry, Ball State University), Timea Gerczei (Chemistry, Ball State University)

Abstract:
Riboswitches are RNA aptamers that are involved in cellular regulatory functions. They fold in a specific 3D shape and often undergo structural transition when bound to the target molecule. This allosteric structural transition typically allows or prevents the downstream translation of a protein. Here, we study the ykkCD riboswitch, which recognizes the antibiotic tetracycline and regulates expression of a multidrug resistant efflux pump in Bacillus subtillis. Our goal is to use SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry to identify structural change that takes place upon tetracycline binding by the ykkCD riboswitch. We will also examine whether there is any difference in structure when the ykkCD RNA is made in vivo versus in vitro.

Keywords: SHAPE, tetracycline, riboswitch

20. Uncovering the RNA binding protein machinery associated with age and gender during liver development

Praneet Chaturvedi (Department of Biohealth Informatics, School of Informatics & Computing, Indiana University-Purdue University, Indianapolis), Yaseswini Neelamraju (Department of Biohealth Informatics, School of Informatics & Computing, Indiana University-Purdue University, Indianapolis), Waqar Arif (Departments of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Departments of Biochemistry and Medical Biochemistry, University of Illinois, Urbana-Champaign), Sarath Chandra Janga (Department of Biohealth Informatics, School of Informatics & Computing, Indiana University-Purdue University, Indianapolis)

Abstract:
Alterations in the expression of genes are known to be associated with changing age and gender although the mechanisms mediating such a change remain unclear. In the present study, we perform an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Majority (~60%) of the age-associated RBPs were found to be increasing in their expression levels with age. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations state that RBPs are important developmental regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. Thus, the compendium of RBPs and this study will not only help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes but also provide a foundation for identifying the regulators contributing to the splicing eQTLs increasingly being discovered in association studies.

References:
1) Innocenti, F., Cooper, G.M., Stanaway, I.B., Gamazon, E.R., Smith, J.D., Mirkov, S., Ramirez, J., Liu, W., Lin, Y.S., Moloney, C. et al. (2011) Identification, replication, and functional fine-mapping of expression quantitative trait loci in primary human liver tissue. PLoS genetics, 7, e1002078.
2) Schroder, A., Klein, K., Winter, S., Schwab, M., Bonin, M., Zell, A. and Zanger, U.M. (2013) Genomics of ADME gene expression: mapping expression quantitative trait loci relevant for absorption, distribution, metabolism and excretion of drugs in human liver. The pharmacogenomics journal, 13, 12-20.
3) Castello, A., Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L.M., Krijgsveld, J. and Hentze, M.W. (2013) System-wide identification of RNA-binding proteins by interactome capture. Nature protocols, 8, 491-500.

Keywords: Regulatory Proteins, Liver Development, Genome-wide Associations

21. A-to-I editing is cancer subtype specific

Cai Chen (Biophysics Graduate Program, The Ohio State University), Ralf Bundschuh (Departments of Physics and Chemistry&Biochemistry and Division of Hematology, The Ohio State University)

Abstract not available online - please check the printed booklet.

22. Characterization of theProRS/YbaK/tRNAPro ternary complex by analytical ultracentrifugation and native mass spectrometry

Lin Chen (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Akiko Tanimoto (Department of Chemistry and Biochemistry, The Ohio State University), Marina BaKhtina (Department of Chemistry and Biochemistry and Center for RNA Biology,The Ohio State University), Vicki Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

23. Mass spectrometry-aided identification of substrate contact sites in a proteinaceous RNase P, a tRNA processing enzyme

Tien-Hao Chen (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Akiko Tanimoto (Department of Chemistry and Biochemistry,The Ohio State University, Columbus, OH 43210), Vicki H. Wysocki (Department of Chemistry and Biochemistry,The Ohio State University, Columbus, OH 43210), Venkat Gopalan (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract not available online - please check the printed booklet.

24. The DEAD-box helicase Dhh1p monitors translational elongation rate to simulate mRNA decay

Ying-Hsin Chen (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland), Sophie Martin (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland), Vladimir Presnyak (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland), Najwa Al Husaini (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland), Jeff Coller (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland)

Abstract:
mRNA decay is a cellular process that controls the steady-state levels of mRNAs. Every mRNA has a different half-life. In eukaryotic cells, the major pathway of mRNA decay initiates with digestion of the 3’ poly (A) tail (deadenylation), followed by removal of the 5’ cap (decapping) and exonucleolytic digestion in a 5’ to 3’ direction. Previous work in our lab indicated that mRNA decay occurs co-translationally and the insertion of rare codons in a PGK1 reporter, which slowed the elongation rate, resulted in destabilization of the mRNA (Hu et al, 2009). To understand how translational elongation affects the mRNA decay rate, we globally measured mRNA decay rates and investigated the correlation between mRNA half-life and codon usage.

We found that mRNA stability correlates well with codon usage and that transcripts with a higher ratio of optimal codons are more stable than transcripts with a higher ratio of non-optimal codons. As a way to verify that codon usage affects mRNA stability, we designed two synthetic synonymous reporters that encoded the same amino acid sequence but that were comprised of either all optimal or all non-optimal codons. We found that the reporter that consisted of all optimal codons had a longer half-life than the reporter that consisted of all non-optimal codons. Optimal codons are translated faster and more accurately by the ribosome, while non-optimal codons slow down translational elongation rate. Our data strongly suggest that translational elongation rate impacts mRNA stability. We also found that deletion of decay factors including DCP2, PAT1, LSM1, and CCR4 increased the half-lives of both the optimal and non-optimal reporters, while deletion of DHH1 only stabilized the non-optimal reporter. 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 from our lab have shown that endogenous Dhh1p can associate with slowly moving ribosomes (Sweet et al, 2012). We therefore propose that Dhh1p senses slowly translocating ribosomes on transcripts and drives these transcripts into decay.

References:
1. Hu, W., Sweet, T. J., Chamnongpol, S., Baker, K. E. & Coller, J. Co-translational mRNA decay in Saccharomyces cerevisiae. Nature 461, 225-229, doi:10.1038/nature08265 (2009).
2. Sweet, T., Kovalak, C. & Coller, J. The DEAD-box protein Dhh1 promotes decapping by slowing ribosome movement. PLoS Biol 10, e1001342, doi:10.1371/journal.pbio.1001342 (2012).

Keywords: DHH1, translational elongation, mRNA decay

25. Elements in the 3’UTR of Selenoprotein S regulate Selenocysteine insertion

Eric Cockman (Department of Cellular and Molecular Medicine, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH), Jodi Bubenik (Department of Cellular and Molecular Medicine, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH), Donna Driscoll (Department of Cellular and Molecular Medicine, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH)

Abstract:
Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved at the translational level by the recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNA transcripts possess a 3’UTR sequence, known as the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. The SECIS elements of selenoproteins differ in sequence and structure and vary in efficiency of Sec insertion (1). The dogma in the field is that the SECIS element is the major RNA sequence that determines Sec incorporation. However, our lab has shown that the full 3’UTR of Selenoprotein S (SelS) has a much lower recoding efficiency than the SelS SECIS alone, suggesting that there are other elements in the 3’UTR that regulate the insertion of Sec (2).
Using a luciferase reporter assay in rabbit reticulocyte lysate (RRL), we have shown that a deletion of the last 101 nucleotides of the SelS 3’UTR restored Sec incorporation efficiencies to the level of the SelS SECIS alone. Alignments of this region across species revealed three conserved sequences. Deletion of the distal most sequence rescued recoding efficiency of the SelS 3’UTR in the RRL system. Using UV-crosslinking, we observed the binding of two proteins, approximately 40-45 kDa in size, that are present in multiple cell types. Based on competition experiments, the two proteins require the distal most sequence for high affinity binding. Thus, the protein binding correlates with the recoding results. Currently, RNAase-assisted affinity purification is being performed to isolate and identify the binding proteins.

References:
1) Latreche L, Jean-Jean O, Driscoll DM, Chavatte L (2009) Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine. Nucleic Acids Res 37: 5868–5880gkp635
2) Bubenik JL,Miniard AC, Driscoll DM , Alternative Transcripts and 3’ UTR Elements Govern the Incorporation of Selenocysteine into SelenoproteinS PLoS One. 2013 Apr 16;8(4):e62102. doi: 10.1371/journal.pone.0062102. Print 2013

Keywords: Selenoproteins, RNA-Protein Interaction, Selenocysteine

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, Columbus, OH 43210 ), 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, Columbus, OH 43210 ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH 43210 )

Abstract:
Human tRNALys3 serves as the primer for reverse transcription in human immunodeficiency virus type-1 (HIV-1). All three tRNALys isoacceptors are selectively packaged into virions through specific interactions between human lysyl-tRNA synthetase (hLysRS) and the viral Gag protein. tRNALys3 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. We have proposed that this release is facilitated, in part, 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 similar to that of tRNALys3. 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 TLE/tRNA mimicry is conserved across various subtypes of HIV-1. Here, we study the PBS region of the HIV-1 MAL isolate, which contains a 23-nt insertion that is known to result in an alternative secondary structure of the 5´-UTR (3). Fluorescence anisotropy assays used to investigate the binding of hLysRS to MAL-derived RNA constructs revealed moderate to high-affinity binding of hLysRS to MAL 123-218 (Kd = 499 ± 119 nM) and MAL 123-349 (Kd < 50 nM). To locate the sequence elements responsible for high-affinity binding, truncated RNAs and mutants of MAL 123-218 were tested. The experiments revealed decreased binding of hLysRS to the truncated RNAs, while more subtle mutations of MAL 123-218 did not significantly affect binding. The tertiary structure of MAL 123-218 was also analyzed using SAXS, revealing that this RNA shows remarkable structural similarity to the NL4-3 PBS-TLE region. The results obtained to date suggest that specific recognition of the TLE is based on overall 3D topology.

References:
1. Jones, C. P., Saadatmand, J., Kleiman, L., and Musier-Forsyth, K. (2013) Molecular mimicry of human tRNALys 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 U S A 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. Title not available online - please see the printed booklet.

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. Mapping of Structural Changes to the ykkCD Antibiotic Sensor RNA Caused by Tetracycline Binding

Caroline Conley (Department of Chemistry Ball State University Muncie, Indiana), Timea Gerczei (Department of Chemistry Ball State University Muncie, Indiana)

Abstract:
Mapping of Structural Changes to the ykkCD Antibiotic Sensor RNA Caused by Tetracycline Binding

Abstract:

A riboswitch is a noncoding RNA that controls gene expression response to changes in the cellular environment. Most riboswitches regulate genes responsible for transporting, synthesizing or recycling small metabolites in bacteria.1 These riboswitches, primarily found at the 5’ region of messenger RNA, recognize the metabolic product of the gene being regulated. When a metabolite concentration threshold is reached, expression is turned off. In contrast, the ykkCD putative riboswitch increases production of an efflux pump that ejects toxins from the cell by binding to the antibiotic tetracycline. As tetracycline binds to ykkCD riboswitch, structural changes take place that trigger the production of the efflux pump.2 We will present terbium fragmentation and other nucleic acid footprinting methods to map the tetracycline site and to visualize the allosteric changes that take place due to tetracycline binding.

References:
References:

1. Barrick, J.E.: Edited by Alexander Serganov. Department of Structural Biology, New York, NY. In Riboswitches Methods and Protocols: Predicting Riboswitch Regulation on a Genomic Scale pp1-14

2. Howell, L. A. Mapping the structural change caused by tetracycline binding to the ykkCD antibiotic sensor RNA. Ball State University, Muncie, IN, 2013

Keywords: riboswitch, efflux pump, ykkCD

29. Muscleblind-like 1 regulates alternative splicing and transforming growth factor beta signaling during heart valve development

Ryan J. Coram (Department of Cellular & Molecular Medicine, Cleveland Clinic), Samantha S. Stillwagon (Department of Cellular & Molecular Medicine, Cleveland Clinic), Anuradha Guggilam (Department of Cellular & Molecular Medicine, Cleveland Clinic), Stanley L. Hazen (Department of Cellular & Molecular Medicine, Cleveland Clinic), Maurice S. Swanson (Department of Molecular Genetics & Microbiology, University of Florida, Gainesville), Andrea N. Ladd (Department of Cellular & Molecular Medicine, Cleveland Clinic)

Abstract:
Approximately 5% of infants are born with heart defects, among the most common of which are valve defects. The heart valves develop through the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). A subpopulation of endocardial cells undergoes an epithelial-mesenchymal transition (EMT) to give rise to invasive mesenchyme, which later differentiates into fibroblasts that establish and maintain the stratified matrix of the mature heart valves. Endocardial cushion EMT is induced by transforming growth factor beta (TGFb). TGFb signaling has also been linked with post-EMT cushion remodeling and valve maintenance, but little is known about how TGFb signaling is regulated. Muscleblind-like 1 (MBNL1) is a highly conserved RNA binding protein. We previously demonstrated that in the chick, MBNL1 is highly expressed in AVC and OFT endocardium, and knockdown of MBNL1 promotes TGFb signaling and invasive mesenchyme production. As in the chick, we found that MBNL1 is highly expressed in AVC and OFT endocardium and mesenchyme in the mouse embryo. Loss of MBNL1 enhances EMT ex vivo in mouse endocardial cushion explants, and induces precocious EMT in vivo in Mbnl1-null mice. MBNL1 expression continues in post-EMT cushions and adult heart valves, and adult Mbnl1-null mice exhibit valve dysmorphia and dysfunction, enhanced TGFb signaling, and altered extracellular matrix composition. Regulation of TGFb activity has been linked to alternative splicing of a MBNL1 target, fibronectin (Fn), a matrix protein with critical roles in cell adhesion and migration. We have found that MBNL1-dependent Fn alternative splicing changes correlate with changes in TGFb and mesenchymal markers in a murine cell culture model of TGFb-dependent EMT. Together, these data indicate that MBNL1 plays a conserved role as a regulator of TGFb signaling, EMT, and valvulogenesis during heart development.

Keywords: MBNL1, EMT, heart development

30. The nucleosome acidic patch is required for histone H2B lysine 123 monoubiquitylation and downstream transcription-coupled histone modifications

Christine E. Cucinotta (Biological Sciences, University of Pittsburgh), Alexandria N. Young (Biological Sciences, University of Pittsburgh), Karen M. Arndt (Biological Sciences, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

31. Mapping elements that confer specific acceptor stem recognition of Ala-tRNAPro by a bacterial trans-editing domain

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

Abstract:
Aminoacyl-tRNA synthetases (ARS) catalyze the attachment of specific amino acids to cognate tRNAs. Mistakes in this process lead to errors in protein synthesis that can be deleterious to cells. Prolyl-tRNA synthetase (ProRS) mischarges tRNAPro with Ala, which is hydrolyzed by a cis-editing domain (INS) in most bacteria. However, some bacteria lacking the INS domain encode a homologous free-standing trans-editing domain known as ProXp-ala that functions to clear Ala-tRNAPro. Previous studies identified acceptor stem elements of tRNAPro (G72 and A73) that are critical for ProXp-ala activity. In fact, a microhelix that mimics the acceptor stem of tRNAPro is a good substrate for Ala deacylation. To define the elements in ProXp-ala that confer acceptor stem specificity, NMR mapping studies were carried out with an uncharged microhelixPro and with a non-hydrolyzable, amide-linked Ala-microhelixPro mimic. We observe similar but significantly stronger chemical shift perturbations in the presence of the charged microhelix, which also displays 5-fold higher affinity for binding to ProXp-ala, as measured by analytical ultracentrifugation (AUC). Four main regions of interaction were identified: a %beta-strand (aa 43-49) within the active site, a region (aa 68-70; 80-84) that we propose is the G72/A73 interacting domain, a dynamic helix at the top of the active site pocket (aa 27-30), and amino acids proximal to this helix (aa 128-134). Mutagenesis and AUC studies are consistent with the critical nature of conserved residues R80 and F83 for substrate binding. Mutation of conserved active site residues K45 and N46 also caused severe (>10-fold) losses in activity, whereas mutation of conserved residues V29, H130, and N134 resulted in more modest decreases (3- to 6-fold). The combined results of these studies allow us to propose a mechanism for Ala-microhelixPro recognition that involves induced fit binding, specific protein-RNA interactions, and key contributions from the Ala moiety.

Keywords: tRNA, NMR, AUC

32. Genome-wide analysis of stability & translatability in CAP-associated RNAs in Arabidopsis

Laura de Lorenzo (Department of Plant and Soil Sciences. University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences. University of Kentucky)

Abstract:
Genome-wide analysis of stability & translatability in CAP-associated RNAs in Arabidopsis
Laura de Lorenzo & Arthur G. Hunt
Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546-0312, USA.

Key words: Alternative polyadenylation, PAT-seq, stability and/or translation.


Posttranscriptional regulation of mRNA plays an important role in gene expression. One mode of control that is accomplished through RNA processing is the alternative polyadenylation (APA) (1). Polyadenylation can occur upstream, downstream and within open reading frames. Among them, coding region-APA (CAP) is the least understood. Such events should lead to transcripts without an in-frame stop codon, and affected mRNAs should be subject to rapid degradation via the nonstop decay mechanism. Thus, CAP-associated RNAs should be relatively unstable, and their translation products likewise short-lived. In eukaryotes, non-stop RNAs are subject to a battery of negative regulatory processes. In yeast, non-stop RNAs are rapidly degraded by the exosome, and the protein products of translation of non-stop mRNAs are degraded by the proteasome (2). In mammals, translation of non-stop RNAs is inhibited at a step after initiation but before the end of the non-stop RNA is reached (3).
In Arabidopsis, the paradoxical outcomes of polyadenylation within coding regions, raises questions as to if coding-region polyadenylation is linked with non-stop decay. To investigate this, we are conducting a study to analyze the stability and translatability of CAPs-associated RNAs using a genome-scale approach. In order to test the stability of RNA that end at CAPs in relation to their full-length counterparts, the genome-wide poly(A) profiles in samples treated with cordycepin (or with buffer, for controls) have been determined and the relative poly(A) site usage assessed on a gene-by-gene basis. In the same way, the translatability of CAP-associated RNAs have been tested by assessing the 3’ end profiles of mRNAs associated with polysomes (or total RNA, as control). To confirm the global poly(A) result, RT/PCR of candidates genes were carried out. Our results show that CAP-associated RNAs are stable and are found associated with polysomes (as in other eukaryotes). Future analyses will be carried out to evaluate if CAP-associated RNAs are less stable that RNAs encoded by the same gene that end at “normal” poly(A) site; and to analyze the activity and degradation of the translated protein. This will support the hypothesis that CAP utilization is a mechanism of negative regulation.

References:
1. Hunt, A.G (2014) The Arabidopsis polyadenylation factor subunit CPSF30 as conceptual link between mRNA polyadenylation and cellular signaling. Current Opinion in Plant Biology. 21: 128-132.
2. Wilson, M.A., S. Meaux, and A. van Hoof (2008). Diverse aberrancies target yeast mRNAs to cytoplasmic mRNA surveillance pathways. Biochim Biophys Acta. 1779: 550-557.
3. Akimitsu, N., J. Tanaka, and J. Pelletier (2007). Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells. EMBO Journal. 26: 2327-2338.

Keywords: Alternative polyadenylation, PAT-seq, stability and translation

33. Regulation of the 3' UTR in BDNF mRNA from the DNA level

Brett DeMarco (Duquesne University, Chemistry and Biochemistry), Rita Mihailescu (Duquesne University, Chemistry and Biochemistry )

Abstract not available online - please check the printed booklet.

34. Single Molecule Fluorescence Studies on Conformation of Backbone Branched RNA

Sourav K. Dey (Carnegie Mellon University), Linda A. Peteanu (Carnegie Mellon University), Subha R. Das (Carnegie Mellon University)

Abstract:
Lariat RNAs generated during splicing include a branch-point adenosine residue which has a unique 2'-5'-phosphodiester linkage. This 2'-5' linkage in the lariat RNA backbone can be specifically cleaved by the lariat debranching enzyme (Dbr1p). Following debranching, some lariat introns participate in important biological processes like snoRNA or microRNA biogenesis. We have developed a solid phase synthesis method to generate 2'-5'-phosphodiester linked RNAs which are mimics of the lariat RNAs. Our method of backbone branched RNA (bbRNA) synthesis allows us to incorporate modifications into the 5'- and 3'- ends and within the branch. A bbRNA, dual-labeled with fluorescent dyes (Alexa488 and Alexa594) will allow us to examine fluctuations in its conformation using single molecule FRET (smFRET). We have also synthesized an unnatural 'click-branched' RNA where the native 2'-5'-phosphodiester bond is replaced by a triazole linkage that is non-cleavable by Dbr1p. This non-native bbRNA with the same dual labels can be used to identify conformational changes due to binding of Dbr1p. Together, these bbRNAs with native and non-cleavable linkages will be used to study the conformations and dynamics pertaining to the Dbr1p debranching reaction at the single molecule level.

Keywords: Backbone branched RNA, Dbr1p, Single molecule FRET

35. AFP anti-sense transcripts in mouse liver and their potential role in gene regulation

Maria M. Dixon (Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine), Guofang Qiu, Lilia Turcios, Brett T. Spear and Martha L. Peterson (Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine)

Abstract not available online - please check the printed booklet.

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

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

Abstract not available online - please check the printed booklet.

37. Examination of human lysyl-tRNA synthetase/tRNAlys primer recruitment and packaging into HIV

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

Abstract:
The primer for reverse transcription in HIV-1, human tRNA Lys3, is selectively packaged into virions along with tRNA Lys1,2. Human lysyl-tRNA synthetase (LysRS) , the only cellular factor known to interact specifically with all three tRNA Lys isoacceptors, is also packaged into HIV-1. Selective packaging of tRNA Lys depends on the ability of the tRNA to bind to LysRS and the presence of both host cell factors is required for optimal viral infectivity. LysRS is part of a dynamic mammalian multisynthetase complex (MSC) and has been shown to be mobilized from the MSC and to function in a wide variety of non-translational pathways. While some aspects of tRNA primer packaging into HIV-1 particles are now understood, the mechanism by which the LysRS/tRNA complex is diverted from its normal function in translation and recruited into viral particles is unclear. Here, we show that the expression of LysRS is unaltered upon HIV-1 infection, suggesting that the LysRS species packaged is recruited from an existing pool of LysRS. Using immunofluorescence and confocal microscopy, we find that LysRS trafficking is altered upon HIV-1 infection with more LysRS localized to the nucleus. LysRS-Gag and LysRS-genomic RNA co-localization studies are currently underway. Our studies also indicate that phosphorylation of LysRS on Ser/Thr occurs only after HIV-1 infection. We hypothesize that LysRS phosphorylation results in release from the MSC and nuclear entry. Studies to understand the significance of these findings for HIV infectivity are in progress. A recent study suggests that HIV-1 Matrix (MA), a viral protein that functions in assembly by targeting Gag to the plasma membrane, preferentially binds certain cellular tRNAs, including tRNALys, over viral RNA. Studies to determine the specificity of MA binding in vitro are also underway.

Keywords: HIV-1 , LysRS, Assembly

38. Substrate recognition by the tRNAHis-guanylyltransferase: a kinetic investigation with model RNA substrates

Lauren Duff (Chemistry and Biochemistry Dept. The Ohio State Univeristy), Krishna Patel (Chemistry and Biochemistry Dept. The Ohio State Univeristy), Dr. Jane Jackman (Chemistry and Biochemistry Dept. The Ohio State Univeristy)

Abstract not available online - please check the printed booklet.

39. Regulation of Androgen Receptor expression through its 3’ UTR

Jey Sabith Ebron (Center for Gene regulation in Health and Disease, Department of Biological, Geo and Evs Sciences, Cleveland State University), Kavleen Sikand , Girish C. Shukla (Center for Gene regulation in Health and Disease, Department of Biological, Geo and Evs Sciences, Cleveland State University)

Abstract:
Androgen receptor (AR) is a member of the steroid receptor family which plays a significant role in the regulation of both prostate cell proliferation and growth suppression. AR is a target for Prostate Cancer (PCa) treatment. The 3’UTR of AR mRNA is 6.8 kb long and is a potential target of many miRNAs. We have identified a novel regulation of AR signaling pathway and invasion through regulation of AR gene expression using miRNAs. In this study we show that AR is a direct target of hsa-miR-644 and hsa-miR-644. Our results demonstrate that hsa-miR-644 targets AR signaling pathway as well as migration and invasion of PCa cells. Further, we show the synergistic and combinatorial action of hsa-miR-488* and hsa-miR-644 on AR gene expression in prostate cancer cells. In addition, we have explored the inhibitory function of RNA binding protein HuR on the miRNA mediated repression of AR expression in PCa cells. Our preliminary results indicate that HuR affects the downregulation of AR presumably by competing for miRNA binding target sites within the 3’ UTR. These results demonstrate a complex posttranscriptional regulation of AR that is mediated via miRNAs and HuR protein antagonistic action in PCa cells.

Keywords: Androgen receptor, miRNA, Prostate Cancer

40. Discovery of a new processive anti termination mechanism

Grace Ellis ( University of Maryland College Park, University of Texas Southwestern Medical Center), Carole Vargas-Bautista (Texas A&M), Irnov Irnov (Yale University), Jonathan Goodson (University of Maryland College Park), Paul Straight (Texas A&M), Wade Winkler (University of Maryland College Park)

Abstract not available online - please check the printed booklet.

41. An Investigation into RNA Oxidation Using Radical Precursors

Matthew W. Ellis (Medicinal & Biological Chemistry, The University of Toledo), Raziya Shaik (Medicinal & Biological Chemistry, The University of Toledo), Matthew J. Starr (Medicinal & Biological Chemistry, The University of Toledo), Dr. Amanda Bryant-Friedrich (Medicinal & Biological Chemistry, The University of Toledo)

Abstract:
Reactive oxygen species can cause oxidative damage to DNA, RNA, and proteins. It has been shown that oxidative damage to RNA plays a role in neurodegenerative diseases, such as Alzheimer’s. Modified uridines were synthesized and subjected to photolytic activation, generating a C5’- uridinyl radical mimicking hydrogen atom abstraction under conditions of oxidative stress. These studies were done under anaerobic conditions in the presence or absence of glutathione to the diversity of damage products that may form depending upon the cellular environment. Identification of these damage products may assist in the development of new assays based on biomarkers of oxidative damage. Preliminary monomer studies indicate that the base elimination product uracil is the major product when the radical of interest is generated in the absence of glutathione while the expected reduction product uridine is the major constituent formed in the presence of glutathione.

References:
Nunomura, Akihiko et. al. "RNA Oxidation in Neurodegenerative Diseases: Sublethal Damage as a Mechanism for Chronic Degeneration". Yamanashi Med. J. 23 (4), 75-95, 2008.

Keywords: Oxidative Damage, Ribose, Organic Synthesis

42. Thermodynamic analysis of DEAD-box proteins reveal a temperature-dependent change

Haley Englert (Department of Chemistry, Allegheny College), Ivelitza Garcia (Department of Chemistry, Allegheny College), Ericka Bell (Department of Chemistry, Allegheny College)

Abstract not available online - please check the printed booklet.

43. The Riboswitch Calculator: automated physics-based design of synthetic riboswitches from diverse RNA aptamers

Amin Espah Borujeni (Chemical Engineering, Penn State University), Dennis M. Mishler (Molecular Biosciences, University of Texas at Austin), Jingzhi Wang (Chemistry, Emory University), Walker Huso (Chemical Engineering, Penn State University), Justin P. Gallivan (Chemistry, Emory University), Howard M. Salis (Chemical Engineering and Biological Engineering, Penn State University)

Abstract:
Riboswitches are RNA-based biosensors that regulate gene expression by employing an RNA aptamer that senses chemicals, changes its shape, and alters mRNA’s transcription or translation rate. Riboswitches can be applied in various disciplines such as in medical diagnosis, metabolic engineering, environmental remediation, and human health. Although hundreds of RNA aptamers have been generated to bind important ligands, only a few of these aptamers were converted into riboswitches, mainly due to the need for expert design or high-throughput screening, both of which require experience and human intuition. To overcome this challenge, we have developed a biophysical model and an automated design method to convert aptamers into translation-regulating riboswitches. This biophysical model predicts the sequence-structure-function relationship for the riboswitches directly from their aptamer’s specificities, aptamer’s surrounding sequences, and ligand and mRNA concentrations. To validate our intuition-free approach, we designed 52 riboswitches from the theophylline, TMR, and fluoride aptamers and measured their activation in 136 in vivo and in vitro conditions. Our automated design method reliably generated functional riboswitches with high activation ratios, up to 383-fold. We demonstrated the capability of our method in designing riboswitches that use aptamers with pseudoknot structures (by testing fluoride aptamer) and for ligands that must be sensed at very low concentrations (by testing TMR aptamer). We also elucidated the key reasons why some riboswitches fail to regulate gene expression, and thus developed quantitative criteria to predict whether a riboswitch is thermodynamically driven or kinetically trapped. We next showed that increasing mRNA concentration has an opposite effect on the riboswitch function in in vivo versus in vitro conditions. We related this counter-intuitive observation to the mRNA’s excluded volume effect within the crowded cellular environments versus diluted in vitro systems. Finally, we incorporated our design method into a user-friendly web interface, called the Riboswitch Calculator, enabling the scientific community and other non-experts to easily design and build riboswitches for their own applications.

Keywords: Synthetic riboswitch, Biophysics, Automated design

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

Ian M.C. Fleming (Department of Microbiology and Center for RNA Biology, The Ohio State University), Mary Anne T. Rubio (Department of Microbiology and Center for RNA Biology, The Ohio State University), Zdenek Paris (Institute of Parasitology, Academy of Sciences of the Czech Republic), Kirk W. Gaston (Department of Chemistry, Rieveschl Laboratories for Mass Spectrometry, University of Cincinnati), Juan D. Alfonzo (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

45. Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus

Shawn W. Foley (Cellular and Molecular Biology Graduate Group & Department of Biology, University of Pennsylvania, Philadelphia, PA), Sager J. Gosai, Nur Selamoglu, Fevzi Daldal (Department of Biology, University of Pennsylvania, Philadelphia, PA), Dongxue Wang, Roger B. Deal (Department of Biology, Emory University, Atlanta, GA), Ian M. Silverman (Cellular and Molecular Biology Graduate Group & Department of Biology, University of Pennsylvania, Philadelphia, PA), Andrew D.L. Nelson, Mark A. Beilstein (School of Plant Sciences, University of Arizona, Tucson, AZ), Brian D. Gregory (Cellular and Molecular Biology Graduate Group & Department of Biology, University of Pennsylvania, Philadelphia, PA)

Abstract not available online - please check the printed booklet.

46. Enhanced tools for manipulating and analyzing gene function in a sea star larval model to explore regenerative biology

Munira Fouz (Department of Chemistry, Carnegie Mellon University), Lauren Xu (Department of Chemistry, Carnegie Mellon University), Veronica Hinman (Department of Bioloical Sciences, Carnegie Mellon University), Danith Ly (Department of Chemistry, Carnegie Mellon University), Bruce Armitage (Department of Chemistry, Carnegie Mellon University)

Abstract:
Humans have little capacity to regenerate. However, human tissues could potentially be reengineered if the mechanisms controlling this process in animals that do regenerate are properly understood. While vertebrate animals and humans in particular have generally poor regenerative capabilities, members of their closest invertebrate phylum, sea stars, have extraordinary abilities to regenerate. The sea star larva is a newly emerged model system for understanding the regulatory mechanisms of cell specification during early development. Here we develop enhanced fluorescent detection tools using gamma modified Peptide Nucleic Acid (PNA) mini probes, to assay small RNA expression and to perturb gene function. These mini probes will be powerful detection and signal amplification agents for Whole Mount InSitu Hybridization (WMISH) to visualize spatial patterns of microRNA expression. The melting curves of the ternary duplexes formed by the mini probes and a synthetic analog of a representative micro RNA show highly cooperative affinity towards the target and a significant destabilization with a single mismatch RNA sequence; an important feature to discriminate among closely related miRNA family members. These tools are likely to be widely useful for any developmental model system.

References:
Goldman, J. M.; Zhang, L. A.; Manna, A.; Armitage, B. a; Ly, D. H.; Schneider, J. W. High Affinity γPNA Sandwich Hybridization Assay for Rapid Detection of Short Nucleic Acid Targets with Single Mismatch Discrimination. Biomacromolecules 2013, 14, 2253–2261.

Keywords: Regenerative biology, Peptide Nucleic Acids (PNA), Whole Mount InSitu Hybridization (WMISH)

47. Characterization of RNA and Magnesium Ion Partitioning in Polyamine-Nucleotide Coacervates

Erica A. Frankel (Department of Chemistry and Center for RNA Molecular Biology, Pennsylvania State University), Christine D. Keating (Department of Chemistry, Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry and Center for RNA Molecular Biology, Pennsylvania State University)

Abstract not available online - please check the printed booklet.

48. DICER-Like 3 is required for 24nt small RNA biogenesis and paramutation in Zea mays

Janelle M. Gabriel (Department of Molecular Genetics, The Ohio State University), Ankur Narain, Irene T. Liao (Department of Plant and Microbial Biology, University of California, Berkeley), Joy-El R.B. Talbot (Department of Molecular Genetics, The Ohio State University and Department of Plant and Microbial Biology, University of California, Berkeley), Stacey A. Simon (Delaware Biotechnology Institute, University of Delaware), Blake C. Meyers (Delaware Biotechnology Institute, University of Delaware), Jay B. Hollick (Department of Molecular Genetics, The Ohio State University and Department of Plant and Microbial Biology, University of California, Berkeley)

Abstract not available online - please check the printed booklet.

49. The DEAD-box RNA helicases Ded1p and eIF4A function simultaneously on the eukaryotic initiation factor 4F

Zhaofeng Gao (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Heath Bowers (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Andrea Putnam (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Ulfpeter Gunther (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106)

Abstract:
The eukaryotic translation initiation factor 4F (eIF4F) consists of the large scaffolding and RNA binding protein eIF4G, the cap-binding protein eIF4E, and the loosely associated DEAD-box RNA helicase eIF4A. In Saccharomyces cerevisiae, another DEAD-box RNA helicase, Ded1p also interacts with eIF4G. Here, we show that the two DEAD-box helicases are functionally coupled to stimulate remodeling of structured RNA.
Using recombinant purified proteins, we show that Ded1p directly interacts with eIF4A, and that eIF4A stimulates RNA unwinding by Ded1p. Ded1p also directly interacts with eIF4G/E, and eIF4G/E promotes RNA binding by Ded1p, but inhibits RNA unwinding by Ded1p by interfering with oligomerization of the helicase. Addition of eIF4A to the eIF4G/4E-Ded1p complex stimulates the RNA unwinding well above the level of stimulation seen for Ded1p with eIF4A alone. The eIF4G/E/A-Ded1p complex shows a strong preference for remodeling of substrates with 3' unpaired tails, over those with 5' tails, a feature not seen with both helicases alone. Moreover, Ded1p significantly enhances the affinity of eIF4F for RNA with a 5' cap.
Collectively, our results reveal intricate functional coupling between Ded1p and the eIF4F components. The interactions rationalize previously observed genetic interactions between the eIF4F components and Ded1p, and indicate that both DEAD-box helicases, eIF4A and Ded1p function simultaneously in the eIF4F complex.

Keywords: DEAD-box RNA helicase, translation initiation, eIF4F

50. Identifying potential substrates of the reverse polymerase, BtTLP, by developing a variation on RNA-Seq

Spencer Gardner (Biomedical Engineering, The Ohio State University), Jane Jackman (Chemistry and Biochemistry, The Ohio State University), Ralf Bundschuh (Biophysics, The Ohio State University)

Abstract:
In this study, a variation on RNA-seq is being developed both from the bench and from the computer lab to discover potential small RNA substrates for the Thg1-like-protein (TLP) from Bacillus thuringiensis, called BtTLP. Thg1 and TLPs are a large family of reverse (3'-5') polymerases with various functions that revolve around tRNA repair and editing. Thg1 (tRNAHis guanylyltransferase) was discovered initially and its function is to add G-1 to tRNAHis in a non-Watson-Crick (W-C) manner, but TLPs prefer W-C polymerization, which may correspond to an ancestral activity found in the earlier members of this enzyme family. TLPs, being orthologs of Thg1, have been found in organisms from all three domains of life, including those that already contain a genomically encoded G-1 on tRNAHis. Surprisingly however, TLPs, including BtTLP, catalyze nucleotide addition in vitro on other 5' truncated RNAs that are not related to tRNAs in overall structure, such as 5S rRNA. This reaction begs the question of what the cellular substrates of 3'-5' reverse polymerases (BtTLP) are. The overall goal of this study is to investigate the roles of 3'-5' polymerases in biology.
This question is being investigated by isolating small RNA (<200 bp) from a Saccharomyces cerevisiae (baker's yeast) strain that has been engineered to express BtTLP. The resulting RNA is to be analyzed by high throughput deep sequencing, through a technique known as RNA-Seq. The massive amount of data (>106 reads per sample) that comes from this type of sequencing must be dealt with through a pipeline of various programs to process the data. The resultant processed genomic positions are analyzed on the UCSC Genome Browser to find peaks corresponding to different 5' locations on particular groups of reads. This approach is advantageous because it allows for essentially every RNA in the cell to be tested in a single experiment, rather than testing each RNA individually to determine whether it is a substrate for BtTLP-catalyzed nucleotide addition. This approach utilizes a novel way to use RNA-seq; which traditionally has been used to determine sequences that can be mapped back to the genome, here it is being used to discover post-transcriptional 5' nucleotide addition to small and perhaps 5'-truncated RNA molecules.

References:
1. Abad, Maria G., Bhalchandra S. Rao, and Jane E. Jackman. “Templated-dependent 3’-5’ nucleotide addition is a shared feature of tRNAHis guanylytransferase enzymes from multiple domains of life.” PNAS 107.2 (2010): 674-79. Print.
2. Jackman, Jane E., Jonatha M. Gott, and Michael W. Gray. “Doing it in reverse: 3’-to-5’ polymerization by the Thg1 superfamily.” RNA (2012): 886-99. Print.
3. Jackman, Jane E., and Eric M. Phizicky. “tRNAHis guanylyltransferase catalyzes a 3’-5’ polymerization reaction that is distinct from G-1 addition.” PNAS 103.23 (2006): 8640-45. Print.
4. Rao, Bhalchandra S., Emily L. Maris, and Jane E. Jackman. “tRNA 5’-end repair activates of tRNAHis guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea.” Nucleic Acids Research (2010): 1-10. Print.

Keywords: RNA-Seq, Reverse Polymerase, Bioinformatics

51. A Riboswitch-Containing sRNA Controls Gene Expression by Sequestration of a Response Regulator

Margo Gebbie (University of Maryland), Sruti Debroy (University of Texas Health Science Center at Houston), Jonathan Goodson (University of Maryland), Arati Ramesh (University of Texas Southwestern Medical Center at Dallas), Danielle Garsin (University of Texas Health Science Center at Houston), Wade Winkler (University of Maryland)

Abstract not available online - please check the printed booklet.

52. Prototyping a novel cheap electroelution device assembled from readily available materials

Jacob Gordon (Chemistry, Carnegie Mellon University), Subha Das (Chemistry, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

53. Defining the role of MRB10130 in uridine insertion and deletion RNA Editing in trypanosomes

Gregory L. Harrison Jr. (Department of Microbiology and Immunology, School of Medicine, State University of New York at Buffalo), Michelle L. Ammerman (Department of Microbiology and Immunology, School of Medicine, State University of New York at Buffalo), Laurie K. Read (Department of Microbiology and Immunology, School of Medicine, State University of New York at Buffalo)

Abstract:
In Trypanosoma brucei, a majority of the mitochondrially encoded genes are encrypted and require the post-transcriptional addition and deletion of uridines to create translatable open reading frames. This process is referred to as Uridine Insertion and Deletion RNA Editing (RNA editing) and is essential in T. brucei. RNA editing is catalyzed by the RNA Editing Core Complex (RECC); however, in vitro and in vivo studies suggest additional factors are required for the processivity, specificity, and efficiency of this process. One of these factors is the Mitochondrial RNA Binding Complex (MRB1), which consists of a dynamic network of protein-protein and protein-RNA interactions. To begin to define MRB1 architecture, our lab previously conducted a comprehensive yeast two-hybrid (Y2H) screen, followed by in vivo immunoprecipitation and mass spectrometry. These studies identified an MRB1 core and at least two TbRGG2 subcomplexes. 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 interactions with several MRB1 components were enhanced in the presence of RNA. According to its predicted structure, MRB10130 is composed almost entirely of alpha-helical repeats that resemble ARM/HEAT repeats. Proteins containing these types of repeats often act as organizers and mediators of protein-protein interactions. The involvement of numerous proteins and RNA species in RNA editing presents the need for spatial and temporal regulation of these interactions. We hypothesize that MRB10130 is involved in coordinating interactions within the MRB1 complex. Here we show that repression of MRB10130 leads to a significant RNA editing defect and growth phenotype. We are currently purifying MRB10130-containing complexes by tandem affinity purification followed by glycerol gradient sedimentation to more precisely define the interactions of this protein with MRB1 subcomplexes. In addition, we are using a series of knockdown and epitope-tagged cell lines to determine the role of MRB10130 in maintaining interactions within and between MRB1 subcomplexes. These studies will provide insight into the mechanisms underlying coordination of MRB1 complex function in trypanosome RNA editing.

Keywords: RNA editing, trypanosome

54. DNA computation in mammalian cells: microRNA logic operations

James Hemphill (Dept of Chemistry, University of Pittsburgh), Alexander Deiters (Dept of Chemistry, University of Pittsburgh)

Abstract:
DNA computation can utilize logic gates as modules to create molecular computers with biological inputs. Modular circuits that recognize nucleic acid inputs through strand hybridization activate computation cascades to produce controlled outputs. This allows for the construction of synthetic circuits that can be interfaced with cellular environments. We have engineered oligonucleotide AND gates to respond to specific microRNA (miRNA) inputs in live mammalian cells. Both single and dual-sensing miRNA-based computation devices were synthesized for the cell-specific identification of endogenous miR-21 and miR-122. A logic gate response was observed with miRNA expression regulators, exhibiting molecular recognition of miRNA profile changes. Nucleic acid logic gates that are functional in a cellular environment and recognize endogenous inputs significantly expand the potential of DNA computation to monitor, image, and respond to cell-specific markers.

Keywords: microRNA

55. Nucleoside Triphosphate Phosphohydrolase I (NPH I) in vaccinia early gene transcription termination.

Ryan Hindman (Department of Biological Sciences, The State University of New York at Buffalo. ), Paul Gollnick (Department of Biological Sciences, The State University of New York at Buffalo. )

Abstract not available online - please check the printed booklet.

56. The roles of DDX41 in the spliceosome and myeloid neoplasms

James M. Hiznay (Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH), Chantana Polprasert, Hideki Makishima (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH), Isabell Schulze, Carsten Muller-Tidow (Department of Hematology and Oncology, University of Halle, Halle, Germany), Jaroslaw P. Maciejewski (Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH), Richard A. Padgett (Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH)

Abstract:
Myelodysplastic syndrome (MDS) encompasses a group of bone marrow failure disease characterized as hematological pre-malignancies that often further transform to acute myeloid leukemia (AML). A recent surprising finding in patients with MDS or AML is recurrent mutations of components of the spliceosome, the nuclear complex responsible for the process of pre-mRNA splicing during gene expression. Spliceosomal protein genes such as SF3B1, U2AF1, and SRSF2 are found to have somatic (i.e. tumor) mutations at specific “hot spots.” Whole exome sequencing of MDS patients’ genomes and bone marrow has identified novel mutations in another spliceosome factor gene named DDX41. In this case, both germline and somatic mutations were found. This unique combination results in lower expression of DDX41 and confers a more advanced stage of disease and a lower patient survival outcome. DDX41 is a member of the DEAD-box family of RNA helicases, many of which are involved in splicing. Although proteomics has identified DDX41 as a component of the spliceosome catalytic core (complex C), no information is available on the exact role the protein plays in splicing or on its interaction partners in the spliceosome. We hypothesize that DDX41 interacts with one or more of the complex C snRNAs (U2, U5, U6) and/or with the splice sites. Our work has focused on determining the RNA interaction sequences of wild type and mutant DDX41 in vivo using RNA-protein crosslinking, complex immunoprecipitation, and high-throughput sequencing (collectively known as CLIP-seq). For this study, we have generated 293 cells expressing V5-tagged DDX41. Observed sensitivity to RNase A titration following crosslinking adds credence to the hypothesis that DDX41 is an RNA-binding protein. Preparation of cDNA libraries generated from RNA isolated during a CLIP is currently underway. Future work on this project includes the analysis of changes in RNA binding partners of mutant DDX41 as well as the generation of depleted nuclear extracts for in vitro biochemical studies.

References:
Maciejewski JP, Padgett RA. Br J Haematol. 2012 Jul;158(2):165-73.
Makishima H et al. Blood. 2012 Apr 5;119(14):3203-10.
Padgett RA. Trends Genet. 2012 Apr;28(4):147-54.

Keywords: DDX41, Pre-mRNA splicing, Myeloid neoplasm

57. Enzymatic generation of 3’-phosphate from 2’,3’-cyclic phosphate endonuclease digestion products of recombinant RNase U2 for improved LC/MS analysis of RNAs

Whitney M. Houser (Chemistry Department, University of Cincinnati), Annika B. Kotter (University of Antwerp), Balasubrahmanym Addepalli (Chemistry Department, University of Cincinnati), Patrick Limbach (Chemistry Department, University of Cincinnati)

Abstract:
De novo sequencing of RNAs using LC-MS involves RNA mass mapping, which employs the use of endoribonucleases to digest the nucleic acid strand into detectable oligonucleotides. Ribonuclease U2 cleaves at the 3’ end of purine bases, but occasionally leaves a 2’,3’-cyclic phosphate at the 3’ end of its digestion products. This complicates mass spectra, resulting in overlapping peaks, lowered peak intensities, and reduced CID scans. An enzymatic approach for reducing the amount of these cyclic phosphate-ending digestion products is used. Bacteriophage lambda protein phosphatase (LPP) is used as a post-RNase U2 step in order to hydrolyze cyclic phosphate bonds, and for labelling purposes, as a means of introducing the label into the sample. These digestion products are then separated using reverse-phase HPLC, and analyzed using ESI-MS/MS. A dramatic reduction in the number of cyclic phosphate-ending digestion products is observed after treatment with LPP. Additionally, LPP is shown to be capable of introducing 18O label into oligoribonucleotides via digestion in the presence of heavy oxygen-containing water, allowing for the extension of current CARD methods to include digestion products ending in adenosines.

References:
1. Hossain, M., Limbach, P. A. RNA, 2007, 13, 295-303.
2. Uchida, T. et al. J. Biochem. 1972, 67:1, 91-102.
3. Egami, Uchida. Mol. Bio. Biochem. Biophys. 1980, 32:250-270.
4. Meng, Limbach, Int. J. Mass Spectrom. 2004, 234:37-44.
5. Voegtli, W.C., et al. Biochemistry. 2000, 39, 15365-15374.

Keywords: Mass spectrometry, Enzymatic Digestion, RNA sequencing

58. Identifying mitochondrial DNA-associated trans-acting factors required for mtRNA editing in Physarum polycephalum

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

Abstract:
Proper gene expression in the mitochondria of the acellular slime mold Physarum polycephalum requires extensive RNA editing. RNA editing refers to a change in nucleotide sequence from that encoded in DNA and includes both nucleobase substitutions or conversions as well as nucleotide insertions and deletions. While both types of RNA editing occur in P. polycephalum mitochondria, the overwhelming majority (94%) of editing events comprise single cytosine (C) insertions. While it is known that this insertional editing is co-transcriptional (i.e., nucleotides are added at the 3' end of the nascent RNA during transcription), it is unclear both how editing sites are recognized as well as how the insertion of non-templated nucleotides is achieved. Preliminary data implicate at least one trans-acting protein factor is required for insertional editing. As efforts to uncouple editing from transcription have thus far been unsuccessful, it remains unclear whether this trans-acting factor associates with the mitochondrial DNA (mtDNA) or with both the mtDNA and mitochondrial RNA polymerase (mtRNAP). While our initial efforts to enrich for proteins associated with active transcription complexes via affinity selection using a nascent RNA handle were unsuccessful, we have more recently focused on enrichment of mtDNA template-associated proteins.
Previous work in our lab has yielded a mitochondrial lysate fractionation method which reduces the protein complement of the lysate by greater than 90%. The excluded mtDNA and associated protein factors support both transcription (and thus we refer to it as a mitochondrial Ttranscription Eelongation Complex, mtTEC) as well as mtRNA editing (to approximately 50% efficiency compared to intact mitochondria). The DNA in mtTEC preparations also serves as a substrate for restriction digestion, and our current aim is to enrich for protein factors associated with a discreet region of the mitochondrial genome that is actively transcribed and whose transcript is also edited. Restriction-digested mtTEC fragments are then substrates for affinity selection once filled-in or tailed with biotinylated deoxynucleotide triphosphates (dNTPs) and immobilized on streptavidin-coated magnetic beads.

Keywords: Physarum polycephalum, RNA Editing, mitochondria

59. Prolyl-tRNA synthetase–like editing domains: Exploring cellular functions in E. coli.

C. Bradley Howard (Department of Chemistry and Biochemistry and the Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Mom Das (Department of Chemistry and Biochemistry and the Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and the Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
Despite the importance of protein fidelity in translation for proper cellular function, prolyl-tRNA synthetase (ProRS) has been shown to mischarge amino acids Ala and Cys, which are smaller or similar in size to cognate Pro. Fidelity is maintained via tRNA editing. In most bacteria, the Ala-specific editing activity resides in the INS domain of ProRS, whereas YbaK, a single domain trans-editing INS homolog, is responsible for Cys-tRNAPro deacylation. Four other INS homologs (ProXp-ala, ProXp-ST1, ProXp-ST2, and ProXp-abu) have been identified and shown to have in vitro deacylation activity.The physiological roles of INS homologs have not been characterized in vivo. Therefore, the goal of this work is to identify potential cellular functions and pathways in which INS homologs participate. Published transcriptomic data correlates the expression of these proteins with cellular stresses (e.g., oxidative, heat, and cold). Using the comparative genomic database SEED, these homologs cluster in many bacteria with periplasmic proteins and amino acid metabolic proteins, suggesting periplasmic localization and potentially the regulation of amino acid levels. INS homologs are common among metabolically diverse bacteria, including soil bacteria and pathogens. Tandem-affinity chromatography in E. coli has linked YbaK and ProXp-ST1 with glycolysis, amino acid biosynthesis, translation, and aminoacylation. Amino acid misincorporation will be examined in YbaK and ProXp-ST1 knockout strains under a variety of stress conditions using a gain-of-activity misincorporation GFP reporter. In a parallel approach, mistranslation levels will be measured via mass spectrometry analysis of a Pro-rich protein expressed in the null strains. Since ProRS-like trans-editing domains are not present in higher eukaryotes including humans, identifying the conditions in which these proteins are essential may lead to new avenues for antibiotic development.

Keywords: aminoacyl-tRNA synthetases, tRNA editing, proline

60. Molecular Recognition and Mechanistic Insights into Protein-only RNase P

Michael Howard (University of Michigan), Bradley Klemm (University of Michigan), Markos Koutmos (Uniformed Health Services ), Carol Fierke (University of Michigan )

Abstract not available online - please check the printed booklet.

61. Structure and Assembly Dynamics of the TRAP-Anti-TRAP Regulatory System

Elihu C. Ihms (Department of Chemistry & Biochemistry, The Ohio State University), Mowei Zhou (Department of Chemistry & Biochemistry, The Ohio State University), Courtney Szyjka (Department of Biological Sciences, State University of New York at Buffalo), Vicki H. Wysocki (Department of Chemistry & Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo), Mark P. Foster (Department of Chemistry & Biochemistry, The Ohio State University)

Abstract:
In Bacillus spp., the undecameric (11-mer) protein TRAP is activated by elevated intracellular tryptophan concentration, becoming competent to silence several mRNA transcripts implicated in tryptophan biosynthesis. The trimeric protein Anti-TRAP (AT), upregulated during tryptophan starvation, competes for TRAP’s RNA binding sites and restores tryptophan production. Because of their homooligomeric configuration, Both TRAP and AT possess multiple binding sites for their respective partners, raising the possibility that cooperative interactions play an important role in tuning the regulatory circuit.

We investigated this possibility through a battery of bulk structural methods, including sedimentation, small-angle x-ray scattering, and native mass spectrometry. These revealed an unprecedented discovery for regulatory proteins: the polyvalent nature of TRAP and AT enables the formation of irregular, polymeric clusters containing multiple structurally intact copies of both components. This intriguing process suggests a clear mechanism by which small AT trimers compete effectively with the much larger TRAP-RNA interaction surface.

We extended the structural studies by investigating the thermodynamics and kinetics of the TRAP-AT interaction; to deconvolute the data, we engineered AT trimers incompetent to condense TRAP oligomers, and performed stopped-flow FRET experiments to probe the association rates of TRAP+AT and TRAP oligomers. Component binding thermodynamics were investigated by calorimetry, revealing strong negative cooperativity of binding for subsequent AT trimers to TRAP. These experimental parameters were used to build and test a variety of model system behaviors via stochastic simulations, ultimately concluding that TRAP-AT chaining plays an essential role. Consistent with this prediction, RNA competition assays utilizing the chaining-incompetent AT revealed that a higher concentration of this AT was required in order to achieve the same regulatory effect.

References:
Ihms, Elihu C., et al. "Gene regulation by substoichiometric heterocomplex formation of undecameric TRAP and trimeric anti-TRAP." Proceedings of the National Academy of Sciences 111.9 (2014): 3442-3447.

Keywords: gene regulation, reversible oligomerization, hybrid methods

62. Regulation of MDM2 alternative splicing: balance of positive and negative interactions between cis elements and trans factors.

Aishwarya G. Jacob (MCDB, The Ohio State University), Ravi K. Singh (MCDB, The Ohio State University), Fuad Mohammad (Department of pediatric cancer, Nationwide Childrens Hospital), Dawn S. Chandler (Department of pediatric cancer, Nationwide Childrens Hospital)

Abstract not available online - please check the printed booklet.

63. RNA as a boiling-resistant anionic polymer material to build robust polygons with tunable angle, size, stoichiometry and functionality

Daniel L. Jasinski (Nanobiotechnology Center, Markey Cancer Center, and College of Pharmacy, University of Kentucky), Emil F. Khisamutdinov (Department of Chemistry, Ball State University), Peixuan Guo (Nanobiotechnology Center, Markey Cancer Center, and College of Pharmacy, University of Kentucky)

Abstract:
RNA, a special class of anionic polymer, has recently emerged as an important nanotechnology platform due to its diversity, versatility and thermostability. RNA nanoparticles can be fabricated with precision characteristic of DNA while possessing adaptable tertiary structures and catalytic functions that can mimic some forms of proteins. RNA is unique in comparison to DNA by virtue of its high thermodynamic stability, ability to form both canonical and noncanonical base pairings, capability of base stacking, and unique in vivo attributes in therapeutic applications. RNA nanotechnology involves bottom-up approaches to assemble nanometer-scaled particles composed of primarily RNA. Utilizing the three-way junction (3WJ) motif from the phi29 motor pRNA we have constructed an assortment of thermodynamically and chemically stable RNA nanoparticles that carry multiple therapeutics, fluorogenic motifs, and other functional modules. The nanoparticles self-assemble with high efficiency, are resistant to RNase degradation and 8M urea denaturation, and remain intact after injection into the body. Ribozyme, siRNA, miRNA, or aptamers can be fused to the nanoparticles without affecting the folding of the core or the functionality of the modules. RNA can serve as a boiling-resistant anionic polymer material to build robust polygons with tunable angles, sizes, stoichiometry and functionality. The angle of the pRNA 3WJ can be tuned from 60o to 90o or 108o, transitioning from triangle to square or pentagon. The size of RNA nanoparticles can be easily increased or decreased as shown by the construction of 5, 10, and 20 nm square nanoparticles. The vertices of the angular nanoparticles were used to incorporate siRNA, ribozyme, and fluorogenic RNA motifs. Triangular nanoparticles were used to construct supramolecular hexamer constructs as well as honeycomb shaped arrays. This technique for simple design to finely tune RNA architectures adds a new angle to RNA nanotechnology.

References:
1. Jasinski DL, et al. and Guo P. Physicochemically tunable poly-functionalized RNA square architecture with fluorogenic and ribozymatic properties. ACS Nano. 2014. 8: 7620.
2. Khisamutdinov E, et al. and Guo P. Enhancing immunomodulation on innate immunity by shape transition among RNA triangle, square and pentagon nanovehicles. Nucleic Acids Research. 2014. 42: 9996.
3. Khisamutdinov E, et al. and Guo P. RNA as a Boiling-Resistant Anionic Polymer Material To Build Robust Structures with Defined Shape and Stoichiometry. ACS Nano. 2014. 8: 4771.
4. Shu Y, et al. and Guo P. Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs. RNA. 2013.19:767.
5. Guo P. The emerging field of RNA nanotechnology. Nature Nanotech. 2010. 5: 833.

Keywords: Bionanotechnology, RNA Nanoparticles, Tunable Size and Properties

64. Novel functions of cytochrome C in the cellular response to stress

Raul Jobava (Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Mridusmita Saikia (Department of Pharmacology, Case Western Reserve University, Cleveland, OH ), Andrea Putnam (RNA Center/Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Tao Pan (Department of Biochemistry and Biophysics, University of Chicago, Chicago, IL), Eckhard Jankowsky (RNA Center/Department of Biochemistry, Case Western Reserve University, Cleveland, OH ), Maria Hatzoglou (Department of Pharmacology, Case Western Reserve University, Cleveland, OH )

Abstract:
Regulation of cell volume in response to changes of extracellular tonicity is essential for cell survival. Hyperosmotic stress is associated with inflammation, diabetes and other diseases. Severe hyperosmotic stress induces cytochrome C release from the mitochondria, formation of the apoptosome and initiation of apoptosis. Hyperosmotic stress also activates the ribonuclease Angiogenin, which cleaves a small pool of tRNAs in the anticodon loop producing 30-45 nt long half tRNA molecules known as tiRNAs (tRNA-derived stress-induced fragments). The subcellular localization and molecular function of these small RNAs is largely unknown. Here we demonstrate that certain tiRNAs bind cytochrome C in the cytoplasm after it is released from mitochondria. Next generation sequencing revealed that twenty tiRNAs are highly enriched in this cytochrome C ribonucleoprotein complex. Direct binding of cytochrome C to tiRNAs within the cytochrome C ribonucleoprotein complex was confirmed using RNA gel-shift analysis. Our preliminary data suggest, that certain tiRNAs have prosurvival functions during hyperosmotic stress by inhibiting apoptosome formation. Our findings also reveal novel functions of cytochrome C during hyperosmotic stress which can be further explored for therapeutic purposes of protecting healthy cells from apoptosis.

Keywords: tiRNA, cytochrome C, stress

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

Evan Jones (Department of Chemistry, Ball State University), Timea Gerczei (Department of Chemistry, Ball State University)

Abstract not available online - please check the printed booklet.

66. The effect of arginine methylation on MRB1 core component function in trypanosome

Lucie Kafkova (Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, NY, USA), John C. Fisk (Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, NY, USA), Rachel Simpson (Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, NY, USA), Laurie K. Read (Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, NY, USA)

Abstract not available online - please check the printed booklet.

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

Alan Kessler (Dept. of Microbiology and OSU Center for RNA Biology, The Ohio State University), Zdenk Paris (Dept. of Microbiology and OSU Center for RNA Biology, The Ohio State University), Juan Alfonzo (Dept. of Microbiology and OSU Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

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

Prakash Kharel (Department of Chemistry and Biochemistry, Kent State University), Jennifer McDonough (Department of Biological Sciences, Kent State University), Soumitra Basu (Department of Chemistry and Biochemistry, Kent State University)

Abstract not available online - please check the printed booklet.

69. Active Site Architecture and Substrate Binding Interactions in an Arabidopsis thaliana Proteinaceous Rnase P

Bradley Klemm (Department of Biological Chemistry, University of Michigan), Michael Howard (Department of Biological Chemistry, University of Michigan), Carol Fierke (Departments of Chemistry and Biological Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

70. Distinct functions of the discrete regions of the multifunctional ribosome assembly protein Erb1 in yeast 60S subunit biogenesis

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.

71. Effects of guanine quadruplex mutagenesis on yeast ribosome biosynthesis

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

Abstract not available online - please check the printed booklet.

72. Interactions between RNA components of eukaryotic RNases P/MRP RNPs and Pop1, the largest RNase P/MRP protein

Robert Fagerlund (Biochemistry and Molecular Biology, Pennsylvania State University), Anna Perederina (Biochemistry and Molecular Biology, Pennsylvania State University), Igor Berezin (Biochemistry and Molecular Biology, Pennsylvania State University), Andrey S. Krasilnikov (Biochemistry and Molecular Biology, Pennsylvania State University)

Abstract not available online - please check the printed booklet.

73. Studying the catalytic mechanism of the m1G9/m1A9 methyltransferase (Trm10) family enzymes

Aiswarya Krishnamohan (Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Chemistry and Biochemistry, The Ohio State University)

Abstract:
The tRNA m1G9 methyltransferase (Trm10) methylates the guanosine residue at the 9th position (G9) of some tRNAs using S-adenosyl methionine (SAM) as the methyl group donor. Trm10 was originally discovered in Saccharomyces cerevisiae but is nearly ubiquitous in Archaea and Eukarya, and has been classified based on limited sequence similarity as a member of the SpoU-TrmD (SPOUT) family of methyltransferases. Intriguingly, some Trm10 orthologs also methylate A9 residues, representing an expansion of nucleotide substrate specificity not observed in any other methyltransferase family, and which is expected to require different catalytic groups based on the very different pKa values of the N1 atom of guanosine vs. adenosine nucleotides. Although a crystal structure of fungal Trm10 was recently determined, a mechanism of action that explains the dual specificity of these enzymes has not yet been elucidated. Thus, this mechanism is of interest as Trm10 enzymes likely use a novel mechanism to carry out N-1 methylation compared to other well-studied methyltransferases. Moreover, a complete understanding of the biological significance of Trm10 family enzymes also remains elusive, as deletion of the Trm10 gene in Saccharomyces cerevisiae does not result in an obvious phenotype, but mutations in a human homolog, TRMT10A, were recently implicated in numerous abnormalities including defects in glucose metabolism and neurological dysfunction. This direct correlation to human health has also elevated the importance of studying this unique group of enzymes. Here we use site-directed mutagenesis to mutate conserved residues that are implicated in Trm10 catalytic activity based on their positions in the crystal structure and utilize kinetic assays to evaluate their resulting activities. Although the SAM-binding site predicted by the crystal structure is supported by this analysis, the evidence argues against the proposed involvement of a conserved aspartate as a general base in the reaction. Analysis of additional Trm10 variants is underway with the ultimate goal of elucidating the complete catalytic mechanism of this unusual methyltransferase enzyme family.

Keywords: tRNA, methyltransferase, Trm10

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

Roni M. Lahr (University of Pittsburgh, Department of Biological Sciences), Seshat M. Mack (University of Pittsburgh, Department of Biological Sciences), Benjamin P. Robinson (University of Pittsburgh, Department of Biological Sciences), Sarah P. Blagden (2Imperial College London, Hammersmith Campus), Andrea J. Berman (University of Pittsburgh, Department of Biological Sciences)

Abstract not available online - please check the printed booklet.

75. Insights Into the Folding of RNA Under In vivo-Like Conditions

Kathleen A. Leamy (Pennsylvania State University, Department of Chemistry and Center for RNA Molecular Biology), Elisabeth E. Whitman, Philip C. Bevilacqua (Pennsylvania State University, Department of Chemistry and Center for RNA Molecular Biology)

Abstract not available online - please check the printed booklet.

76. Delineation of lncRNA recognition sites by NMR: SHARP RRM binding to SRA1 STR7.

Thomas C. Leeper (Department of Chemistry and Biochemistry, University of Akron), Joel Caporoso (Department of Chemistry and Biochemistry, University of Akron), Gregory Buchan (Departments of Immunology and Virology, University of Pittsburgh School of Medicine), Caroline Davis (Department of Chemistry and Biochemistry, University of Akron)

Abstract not available online - please check the printed booklet.

77. Towards Developing a Structure Based Mechanism of Splicing Repression by hnRNP A1 at ssA7 on HIV-1

Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University), Jennifer Meagher (Life Sciences Institute, University of Michigan), Jeanne Stuckey (Life Sciences Institute, University of Michigan), Blanton S. Tolbert (Department of 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. One such site regulated by hnRNP A1 is the conserved 3’ acceptor splice site A7 (ssA7). Silencing of splicing at this site is necessary in order to retain the Rev Responsive Element (RRE) in the adjacent tat/rev intron. The RRE is responsible for nuclear export of unspliced and partially spliced transcripts.
Our research seeks to clarify the binding determinant of hnRNP A1 on ssA7 by developing a structural model that will correlate ssA7 structure to its splicing function. The ssA7 RNA composes three stem loops, and a divide and conquer approach has been taken to determine the method by which hnRNP A1 binds and propagates along its RNA substrate. The first domain of ssA7 that is being studied is SL3, which contains the ESS3 binding motif for hnRNP A1. The 3D solution structure of SL3 was solved by NMR, and it showed the UAG motif of ESS3 to be located in a terminal heptaloop.
Building on the solution structure of SL3, experiments have been undertaken to solve the co-structure of hnRNP A1 with SL3. Most of this work has been done using UP1, a protein that consists of the two RRM motifs of hnRNP A1, but lacks the C-terminal glycine rich domain that is responsible for oligomerization of the full length protein. A crystal structure has been obtained of UP1 bound with the trinucleotide AGU, a sequence equivalent to a portion of the SL3 heptaloop. This structure shows the RNA is bound in a pocket formed by aromatic residues of RRM1 and residues from the linker between the two RRM domains. Elements of this binding are supported by 1D saturation transfer difference and NOESY NMR experiments. A variety of strategies are currently being employed to try and obtain crystals of full length SL3 bound by UP1.

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.D., 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.

Keywords: RRM, Crystal Structure, HIV

78. Structure-mediated cooperativity between single-stranded RNA binding partners on 5' and 3'UTRs

Yi-Hsuan Lin (Department of Physics, Center for RNA Biology, The Ohio State University), Ralf Bundschuh (Department of Physics, Department of Chemistry and Biochemistry, Division of Hematology, Center for RNA Biology, The Ohio State University)

Abstract:
In post-transcriptional regulation, many proteins and/or microRNAs bind to an RNA molecule to modulate its biological functions. Such process comprises multiple binding reactions, and in order to enable combinatorial gene regulation it is necessary that these binding partners of RNA communicate with each other, or in other words that their binding to the RNA exhibits cooperativity. Even in the absence of direct physical interactions between the binding partners, such cooperativity can be mediated through the secondary structure of the RNA molecule, since the secondary structure affects accessibility to the various binding sites. Here we propose a quantitative measure of this structure-mediated cooperativity that can be calculated for an arbitrary RNA sequence using computational secondary structure prediction methods. Focusing on an RNA with two binding sites, we derive a characteristic free energy difference as the measurement for describing how the binding strength of one site is affected by the occupancy of the other one. We apply this measurement to a large number of human and C. elegans mRNA sequences, and find that structure-mediated cooperativity is a generic feature of mRNA sequences. By comparing the measurements of human and C. elegans mRNAs, we find that the strength of the cooperativity is strongly related to the lengths of UTRs. Moreover, we generate a group of RNA sequences by replacing the coding regions of human mRNAs with a fixed coding region sequence, and discover that the cooperativities of these RNAs have statistical properties very similar to the original human mRNAs. We thus conclude that cooperativity between two proteins bound on the 5' and the 3' UTR, respectively, can be mediated by the secondary structures of the two UTR sequences independently of the intervening coding sequences. We also apply an evolutionary algorithm to several human RNA sequences, and discover that artificially evolved sequences can support extremely strong cooperativities.

Keywords: RNA-protein interactions, cooperativity, RNA structure

79. Cofactor Modulation of T box Riboswitch Recognition: Effects of Mg2+ and Spermidine on the T box Antiterminator RNA-tRNA Complex

Jia Liu (Department of Chemistry & Biochemistry, Ohio University), Shu Zhou (Department of Chemistry & Biochemistry, Ohio University), Jennifer V. Hines (Department of Chemistry & Biochemistry, Ohio University)

Abstract:
The T box riboswitch is a gene-expression controlling mechanism in many Gram-positive bacteria. In the T box mechanism, transcription is controlled by the recognition and binding between tRNA and the antiterminator, a secondary structure of the riboswitch formed in the 5´-UTR region of the mRNA. Cofactor modulation of tRNA-antiterminator complex formation was investigated using antiterminator model RNA AM. The effects of spermidine and Mg2+ on AM and the AM-tRNA complex were tested by ITC and fluorescence steady-state and anisotropy assays. Both spermidine and Mg2+ facilitated tRNA-AM binding. The optimal combination to facilitate tRNA-AM complex formation was determined to be [Mg2+]=2-5 mM, [spermidine]=2-3 mM, indicating that the combination of the two cofactors might lower the requirement for Mg2+ during in vitro transcription antitermination. Simplified tRNA models were also investigated to determine the extent to which the overall tRNA structure affects acceptor end binding to AM.

Keywords: T box riboswitch, spermidine, antiterminator

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

Qi Liu (Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University), Kurt Fredrick (Department of Microbiology, Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

81. Role of the U2AF Homology Motif in Human Tat-SF1 as a Cofactor for HIV-1 Splicing

Sarah Loerch (Center for RNA Biology, University of Rochester Medical Center, Rochester, NY), Clara L. Kielkopf (Center for RNA Biology, University of Rochester Medical Center, Rochester, NY)

Abstract not available online - please check the printed booklet.

82. Multiple functions of a family of 3’-to-5’ polymerases in Dictyostelium discoideum

Yicheng Long (Ohio State Biochemistry Program, The Center for RNA Biology, The Ohio State University), Maria Abad (The Center for RNA Biology, The Ohio State University), Fuad Mohammad (The Center for RNA Biology, The Ohio State University), Erik Olson (Ohio State Biochemistry Program, The Center for RNA Biology, The Ohio State University), Jane Jackman (Ohio State Biochemistry Program, The Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

83. Unravelling the Trl1–like enzyme function in Trypanosoma brucei

Raphael R. S. Lopes (Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro), Gilbert S. Oliveira, Roberta Eitler (Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro), Zdenek Paris (Microbiology Department, The Ohio State University), Juan D. Alfonzo (Microbiology Department; The Ohio State University), Raphael S. Vidal (Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro), Carla Polycarpo (Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro)

Abstract:
Transfer RNAs (tRNAs) play a central role in protein synthesis as translators of the genetic code connecting the information found in genes to that ultimately deposited into proteins. The biosynthesis of mature and functional tRNAs involves many processing steps, including 5’ and 3’ end trimming, incorporation of numerous chemical post-transcriptional modifications and addition of a CCA sequence at the 3’ end. A subset of tRNAs, that varies with different organisms, also contain introns that are cleaved by a tRNA splicing endonuclease, generating exon halves that are subsequently sealed by a splicing-specific tRNA ligase. We have identified a homolog of yeast tRNA ligase (Trl1) in trypanosomatids, that is presumably responsible for the joining of the two tRNA exon halves generated by endonuclease cleavage of tRNATyr; the only intron-containing tRNA in these organisms. In the present work, we have preliminarily characterized the Trl-1 homolog of T. brucei (TbTrl1). We constructed a stable T. brucei RNAi knockdown strain of Tbtrl1 and show that RNAi induction leads to a severe growth defect. Unexpectedly, RNAi of TbTrl1 caused accumulation of intron-containing tRNA, as opposed to unligated exon halves, and almost a complete disappearance of mature tRNA. Furthermore, down-regulation of TbTrl1 also led to pronounced cell-cycle arrest. Taken together these results demonstrate the essentiality of the TbTrl1 homolog for cell viability. The observed accumulation of intron-containing tRNA is discussed in the context of the intra-cellular localization of TbTrl1 and its potential role in tRNA export from the nucleus. The restricted phylogenetic distribution to fungi and trypanosomatids for Trl1-splicing ligases shown in this work makes this family of enzymes promising therapeutic targets for such medically important organisms.

References:
Acknowledgements: This research was supported by NIH,FAPERJ and CNPq

Keywords: tRNA ligase, Trypanosoma, Editing

84. Aging-and Oxidative Stress-Related Alternative Splicing in Drosophila melanogaster

Deborah Makin (Biological Sciences, Carnegie Mellon University), Yevgeniya Monisova (Biological Sciences, Carnegie Mellon University), A. Javier Lopez (Biological Sciences, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

85. Mechanistic Insights into HIV-1 Tat Dependent Release of P-TEFb from the 7SK snRNP Complex

Le Luo (Department of Chemistry, Case Western Reserve University), Uri R. Mbonye (Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University), Jonathan Karn (Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University; Center for AIDS Research, Case Western Reserve University), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University; Center for AIDS Research, Case Western Reserve University)

Abstract:
The HIV-1 protein, Tat, stimulates transcriptional elongation by recruiting P-TEFb to the nascent RNAP II complex during transactivation of the proviral genome. 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. The Tat protein is able to activate P-TEFb by releasing it from the inhibitory complex; however, the series of molecular events that promote the release mechanism is poorly understood. In this study, we demonstrate the specific binding of 7SK stem loops to HEXIM1 and Tat peptides using quantitative electrophoretic mobility shift assay. Both HEXIM1 and Tat can bind 7SK SL1 with nM affinities and HEXIM1 also associates with SL4 in the nanomolar range. Intriguingly, competition experiments between a Tat peptide and HEXIM1 for 7SK SL1 reveal Tat can effectively displace the HEXIM1-7SK complex. Thus, the results presented here offer insights into the mechanism used to promote the release of P-TEFb from the HEXIM1/7SK inhibitory complex.

Keywords: P-TEFb , HEXIM1-7SK, HIV-1 Tat

86. Approaches to Synthetic Mini-lariats for RNA Interference

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

Abstract:
RNA interference is a powerful tool to knockdown gene expression. The RNA-induced silencing complex (RISC) is the endogenous cellular machinery that destroys messenger RNA based on a target complementary that is sequence derived from microRNAs (miRNAs). A large number of miRNAs have been suggested to originate from spliced introns that must be debranched before entering RISC. We are devising mimics for these intronic miRNAs (mirtrons) by synthesizing small RNA lariats that will open up into a linearized strand upon cleavage by debranching enzyme. This strand would mimic the target complementary guide strand of the miRNA and would be taken up by RISC. Solid phase synthesis of the mini-lariats is achieved through 5'-phosphitylation and subsequent selective removal of a 2'-O-photolabile group at the desired branch nucleotide. Following the 2'-O-protecting group removal, coupling of the 5' phosphoroamidite to the free 2'-OH completes mini-lariat synthesis with a 2'-5'-phosphate linkage, a substrate for debranching enzyme. These mini-lariats are being evaluated as agents for RNA interference.

Keywords: RNA interference, micro RNA, mini-lariats

87. Aging-and Oxidative Stress-Related Alternative Splicing in Drosophila melanogaster

Deborah Makin (Biological Sciences, Carnegie Mellon University), Yevgeniya Monisova (Biological Sciences, Carnegie Mellon University), A. Javier Lopez (Biological Sciences, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

88. Effects of electroporation on transcription and editing in isolated physarum mitochondria

Gregory Naegele (Center for RNA Molecular Biology, Case Western Reserve University), Tereena Marks (Center for RNA Molecular Biology, Case Western Reserve University), Jonatha Gott

Abstract not available online - please check the printed booklet.

88. Effects of Electroporation on Transcription and Editing in Isolated Physarum Mitochondria

Gregory Naegele (Center for RNA Molecular Biology Case Western Reserve Univerity), Tereena Marks (Center for RNA Molecular Biology Case Western Reserve Univerity), Jonatha Gott (Center for RNA Molecular Biology Case Western Reserve Univerity)

Abstract:
Please see abstract for Gregory Naegele

Keywords: Physarum polycephalum

89. Codon optimality, translation elongation and the regulation of mRNA stability

Sophie Martin (Center for RNA Molecular Biology, CWRU), Ying-Hsin Chen (Center for RNA Molecular Biology, CWRU), Vladimir Presnyak (Center for RNA Molecular Biology, CWRU), Najwa Al Husaini (Center for RNA Molecular Biology, CWRU), Jeff Coller (Center for RNA Molecular Biology, CWRU)

Abstract:
A tight regulation of mRNA stability is essential in the control of gene expression. It has long been observed that mRNA decay and translation are intimately coupled, with mRNAs which translate being more stable. Translation efficiency depends on the supply of charged tRNAs in the cell and the demand by translating ribosomes (considering mRNA expression levels and the efficiency of tRNA interaction). Therefore, codons are considered “optimal” or “non-optimal” if the availability of cognate tRNAs exceeds or limits their relative usage. In the laboratory, it has been shown that the relative codon composition of an mRNA (optimal or non-optimal) directly influences mRNA stability, with mRNAs containing a high proportion of non-optimal codons being unstable. However, the mechanisms underlying the regulation of mRNA stability through codon decoding are unclear. Here, we sought to understand how translational elongation may be influenced by the codon composition of an mRNA and how this can lead to destabilization of the mRNA. First, we are investigating the relative occupancy of polyribosomes on highly “optimal” or “non-optimal” transcripts in yeast, to establish if mRNA stability is correlated with elongation rate. In conjunction, we are also studying how the decay of the stable PGK1 transcript can be affected by the introduction of synthetic non-optimal codons along the mRNA (different positions from 5’ to 3’), at which elongating ribosomes may be retained. Finally, we are analyzing the polysomal association of artificial optimal and non-optimal transcripts expressed in Drosophila melanogaster S2 cells, to determine if the codon optimality is a conserved determinant of gene expression in higher Eukaryotes. In particular, it is possible that codon composition is sensed during translation elongation, leading to repression of translation (and then decay, storage or transport). This would be a new aspect of gene expression possible differential regulation in distinct cell types, cellular environments or developmental states.

Keywords: mRNA decay, codon optimality, translation elongation

90. RNA trafficking during trypanosome RNA editing

Natalie M. McAdams (Department of Microbiology and Immunology, University at Buffalo School of Medicine), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo School of Medicine)

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 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. For the majority of transcripts, RNA editing is required to create translatable open reading frames, and thus editing is essential for proliferation of both human and insect vector stages of the kinetoplastid, Trypanosoma brucei. Small trans-acting guide RNAs (gRNAs), encoded in the minicircle component of the mitochondrial genome, direct proper U insertion or deletion. The enzymes that catalyze endonucleolytic cleavage of pre-mRNA, U addition, U removal, and resealing by ligation are present in a stable multiprotein structure termed the RNA editing core complex (RECC) or 20S editosome. While RECC structure and enzymology has been probed, the mechanisms by which RECC interacts with mRNAs and gRNAs are almost completely unknown. Recently, we have identified a dynamic multiprotein complex termed the MRB1 complex (mitochondrial RNA binding complex) as a key component of the editing machinery, possibly as the RNA binding component of the editing holoenzyme. Our hypothesis is that the MRB1 complex plays a critical role in RNA trafficking to RECC. Here we look to define the role of two MRB1 subcomplexes in the recruitment of gRNA and pre-mRNA to RECC using UV cross-linking/RNA-immunoprecipitation (CLIP) in cells depleted of specific MRB1 proteins.

Keywords: RNA editing, trypanosome

91. Investigation of the role played by the RNA G-quadruplex structure in ALS/FTD pathology

McAninch, Damian S. (Duquesne University), Mihailescu, Mihaela Rita (Duquesne University)

Abstract:
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in motor neuron loss in brain and spinal cord. Frontotemporal dementia (FTD) is one of the most common forms of young onset dementia and second most common form of dementia overall, after Alzheimer's, resulting in degeneration of temporal lobes along with personality changes and language impairment. ALS and FTD are now recognized as members of a broad continuum of neurodegenerative disorders, linked by similar pathology, mechanisms, and overlapping clinical symptoms. Two RNA-binding proteins of interest that link the two diseases are TAR DNA-binding protein 43 (TDP-43) and the fused in sarcoma/translocated in liposarcoma protein (FUS), which are the major protein components in over 90% of ALS and over 50% of FTD inclusions. We hypothesize that the G-quadruplex RNA structure might play an essential role in the pathogenic mechanisms of FUS in ALS and FTD. In this study, the G-quadruplex RNA binding properties of the wild type and C-terminal NLS mutant FUS protein implicated in ALS/FTD will be analyzed.

Keywords: ALSFTD, RNA Binding, G-Quadruplex

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

Sarah L. Metcalfe (Department of Biology, St. Bonaventure University ), Zachary Mazanek (Department of Biology, St. Bonaventure University ), Meera Babu (Department of Biology, St. Bonaventure University ), Kevin Cilano (Department of Biology, St. Bonaventure University ), Aubrie Russell (Department of Biology, St. Bonaventure University ), Xiao-Ning Zhang (Department of Biology, St. Bonaventure University )

Abstract not available online - please check the printed booklet.

93. Global Intersection of Long Non-Coding RNA (lncRNA) Genes With Processed and Unprocessed Pseudogenes in The Human Genome

Michael Milligan (Wayne State University Center for Molecular Medicine and Genetics), Dr. Leonard Lipovich (Wayne State University Center for Molecular Medicine and Genetics)

Abstract:
Since the completion of the Human Genome Project, a key revelation in post-genomic biology has been the prevalence of non-protein-coding functional elements in the human genome. Highlighted by the ENCODE and FANTOM consortia, these elements include tens of thousands of pseudogenes, as well as comparably numerous long non-coding RNA (lncRNA) genes. Transcription of pseudogenes is still poorly understood, but the field is of great importance for human disease due to the high sequence similarity between pseudogenes and their parental genes, generating the potential for sequence-specific regulation. Recent case studies establish essential functional roles of both pseudogenes and lncRNAs in metazoan systems. Some have even highlighted functional impacts of lncRNA transcription at pseudogene loci on the regulation of the pseudogenes' parental genes. To compute the complete potential regulatory space of integrated pseudogene-lncRNA regulation, we developed and implemented an algorithm, using the PERL programming language, to identify all pseudogenes that are overlapped by lncRNA transcription in both sense and anti-sense orientations. Our algorithm imported three public repositories of pseudogenes: GENCODE v17 (processed and unprocessed; n=15233); UCSC Genome Database (processed only; n=13358) and Yale (processed and unprocessed; n=17216), plus two public lncRNA catalogs: Broad Institute (n=21537) and GENCODE v17 (n=13331). The intersection hence comprised six pseudogene-lncRNA genomewide overlaps. We identified 1167 loci containing genomic-span exon overlaps between all pseudogenes and all lncRNAs with EST and cDNA support. Of these, 374 had bidirectional cDNA support providing evidence of transcription. This project has generated an empirically supported transcriptome list of all pseudogene-lncRNA overlaps in the human genome to serve as a foundation for future curation, parental-gene ontology analysis, and experimental validation of function.

References:
Karro, J. E., Yan, Y., Zheng, D., Zhang, Z., Carriero, N., Cayting, P., . . . Gerstein, M. (2007). Pseudogene.org: A comprehensive database and comparison platform for pseudogene annotation. Nucleic Acids Research, 35(Database issue), D55-D60. doi:10.1093/nar/gkl851

Kalyana-Sundaram, S., Kumar-Sinha, C., Shankar, S., Robinson, D. R., Wu, Y., Cao, X., . . . Chinnaiyan, A. M. (2012). Expressed pseudogenes in the transcriptional landscape of human cancers. Cell, 149(7), 1622-1634. doi:10.1016/j.cell.2012.04.041

Milligan, M. (2014, June). Global Intersection of Long Non-Coding RNA (lncRNA) Genes With Processed and Unprocessed Pseudogenes in The Human Genome. Poster session presented at The 19th Annual Meeting of the RNA Society, Québec City, Canada.

Keywords: LncRNA, Pseudogene, Genomewide

94. Structural and mechanistic insights into hnRNP A1 - RNA recognition

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

Abstract:
In the HIV-1 genome, alternative splicing is a highly regulated process which is controlled through the utilization of individual suboptimal splice sites, where balanced usage of these splice sites is required for complete expression of the viral genome. At splice site acceptor 7 (ssA7), located at the 3’ end of the viral genome, splicing is inhibited through the binding of exon splicing silencer 3 (ESS3) by the hnRNP A1 protein. Many biochemical models suggest that the silencing effects of hnRNP A1 binding occur due to cooperative spreading and unwinding of the RNA; however, recent studies show that this protein binds specific regulatory RNA structural elements present within the HIV genome. In this study, the specificity of UP1 (the two RRM binding domain of the hnRNP A1 protein) to ESS3 is further defined by a crystal structure of the protein in complex with a short RNA oligomer, 5’ – AGU – 3’, analogous to the 3’ portion of the ESS3 apical loop. The structure revealed that UP1 uses a “nucleobase pocket” to make site specific contacts with the AG dinucleotide bases. This nucleobase pocket is formed by the inter-RRM linker and a beta sheet of RRM1 where RRM2 does not contact the RNA. This binding event between UP1 and ESS3 is kinetically characterized through the utilization of BioLayer Interferometry (BLI) and molecular dynamics simulations are performed in an attempt to further elucidate the binding mechanism of hnRNP A1 to RNA. Our kinetic studies show ESS3 mutants adversely affect UP1 association rates with little to no change in the off rates. These results imply that the ESS3 structure drives favorable UP1 interactions. In summary, this study provides critical insights into the biologically important UP1-ESS3 complex.

Keywords: HIV, hnRNP A1, ESS3

95. Shaping of a Genetic Mutation Phenotype by Tissue Specific Alternative Splicing

Daniel Murphy (Biochemistry and Molecular Biology, West Virginia University), Saravanan Kolandaivelu (Biochemistry, Center for Neuroscience, West Virginia University), Visvathan Ramamurthy (Biochemistry, Center for Neuroscience, West Virginia University), Peter Stoilov (Biochemistry and Molecular Biology, West Virginia University)

Abstract not available online - please check the printed booklet.

96. The human RBPome: from genes and proteins to human disease

Yaseswini Neelamraju (Department of Biohealth Informatics,Indiana University-Purdue University,Indianapolis), Seyedsasan Hashemikhabir (Department of Biohealth Informatics,Indiana University-Purdue University,Indianapolis), Sarath Chandra Janga (Department of Biohealth Informatics,Indiana University-Purdue University,Indianapolis)

Abstract:
RNA Binding Proteins (RBPs) play a central role in mediating post transcriptional regulation of genes. However less is understood about them and their regulatory mechanisms. In this study, we construct a repertoire of 1344 RBPs identified from several experimental studies and present a comprehensive meta-analysis to understand their characteristics at a global scale. The domain architecture of RBPs enabled us to classify them into three groups - Classical (29%), Non-classical (19%) and Unclassified (52%). A higher percentage of proteins with unclassified domains reveal the presence of various uncharacterised motifs that can potentially bind RNA. RBPs were found to be highly disordered compared to non-RBPs (p<2.2e-16, Fisher’s exact test), indicating a dynamic regulatory role of RBPs in cellular functioning. Evolutionary analysis in 62 different species showed that RBPs are highly conserved compared to non-RBPs (p<2.2e-16, Wilcox-test), reflecting the conservation of various post-transcriptional processes like mRNA splicing and translational control. Expression patterns of RBPs from human proteome map revealed that majority (~60%) of the RBPs are tissue-specific. RBPs were also seen to be highly associated with several neurological disorders, cancers and inflammatory diseases. Further, anatomical context analysis revealed that B cells, T-cells, Fetal Liver and Fetal Brain are enriched in diseases associated with RBPs implying a prominent role for RBPs in immune responses and different developmental stages. These analyses are made accessible to researchers in the form of a database called RBP expression and disease dynamics database (READ DB: http://darwin.soic.iupui.edu/)

References:
1.Kwon SC, et al. (2013) The RNA-binding protein repertoire of embryonic stem cells. Nature structural & molecular biology 20(9):1122-1130.
2.Ray D, et al. (2013) A compendium of RNA-binding motifs for decoding gene regulation. Nature 499(7457):172-177.
3.Baltz AG, et al. (2012) The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Molecular cell 46(5):674-690.
4.Castello A, et al. (2012) Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149(6):1393-1406.
5.Cook KB, Kazan H, Zuberi K, Morris Q, & Hughes TR (2011) RBPDB: a database of RNA-binding specificities. Nucleic acids research 39(Database issue):D301-308.

Keywords: RNA Binding proteins, post transcriptional regulation

97. Investigating Shared Molecular Recognition By RNA and Protein Subunits of RNase P Using High-Throughput Sequencing Kinetics (HTS-Kin)

Courtney N. Niland (Department of Biochemistry, Case Western Reserve University, Cleveland, OH), Jing Zhao (Department of Biochemistry, Case Western Reserve University, Cleveland, OH), David Anderson (School of Business, CUNY Baruch College), Eckhard Jankowsky (Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, OH), Michael E. Harris (Department of Biochemistry, Case Western Reserve University, Cleveland, OH)

Abstract:
Ribonucleoproteins perform essential functions in biology that rely on their ability to recognize and process many different substrates. Currently an understanding of how the RNA and protein subunits of these enzymes work together to achieve substrate recognition and catalysis is lacking. Ribonuclease P, RNase P, a multi-substrate ribonucleoprotein, is made of a protein subunit and catalytic RNA subunit that removes the 5’ leader from all pre-tRNAs despite their variation in sequence and structure. Previous work from our lab and others indicate that both the protein and RNA subunits of RNase P make specific contacts in the 5’ leader of pre-tRNA. Using a new technique termed High-Throughput Sequencing Kinetics, HTS-Kin, we are able to analyze the effect of substrate variation in the 5’ leader on enzyme processing. This technique allows us to measure the processing rate constant for thousands of substrates in a single reaction and provides context dependence of mutations. The RNase P holoenzyme was placed into multiple turnover reactions with substrate pools randomized in the 5’ leader region contacting the protein subunit (-3 to -8) or the protein and RNA subunits (-1 to -6) and analyzed by HTS-Kin, providing a rate constant for nearly all 4096 substrates. The HTS-Kin results show a correlation between the identity of nucleotides contacting the RNA subunit and identity and contribution of those contacted by the protein subunit, indicative of thermodynamic coupling. Analyses of individual sequence variants is being used to confirm and investigate the basis for this strong thermodynamic coupling between RNA-protein and RNA-RNA interactions. HTS-Kin analysis of these same populations in single turnover reactions will reveal mechanistic detail and monitor the effect of nucleotides in the 5’ leader on catalysis. This information will reveal how RNase P achieves specificity and provide deeper insight into molecular recognition by multi-substrate ribonucleoprotein enzymes.

Keywords: RNase P, Ribonucleoprotein, Enzyme Kinetics

98. The RNA helicase Skiv2l2 works to maintain pluripotency and proliferation in stem cells

Alexis M. Onderak (Biological Sciences, Marquette University ), James T. Anderson (Biological Sciences, Marquette University)

Abstract not available online - please check the printed booklet.

99. DNA Computation: Logic Gates and Amplification Cycles

Alexander Prokup (University of Pittsburgh, Department of Chemistry), James Hemphill (University of Pittsburgh, Department of Chemistry), Alexander Deiters (University of Pittsburgh, Department of Chemistry)

Abstract not available online - please check the printed booklet.

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

Andrei Rajkovic (Microbiology The Ohio State University), Sarah Tyler (Microbiology The Ohio State University), Michael Ibba (Microbiology The Ohio State University)

Abstract not available online - please check the printed booklet.

101. Roles of Eukaryote-Specific rRNA Expansion Segments in Ribosome Biogenesis

Madhumitha Ramesh (Department of Biological Sciences, Carnegie Mellon University), John Woolford (Department of Biological Sciences, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

102. Analysis of ncDNA transcription for roles in regulating gene expression

Elizabeth Raupach (Department of Biological Sciences, University of Pittsburgh), Joseph Martens (Department of Biological Sciences, University of Pittsburgh)

Abstract:
Transcription of non-coding DNA (ncDNA) is widespread in eukaryotes and plays important regulatory roles. Previous studies have elucidated one such regulatory mechanism at the S. cerevisiae gene SER3. The act of transcribing SRG1, a non-coding RNA (ncRNA), across the SER3 promoter positions nucleosomes over the SER3 upstream activating sequences, which serve as a physical barrier to prevent transcription of SER3. The pervasiveness of non-coding transcription suggests that this and other regulatory mechanisms mediated by non-coding transcription may exist throughout the genome. To explore this possibility, I selected six candidate yeast genes expressing unstable ncRNAs over their promoters and analyzed the effects of disrupting intergenic transcription on open reading frame transcript expression. Through this unbiased approach, we identified a previously unknown mechanism of transcription regulation at the ECM3 gene. In contrast to the mechanism of SER3 regulation, intergenic transcription seems to activate ECM3 expression. Further analysis has identified roles for the Paf1 complex in ECM3 activation through the methylation of histone H3K4. Additionally, a longer intergenic transcript is present in the absence of Paf1. Although this transcript awaits further characterization, it may arise from a termination defect and could potentially provide a spatial and/or temporal dimension to ECM3 regulation. Thus, ECM3 is an interesting model gene for elucidation of a novel regulatory mechanism mediated by non-coding transcription.

Keywords: ncRNA, transription, chromatin

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

Nathan Raynard (Department of Biological Chemistry and Genetics Training Program, University of Michigan), Elizabeth Abshire (Department of Biological Chemistry and Chemistry Biology Interface Training Program, University of Michigan), Joel Hrit, Paul Del Rizzo, Adam Petterson (Department of Biological Chemistry, University of Michigan), Ray Trievel (Department of Biological Chemistry and Chemistry Biology Interface Training Program, University of Michigan), Aaron Goldstrohm (Department of Biological Chemistry, Genetics Training Program, and Chemistry Biology Interface Training Program, University of Michigan)

Abstract not available online - please check the printed booklet.

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

Laura E. Ritchey (Department of Chemistry, The Pennsylvania State University, University Park, PA 16802; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, 16802), Zhao SU (Department of Biology, The Pennsylvania State University, University Park, PA 16802; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, 16802), Chun K. Kwok (Department of Chemistry, The Pennsylvania State University, University Park, PA 16802; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, 16802), Sarah M. Assmann (Department of Biology, The Pennsylvania State University, University Park, PA 16802; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, 16802), Philip C. Bevilacqua (Department of Chemistry, The Pennsylvania State University, University Park, PA 16802; Center for RNA Molecular Biology, The Pennsylvania State University, University Park, 16802)

Abstract not available online - please check the printed booklet.

106. Inductive Based Fluidics mass spectrometry for the analysis of modified nucleosides in RNAs

Robert Ross (Department of Chemistry, University of Cincinnati), Drew Sauter (nanoLiter, LLC, Henderson, NV ), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)

Abstract:
Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. These modifications can be present at very low levels and their analysis can be challenging. Thus, newer analytical methods capable of higher sensitivity analysis of modified RNAs would be advantageous. Inductive Based Fluidics (IBF) as a means for RNA sample introduction could be a way to increase sensitivity. Inductive Based Fluidics (IBF) uses inductive charging to produce nanoliter-sized droplets. Here we have compared IBF with nano electrospray ionization (nESI) and micro-ESI for the LC-MS/MS analysis of enzymatically digested RNA. At flow rates < 2 μL/min, the analyte signal to noise with IBF was found to be comparable or higher to nESI. The analyte S/N for IBF was found to decrease below that obtained from ESI-based sources at higher flow rates, likely due to limited desolvation. The analytical advantages of IBF include ease of use and an improved ability to handle “salty” oligonucleotide samples without capillary plugging. Further, preliminary results suggest improved detection of modified nucleosides of low proton affinity, such as modified uridines, for IBF over nESI.

Keywords: Inductive Based Fluidics, nucleosides, modification

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

Bappaditya Roy (Department of Microbiology, The Ohio State University), Kurt Fredrick (Department of Microbiology, The Ohio State University)

Abstract not available online - please check the printed booklet.

108. Understanding the regulatory mechanisms of asparagine and aspartate T box riboswitches in Clostridium acetobutylicum

Nizar Y. Saad (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA), Bettina Schiel (Institute of Microbiology and Biotechnology, Ulm University, D-89069 Ulm. Albert Einstein-Allee 11), Peter Drre (Institute of Microbiology and Biotechnology, Ulm University, D-89069 Ulm. Albert Einstein-Allee 11), Hubert D. Becker (Unite Mixte de Recherche 7156 Genetique Moleculaire Genomique Microbiologie, Centre National de la Recherche Scientifique, Universite de Strasbourg, F-67084 Strasbourg, France), Tina M. Henkin (Department of Microbiology and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA)

Abstract:
The T box riboswitch functions primarily at the level of transcription attenuation and controls the expression of downstream amino acid-related genes by sensing the aminoacylation state of a cognate tRNA [1]. When cells are limited for the cognate amino acid, the tRNA is uncharged and binds to the T box riboswitch, which promotes formation of an antiterminator structure that allows transcription of the downstream gene.
The C. acetobutylicum genome contains two aspartyl-tRNA synthetase genes (aspS2o and aspS2), encoding DRS2o and DRS2, respectively. We previously showed that DRS2o charges tRNAAsp (aspartate) and tRNAAsn (asparagine) with Asp [2, 3]. The mischarging of Asp onto tRNAAsn is followed by Asn-tRNAAsn synthesis through the amidation of Asp by the transamidation pathway [4]. C. acetobutylicum lacks active Asn synthetase, and is unable to synthesize Asn from Asp independent of tRNAAsn [3]. We previously showed that a T box riboswitch regulates the expression of the aspS2o operon, which encodes DRS2o and the amidotransferase complex, forming a functional transamidation pathway [3]. This T box riboswitch senses the tRNAAsn aminoacylation state (NT box). C. acetobutylicum possesses an alternative route for Asn-tRNAAsn synthesis through an AsnRS that directly charges Asn onto tRNAAsn. We recently demonstrated that DRS2 is also capable of charging Asp onto tRNAAsn. Asn and Asp may both affect NT box antitermination, since their availability may affect the aminoacylation state of tRNAAsn. We previously showed that Asn affects the expression of the aspS2o operon through the NT box riboswitch [3], but the effect of Asp availability has not been investigated. We recently observed that expression of the aspS2o operon is up-regulated and that of aspS2 gene is down-regulated in C. acetobutylicum grown in minimal medium containing Asp, relative to their expression during growth in minimal medium lacking all amino acids. The expression of aspS2 is regulated by a T box riboswitch that responds to tRNAAsp (DT box). Transcriptome analysis showed that the aspS2o operon is induced in an aspS2 knock-out strain grown in minimal medium lacking all amino acids. These results suggest that the expression of aspS2 and aspS2o operon may be related. In this study, we investigate in detail the nature of this relation.

References:
1. Henkin, T. M. (2014). The T box riboswitch: A novel regulatory RNA that utilizes tRNA as its ligand. Biochim Biophys Acta.
2. Charron, C., Roy, H., Blaise, M., Giege, R., and Kern, D. (2003). Non-discriminating and discriminating aspartyl-tRNA synthetases differ in the anticodon-binding domain. Embo J 22, 1632-1643.
3. Saad, N. Y., Schiel, B., Braye, M., Heap, J. T., Minton, N. P., Durre, P., and Becker, H. D. (2012). Riboswitch (T-box)-mediated control of tRNA-dependent amidation in Clostridium acetobutylicum rationalizes gene and pathway redundancy for asparagine and asparaginyl-trnaasn synthesis. J Biol Chem 287, 20382-20394.
4. Curnow, A. W., Hong, K., Yuan, R., Kim, S., Martins, O., Winkler, W., Henkin, T. M., and Soll, D. (1997). Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc Natl Acad Sci U S A 94, 11819-11826.

Keywords: T box riboswitch, amino acids, aminoacyl-tRNA synthetases

109. Erythrocytic stage specificity of Plasmodium falciparum telomerase

Samantha Sanford (CNAST, Department of Chemistry, Carnegie Mellon University), Yossi Weizmann (Department of Chemistry, University of Chicago), Kausik Chakrabarti (CNAST, Department of Chemistry, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

110. Determining the kinetic contributions of tertiary interactions to the folding of the thermostable Azoarcus ribozyme using smFRET

Krishnarjun Sarkar (T.C. Jenkins Department of Biophysics, Johns Hopkins University), Boyang Hua (Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign), Taekjip Ha (Department of Physics, University of Illinois at Urbana-Champaign), Sarah A. Woodson (T.C. Jenkins Department of Biophysics, Johns Hopkins University)

Abstract not available online - please check the printed booklet.

111. Probing the role of guanine deprotonation in ribozyme mechanism

Dan Seith (Chemistry at Pennsylvania State University), Jamie Bingaman (Chemistry at Pennsylvania State University), Yin Tang (Center for RNA Molecular Biology at Pennsylvania State University), Phillip C. Bevilacqua (Chemistry at Pennsylvania State University), Sarah Assmann (Biology at Pennsylvania State University)

Abstract:
This work is focused on deprotonation of guanines in functional RNAs. One of the biological motivations for understanding guanine deprotonation is that anionic guanines can act as proton acceptors in ribozyme mechanisms. This is especially true for most of the small ribozymes where guanines appear to be poised in an ideal position in the active site to participate in a chemical step during cleavage. To gain a better understanding of guanine ionization at the N1 position, genome-wide data from dimethyl sulfate (DMS) methylation experiments on Arabidopsis thaliana rRNA were subjected to a scoring procedure to reveal possible correlations between DMS reactivity scores at guanines and RNA secondary structure. The procedure showed what nucleotide positions revealed reverse transcription stops. After investigating supposed guanine N1 reactivity upon DMS treatment in multiple rRNAs, no correlations were found between DMS reactivity at the N1 of guanines and RNA secondary structure. The mechanistic basis for reverse transcription stops one nucleotide before guanine at neutral pH is now being assessed by other models. Also presented in this work is progress that has been made towards developing a new chemical assay for detecting guanine deprotonation in functional ribozymes.

Keywords: ribozyme, guanine

112. Peptides targeting modified helix 69 of 23S rRNA in bacterial ribosomes

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

Abstract:
Due to the rise of antibiotic resistance, there is a need for discovery of new targets and development of novel drugs. In this study, we are targeting helix 69 (H69) of 23S ribosomal RNA (rRNA) in E. coli ribosomes with peptides as potential new antibacterial compounds. This RNA contains highly conserved nucleotides, in addition to three pseudouridine modifications. Helix 69 is located in the 50S subunit of the ribosome, specifically at the interface with the 30S subunit, and plays important roles in translation. Peptides were chosen as possible drug candidates to target modified H69 because they have reasonable stability and can be easily modified. Phage display was performed in order to screen for linear heptapeptides that target H69 under various conditions, such as high and low pH, differing magnesium concentrations, or in the presence of competitor RNAs. After four rounds of selection, the sequences of the bound phage were analyzed and several consensus sequences were identified. These peptides were synthesized by using solid-phase synthesis techniques, purified by HPLC, and characterized by MALDI mass spectrometry. Binding studies such as fluorescent indicator displacement assay, anisotropy and ESI-MS with H69 revealed peptides with moderate affinity. Current studies involve optimization of these peptides for enhanced binding and selectivity for modified H69.

Keywords: helix 69, phage display, binding studies

113. The use of tethered function analysis to investigate the role of Upf1 in targeting substrates to NMD

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

Abstract:
Cells employ multiple quality control mechanisms to ensure the accurate flow of genetic information. Nonsense-mediated mRNA decay (NMD) is a post-transcriptional quality control mechanism responsible for the detection and destruction of transcripts that contain nonsense mutations or other features which cause translation termination to occur in a premature context. The superfamily I RNA helicase Upf1 is a central component of the NMD machinery. Upf1 has been well characterized in vitro. However, relatively little is known about how Upf1 functions to induce rapid mRNA decay in vivo. Here, we use tethered function analysis as an inroad to evaluate the specific requirements for Upf1 to target substrates to NMD.

Initial studies indicate that tethering Upf1 to the 3’ UTR of a reporter mRNA lacking a nonsense codon results in reduction of both steady state mRNA abundance and half-life. Destabilizing activity is lost when Upf1 alleles which contain mutations that result in loss of ATPase and helicase activity are tethered to the mRNA. Interestingly, tethered Upf1 is able to induce reporter destabilization in the absence of Upf2 and Upf3, as well as under conditions where translation is strongly repressed. These initial results indicate that tethered function analysis is a robust and effective assay for examining the requirements for Upf1 to induce rapid mRNA decay during NMD.

Keywords: quality control, NMD, Upf1

114. RNA nanoparticle for specific EpCAM targeting and cancer cell delivery

Ashwani Sharma, Fengmei Pi, Dan Shu (Nanotechnology Center, Department of Pharmaceutical Science, College of Pharmacy, University of Kentucky, Lexington, KY 40536), Piotr Rychahou, B. Mark Evers (Markey Cancer Center, University of Kentucky, Lexington, KY 40536), Peixuan Guo (Nanotechnology Center, Markey Cancer Center, Department of Pharmaceutical Science, College of Pharmacy, University of Kentucky, Lexington, KY 40536)

Abstract:
The epithelial cell adhesion molecule (EpCAM) is a transmembrane protein overexpressed in epithelial cancers. High levels of EpCAM expression has been observed in 97.7% of colon, 90.7% of gastric, 87.2% of prostate cancers and in 63.9% of lung cancers, and has been used as a therapeutic target in cancer therapy. Several targeting ligands have been developed against EpCAM in recent past, but they suffer from limited sensitivity and lack of proficient clinical response. In this study, utilizing state-of-art RNA nanotechnology platform, we generated a serum and thermodynamically stable RNA aptamer against EpCAM from a 2’-Fluoro RNA library. The magnetic bead based SELEX was utilized to select and recover bound RNA sequences using EpCAM protein as target. After several rounds of selection, the library was cloned and sequenced. The selected aptamer was checked for its binding affinity to EpCAM by flow cytometry to colon and breast cancer cells, and was further validated by confocal fluorescence microscopy. The binding constant for the aptamer was calculated to be lower than 55 nM to different human cancer cells. To our knowledge, this is the first RNA aptamer selected utilizing RNA nanotechnology approach that can be used directly for targeted drug delivery. This novel 2’F RNA aptamer against EpCAM was further utilized for specific delivery of RNA interference therapeutics by conjugating siRNA or miRNA to the RNA aptamer.

References:
(1) Guo P. 2010. The Emerging Field of RNA Nanotechnology. Nature Nanotechnology, 5:833-842.
(2) Shu D, Shu Y, Haque F, Abdelmawla S and Guo P. 2011. Thermodynamically stable RNA three-way junction for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology. 6:658-67.
(3) Haque F, Shu D, Shu Y, Shlyakhtenko L, Rychahou P, Evers M, Guo P. 2012 Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers. Nano Today.7:245–57.

Keywords: RNA Nanotechnology, Aptamer, EpCAM protein

115. Biochemical characterization of the human DEAD-box RNA helicase DDX3X

Deepak Sharma (Center for RNA Molecular Biology, Case Western Reserve University), Eckhard Jankowsky (Center for RNA Molecular Biology, Case Western Reserve University)

Abstract:
The human DEAD-box RNA helicase DDX3X is expressed in multiple tissues and has been implicated in transcription regulation, RNA export, translation, and several signal transduction pathways. Mutations and altered expression of DDX3X have been linked to the development of multiple tumors, and DDX3X is directly targeted by several different viruses. Previous studies have qualitatively shown helicase and ATPase activities for DDX3X, but many important biochemical features of this protein are unknown.
Here, we systematically characterize enzymatic activities of DDX3X. We show that DDX3X unwinds RNA-RNA, and RNA-DNA, but not DNA-DNA duplexes. DDX3X shows a significant preference for unwinding RNA-RNA substrates with 3’ unpaired regions over those with 5’ unpaired regions; a notable difference to its yeast ortholog Ded1p, which does not display this pronounced preference, despite more than 95% sequence similarity between both proteins. DDX3X also shows significantly less potent strand annealing activity than Ded1p. With both DDX3X and Ded1p, unwinding rate constants for RNA-RNA duplexes decrease with duplex lengths. However, DDX3X is able to unwind significantly longer duplexes than Ded1p, while Ded1p unwinds shorter duplexes much faster than DDX3X. The ATPase activity of both DDX3X and Ded1p is stimulated by ssRNA, but not by ssDNA. However, ssDNA can bind to both proteins.
Our results show biochemical activities of DDX3X that are typical for DEAD-box RNA helicases. However, the functional differences between Ded1p and DDX3X with respect to substrate preferences, strand annealing, and unwinding of long duplexes, indicate that biochemical features can markedly vary even between highly similar DEAD-box helicase orthologs.

Keywords: DDX3X, DEAD BOX HELICASE, BIOCHEMISTRY

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

Anya Sherwood (Molecular, Cellular, and Developmental Biology Graduate Program, OSU), Elihu Ihms (Biophysics Graduate Program, OSU), Frank Grundy (Department of Microbiology, OSU), Mark Foster (Department of Chemistry and Biochemistry, OSU), Tina Henkin (Department of Microbiology, OSU)

Abstract not available online - please check the printed booklet.

117. Trypanosome RNA Editing Alignment Tool: Using deep sequencing to understand the roles of accessory factors in kinetoplastid RNA editing

Rachel Simpson (Department of Microbiology and Immunology, SUNY Buffalo), Andrew Bruno (New York State Center of Excellence in Bioinformatics and Life Sciences, SUNY Buffalo), Laurie Read (Department of Microbiology and Immunology, SUNY Buffalo)

Abstract not available online - please check the printed booklet.

118. Andro-miRs processing and regulation of Androgen receptor expression in PCa.

Savita Singh (Center for Gene Regulation in Health and Disease,Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH, 44115), Kavleen Sikand (Center for Gene Regulation in Health and Disease,Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH, 44115), Girish C. Shukla (Center for Gene Regulation in Health and Disease,Department of Biological, Geological and Environmental Sciences, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH, 44115)

Abstract not available online - please check the printed booklet.

119. Strategies for cloning 6.8 kb Androgen Receptor 3'UTR into pMIR report.

Mathilda Slaibi (Biological, Geological and Environmental Sciences), Savita Singh (Biological, Geological and Environmental Sciences), Girish C. Shukla Ph.D. (Biological, Geological and Environmental Sciences)

Abstract not available online - please check the printed booklet.

120. Translation of small open reading frames within unannotated RNA transcripts in Saccharomyces cerevisiae

Jenna E. Smith (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Juan R. Alvarez-Dominguez, Wenqian Hu (Whitehead Institute for Biomedical Research, Cambridge, MA), Sarah Geisler, Nicholas Kline, Nathan Huynh (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Jeff Coller (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH), Kristian E. Baker (Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, OH)

Abstract:
High-throughput gene expression analysis has revealed that eukaryotic cells express many previously unidentified and largely uncharacterized transcripts. In most cases, these transcripts are bioinformatically predicted to lack protein-coding capacity. Here, we investigate >1100 unannotated transcripts in yeast predicted to be non-coding. We show that a majority of these RNAs associate with polyribosomes to an extent more similar to mRNAs than classical, well-characterized non-coding RNAs. Furthermore, ribosome profiling demonstrates that many of these RNAs are directly bound by ribosomes, with a number demonstrating periodicity of ribosome footprints indicative of bona fide translation. Location of ribosome footprints facilitated the identification of putative open-reading frames 10-96 codons in length within a number of unannotated RNAs. We directly demonstrate translation of a subset of these open-reading frames through detection of the predicted polypeptide. Consistent with their translation, many unannotated RNAs are targeted for degradation by the translation-dependent, nonsense-mediated RNA decay (NMD) pathway. Based upon ribosome profiling, we predict these RNAs are targets for NMD due to long 3’ ribosome-free regions. Inspection of mammalian lncRNAs reveals that a subset are also sensitive to NMD, indicating translation of non-coding transcripts in higher eukaryotes. These data demonstrate that transcripts considered to lack coding potential do in fact engage translating ribosomes. An intriguing possibility is that this translation serves either as a mechanism to regulate the metabolism or function of these RNAs or to produce short proteins, potentially expanding the proteome of yeast and other eukaryotes.

Keywords: lncRNA, Nonsense-mediated RNA decay, Translation

121. Mutagenesis of RNase U2 for Enhanced Specificity

Beulah Mae Ann Solivio ( University of Cincinnati), Balasubramanyam Addepalli ( University of Cincinnati), Patrick Limbach ( University of Cincinnati)

Abstract:
Given several ribonucleases (RNases) that have specific cleavage on Uridine (U), Cytosine (C), and Guanosine (G), we aim to generate a RNase that has specificity to cleave at Adenosine (A). Due to the availability of RNase U2, which cleaves at both A and G with a preference on A, our goal is to modify the nucleotide binding domain of RNase U2 to be selective for A by rational mutagenesis. In order to achieve this goal, we designed molecular models indicating hydrogen-bonding interactions of RNase U2 complexed with 3’-AMP to serve as a guide for mutagenesis. Mutagenesis has been performed with the use of synthetic gene blocks that were inserted into Pet22b plasmids. Further, recombinant plasmids have been transformed onto E. coli, from which the mutant protein have been overexpressed, extracted, and purified. Specificity was determined by digestion of tRNAPhe and analysis with mass spectrometry.

References:
Czaja, R., Struhalla, M., Hoschler, K., Saenger, W., Strater, N., Hahn, U., (2004)
Matsuzaki, T., Saski, C., Uchida, T., Satow, Y., and Noguchi. S., (1995)

Keywords: RNase U2 , mutagenesis, specificity

122. Ribosome occupancy of RNA cis-regulatory sequences, uORFs, implicated in the simultaneous regulation of gene expression across two levels.

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

Abstract:
It has long been understood that the regulation of gene expression plays a major role in the construction of phenotype from genotype. However, gene regulation has been difficult to study because it is multi-faceted and continuous, involving multiple processes. It has recently become possible to measure regulation at both the transcriptional and translational levels with the advent of ribosome profiling, a deep-sequencing method that allows genome-wide measurement of ribosome occupancy and position on transcripts. Our lab has previously used this approach to identify genes whose regulation has diverged at both levels in two yeast species. One type of this interlevel regulation is 'amplification', where divergence in mRNA abundance is reinforced by post-transcriptional regulation that results in an even greater divergence in ribosome occupancy. Amplified divergence in gene expression may be particularly important for species adaptation. However, the mechanisms responsible for amplifying divergence have yet to be elucidated.
We chose to investigate the mechanisms of divergence using a subset of amplified genes in which both mRNA levels and translation efficiency have evolved reinforcing changes in cis-regulatory sequences (cis-plus-cis genes). By comparing ribosome locations among cis-plus-cis genes, we present evidence suggesting that a single mechanism may be responsible for divergence of both mRNA abundance and translation efficiency. We observed that cis-plus-cis amplified genes are significantly enriched in ribosome-occupied upstream open reading frames (occupied uORFs). Based on our preliminary analysis, cis-plus-cis genes are six times more likely to have occupied uORFs compared to a random gene sample. We propose that evolutionary changes in uORF usage allow for mRNA abundance to be co-regulated with ribosome occupancy by simultaneously modulating mRNA degradation and translation efficiency.

Keywords: regulation of gene expression, uORFs, ribosome profiling

123. Structural and functional characterization of an Aquifex Hfq homolog

Kimberly Stanek (Chemistry, University of Virginia), Jennifer Patterson, Peter Randolph (Chemistry, University of Virginia), AhnThu Nguyen (Biology, University of Virginia), Andrew Holmes (Biomolecular Engineering, University of California Santa Cruz), Todd Lowe (Biomolecular Engineering, University of California Santa Cruz), Cameron Mura (Chemistry, University of Virginia)

Abstract:
The host factor Hfq, as the bacterial branch of the Sm protein superfamily, is an RNA-associated protein that functions in transcript-level regulation of gene expression and mRNA turnover. Hfq acts as an RNA chaperone by facilitating the interactions between mRNAs and small, regulatory noncoding RNAs (sRNAs). Hfq has been shown to form homohexameric rings that feature two distinct faces for RNA-binding. An Hfq homolog has been bioinformatically identified in the genome of the deep-branching thermophile Aquifex aeolicus (Aae), but little is known about its function or structure. Others have demonstrated that Aae Hfq does not complement Hfq-deletion strains of Salmonella enterica, suggesting an independent mechanism of action for Aae Hfq. To elucidate the structure and function of Aae Hfq, we have cloned, over-expressed, purified, crystallized, and biochemically characterized this protein, in vitro and in vivo. We recently determined the crystal structure of Aae Hfq to 1.5 Å resolution. Using fluorescence polarization assays, binding affinities have been determined for the interactions of this Hfq with U- and A-rich RNAs. Notably, recombinant Aae Hfq co-purifies with a collection of small endogenous RNAs; we have recently isolated and characterized these species via RNA-Seq.

References:
Sittka, A., Sharma, C.M., Rolle, K., and Vogel, J. (2009) RNA Biology 6, 226-275.
Franze de Fernandez, M.T., Hayward, W.S., August, J.T. (1972) J. Biol. Chem. 247, 824-831.
Sauter, C., Basquin, J., and Suck, D. (2003) Nucleic Acids Research 31, 4091-4098.
Lenz, D.H., Mok, K.C., Lilley, B.N., Kulkarni, R.V., Wingreen, N.S., and Bassler, B.L. (2004) Cell 118, 69-82.
Kulesus, R.R., Diaz-Perez, K., Slechta, E.S., Eto, D.S., and Mulvey. M.A. (2008) Infection and Immunity 76, 3019-3026.
Soper, T., Mandin, P., Majdalani, Gottesman, S., and Woodsman, S.A. (2010) PNAS 107, 9602-9607.
Ikeda, Y., Yagi, M., Morita, T., Aiba, H. (2011) Mol. Micr. 79(2):419-32.
Patterson, J., Mura, C. (2012) Journal of Visualized Experiments 72: e50225, 1–10.

Keywords: Hfq, Crystallography, RNA-protein interactions

124. Investigating Oxidative Damage in RNA through Pseudouridinyl Radical Precursors

Matthew J. Starr (Department of Medicinal and Biological Chemistry, The University of Toledo), Raziya Shaik (Department of Medicinal and Biological Chemistry, The University of Toledo), Dr. Amanda C. Bryant-Friedrich (Department of Medicinal and Biological Chemistry, The University of Toledo)

Abstract:
Reactive oxygen species can cause damage to proteins, lipids, and nucleic acids through the generation of free radicals. Oxidative damage in DNA has been studied quite extensively, with comparatively little work done on oxidative damage in RNA. The products resulting from radical initiated alterations to RNA have been shown to play a role in Alzheimer’s disease and Parkinson’s disease. The goal of this project is to create a radical precursor that can be used to study oxidative damage in RNA, to determine if the most commonly modified nucleotide found in RNA, pseudouridine, plays a role in the attenuation of oxidative damage. Introduction of a photolabile pivaloyl group at the C3’ and C5’ sites of pseudouridine will allow for the generation of a free radical at these specific positions, to determine if this base modification has any effect on the outcome of oxidative sugar damage in RNA.

References:
1. Shan, Xiu. “Quantification of oxidized RNAs in Alzheimer's disease.” Neurobiology of Aging: 27(5), 2006, 657.
2. Wurtmann, E., & Wolin, S. (2009). “RNA under attack: cellular handling of RNA damage.” Critical Reviews In Biochemistry And Molecular Biology, 44(1), 34-49.

Keywords: pseudouridine, oxidative damage, organic synthesis

125. G quadruplex structures in PSD-95 mRNA as potential regulators of miR-125a seed binding site accessibility

Snezana Stefanovic (Chemistry and Biochemistry, Duquesne University), Mihaela-Rita Mihailescu (Chemistry and Biochemistry, Duquesne University)

Abstract not available online - please check the printed booklet.

126. Identification of Escherichia coli mRNAs with 5’-terminal AUG triplets that act as both leaderless and Shine-Dalgarno led mRNAs.

Sarah R. Steimer (Microbiology Miami University), Ian Fleming (Ohio State University), Heather Beck (Miami University), Gary Janssen (Miami University)

Abstract not available online - please check the printed booklet.

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

Krishna C. Suddala (Biophysics, University of Michigan, Ann Arbor, MI 48109, USA), Malgorzata Michnicka (Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA), Edward P. Nikonowicz (Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA), Nils G. Walter (Chemistry, University of Michigan, Ann Arbor, MI 48109, USA)

Abstract not available online - please check the printed booklet.

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

Julie L. Sutton (Applied & Engineering Physics, Cornell University), Lois Pollack (Applied & Engineering Physics, Cornell University)

Abstract not available online - please check the printed booklet.

129. Investigation of a novel role in transcriptional regulation of the trp operon by TRAP

Courtney Szyjka (Dept. Biological Sciences, SUNY at Buffalo), Natalie Merlino (Dept. Microbiology and Immunology, SUNY at Buffalo), Paul Gollnick (Dept. of Biological Sciences, SUNY 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 termination in the presence of tryptophan, indicating that TRAP may have a more direct role in transcription termination. Through a genetic selection, a TRAP mutant was isolated that is capable of binding RNA and tryptophan at wild-type (WT) levels, but does not terminate transcription. In vitro transcription analysis with B. subtilis RNA polymerase (RNAP) of TRAP proteins from a variety of bacterial species showed a trend of WT levels of termination if TRAP contained an acidic residue at position 60, but not if there was a basic residue in this position. To test the hypothesis that TRAP interacts directly with RNAP to induce termination, we performed label transfer experiments. Our data indicate that TRAP may interact with the alpha subunit of RNAP, a subunit known to have many interactions with transcriptional regulator proteins.

Keywords: transcription, gene regulation

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

Yin Tang (Bioinformatics and Genomics Graduate Program, Pennsylvania State University, University Park), Philip C. Bevilacqua (Department of Chemistry, Pennsylvania State University, University Park), Sarah M. Assmann (Department of Biology, Pennsylvania State University, University Park)

Abstract not available online - please check the printed booklet.

131. Electrostatics of rRNA Hairpins as Determined by Platination Kinetics

Gayani Dedduwa-Mudalige, Supuni Thalalla Gamage, Christine S Chow

Abstract:
Electrostatics play an important role in RNA-ligand interactions. Positively charged drug
molecules and other ligands are attracted to the negatively charged RNA. Monovalent
and divalent ions affect the electrostatic properties and influence these interactions;
however, studying these properties experimentally in solution can be challenging. This
work focuses on the use of transition metal complexes as tools to study the electrostatic
microenvironment, as well as global electrostatics, of RNA. More specifically, positively
charged monoaquated cisplatin and small model ribosomal RNA (rRNA) constructs are
employed. The rates of the platination are determined by using gel shift analysis, and the
electrostatic properties of rRNA are determined from the kinetics of platinum
coordination under varying salt conditions. The rate of platination with RNA is shown to
decrease in the presence of divalent cations. Furthermore, rRNA shows different
electrostatic properties in the presence of monovalent and divalent cations. Overall, the
present work demonstrates the utility of monoaquated cisplatin kinetics as a tool to
compare electrostatic environments of different RNA constructs with varying sequences,
secondary and tertiary structures, or nucleotide modification levels.

Keywords:

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

Meryl Thomas (Department of Chemistry, University of Pittsburgh), Colleen M. Connelly (Department of Chemistry, North Carolina State University), Alexander Deiters (Department of Chemistry, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

133. Rapid clearance of oscillating transcripts during somitogenesis requires the decay adapter Pnrc2

Kiel T. Tietz (Department of Molecular Genetics, The Ohio State University), Thomas L. Gallagher, Nicolas L. Derr (Department of Molecular Genetics, The Ohio State University), Courtney E. French (Department of Plant and Microbial Biology, University of California, Berkeley), Jasmine M. McCammon (Department of Molecular and Cell Biology, University of California, Berkeley), Steven E. Brenner (Department of Molecular and Cell Biology, University of California, Berkeley), Sharon L. Amacher (Department of Molecular Genetics, The Ohio State University)

Abstract:
Vertebrate segmentation is controlled 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 presomitic mesoderm (PSM) and has the same periodicity as that of somite formation. Core cyclic components of the segmentation clock include the hes/her family of transcriptional repressors, but additional transcripts also cycle. Maintenance of the oscillation period requires that transcriptional activation and repression, RNA turnover, translation, and protein degradation are all very rapid as somite pairs quickly develop in all vertebrates, just 30 minutes in the zebrafish model. Our lab isolated a zebrafish segmentation clock mutant, tortugab644; mutant embryos express elevated levels of cyclic mRNAs such as her1 and rhov. We have demonstrated that loss of Proline-rich nuclear receptor coactivator protein Pnrc2 is responsible for cyclic transcript accumulation in tortugab644 mutants. In human cell culture systems, Pnrc2 has been implicated in mRNA decay through interactions with Dcp1a and Upf1. Data from our lab supports a similar mRNA decay function in zebrafish since partial depletion of both Pnrc2 and Upf1 increases her1 and rhov accumulation.

We hypothesize that in pnrc2 mutants, oscillating transcripts accumulate due to inefficient removal of the 5’ cap, thus causing mRNA perdurance. In order to test this hypothesis, we are examining if Pnrc2 regulates the localization, decapping, or deadenylation of her1 and rhov transcripts and if these accumulating transcripts are actively being translated. While the mechanisms of aberrant mRNA decay are well studied, less is understood about pathways regulating decay of non-aberrant transcripts. Our goal is to define mechanisms critical for the rapid turnover of oscillating genes during somitogenesis.

Keywords: Pnrc2, her1 , rhov

134. Determination of the Isoform and Post-translational Modification State of Lysyl-tRNA Synthetase Packaged into HIV-1 Particles by Mass Spectrometry

Nathan P Titkemeier (Department of Chemistry and Biochemistry, The Ohio State University), Michael E Hoover (Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Medical School), Corine St.-Gelais (Center for Retroviral Research, College of Veterinary Medicine, The Ohio State University), Li Wu (Center for Retroviral Research, College of Veterinary Medicine, The Ohio State University), Michael A Freitas (Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Medical School), Karin Musier Forsyth (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Human lysyl-tRNA synthetase (LysRS) is critical for the replication of Human Immunodeficiency Virus 1 (HIV-1), as it directs the selective packaging of host tRNALys3, the primer for HIV-1 reverse transcription, into HIV-1 virions. Like most other aminoacyl-tRNA synthetases (aaRSs), LysRS has been demonstrated to perform a number of non-canonical functions beyond its role in aminoacylating or “charging” amino acids onto their cognate tRNAs. In most of these cases, LysRS becomes activated to perform non-canonical functions through post-translation modifications (PTMs), which cause it to dissociate from the high molecular weight multi-synthetase complex (MSC), where it exists as a homotetramer in complex with 8 other aaRSs and 3 scaffold proteins. In addition, three forms of LysRS are produced from the same gene: the cytoplasmic isoform, an immature pre-mitochondrial isoform, and a processed mitochondrial isoform. The exact isoform(s) packaged into HIV-1 virions has been a point of debate. We are using liquid chromatography-coupled tandem mass spectrometry to identify whether the PTM profile of human LysRS is altered upon HIV-1 infection, as well as to establish the specific isoform(s) of LysRS that is/are packaged into HIV-1 virions. Preliminary data suggests the presence of cytoplasmic LysRS within the virions, and we are employing a multiple reaction monitoring MS/MS approach to definitively identify the packaged isoform. Preliminary data also suggest an increase in phosphoserine on LysRS following HIV-1 infection, but changes in the PTM profile remain to be elucidated through LC-MS and LC-MS/MS. LysRS proteolysis has been optimized to provide better sequence coverage and SILAC studies are planned to confirm changes in LysRS modifications in HIV-1 infected cells.

Keywords: HIV-1, aminoacyl-tRNA synthetase

135. hnRNP A1 RNA recognition motifs induce a conformational change upon binding 7SK RNA stem III

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

Abstract:
Tight regulation of transcription is necessary for cellular survival, homeostasis, and development. Promoter-proximal pausing is a higher eukaryotic phenomenon in which RNA polymerase II pauses shortly after initiation, facilitating the induction of elongation upon phosphorylation by positive transcription elongation factor b (P-TEFb). P-TEFb is, in turn, regulated through association with the 7SK RNA (7SK) to form an inhibitory 7SK ribonucleoprotein (RNP) complex1. When P-TEFb dissociates from 7SK, the RNA forms another set of RNPs with various heterogenous nuclear ribonucleoproteins (hnRNPs), such as hnRNP A1. Here we show biochemically that the RNA recognition motifs of hnRNP A1 (UP1) bind to Stem III of 7SK with ~300 nM affinity. We have found that a base-proximal site is critically important for initiating this interaction. UP1 binding causes a conformational rearrangement in Stem III, exposing sites on the opposite side of the stem. Stem III also binds serine-arginine rich splicing factor 2 (SRSF2), a natural splicing antagonist of hnRNP A1, in the 7SK-P-TEFb RNP2. Together with our results, this suggests a possible mechanism by which Stem III assumes a distinct conformation in each of its RNPs, and the conformational changes arising from protein binding may be important for the switch between the 7SK-P-TEFb RNP and the 7SK-hnRNP RNP. Future work will examine the binding of SRSF2 to Stem III as well as the interplay between SRSF2 and UP1 on Stem III.

References:
1. Guo, J. and D.H. Price, RNA polymerase II transcription elongation control. Chem Rev, 2013. 113(11): p. 8583-603.
2. Ji, X., et al, SR Proteins Collaborate with 7SK and Promoter-Associated Nascent RNA to Release Paused Polymerase. Cell, 2013. 153: p. 855-868

Keywords: 7SK RNA, hnRNP A1, SRSF2

136. Identification of cap methyltransferase as a component of the cytoplasmic capping complex

Jackson B. Trotman (Molecular and Cellular Biochemistry, The Ohio State University), Chandrama Mukherjee (Molecular and Cellular Biochemistry, The Ohio State University), Daniel R. Schoenberg (Molecular and Cellular Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

137. Structure probing of the htrA RNA thermometer from Salmonella enterica

Kelsey A. Ulanowicz (Denison University, Chemistry & Biochemistry ), Casey B. Cempre (Denison University, Chemistry & Biochemistry ), Dr. Rachel M. Mitton-Fry (Denison University, Chemistry & Biochemistry )

Abstract not available online - please check the printed booklet.

138. Bioinformatics analysis of human mRNA coding sequences to identify putative G-quadruplex forming sequences

Aparna Venkataraman (Department of Chemistry & Biochemistry, Kent State University), Gayan Mirihana Arachchilage, Debmalya Bhattacharyya , Soumitra Basu (Department of Chemistry & Biochemistry, Kent State University, Kent, OH)

Abstract:
G-quadruplexes (GQs) are non-canonical secondary structures consisting of stacked G- quartets, which are planar structures of four guanine bases, linked via Hoogsteen hydrogen bonding. GQs are found widespread in various types of RNA, including mRNA, long noncoding RNA, telomeric RNA, and viral genomic RNA. Consequently, RNA GQs are known to play vital roles in RNA biology. G-quadruplexes present within the 5’-UTRs of mRNA have been shown to serve a critical function in translational modulation. While 5’-UTR GQs have been previously identified using bioinformatics approaches, GQs within the coding regions of mRNAs have yet to be analyzed. Here we present our findings on the potential of GQ formation within the coding sequence (CDS) through multi-pronged computational analyses. Our initial screening indicates a sizeable number of putative GQ forming sequences within the CDS. We will also determine codon usage bias against the formation of G-quadruplexes within the CDS of human mRNAs. Given that the presence of secondary structures, including GQs, within the CDS would have a deleterious effect on translation, we believe that this study will provide new insight into an additional regulatory mechanism in protein synthesis.

Keywords: coding sequence, G-quadruplex, bioinformatics

139. Purification and characterization of cytidine-specific ribonuclease, Cusativin, for mapping nucleoside modifications in RNA

Sarah Venus (Department of Chemistry, University of Cincinnati), Patrick Limbach (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati)

Abstract:
A cytidine-specific ribonuclease, known as Cusativin, is partially purified and characterized from cucumber (Cucumis sativus) seeds. The purification involves conventional biochemical procedures initially described by Girbés and coworkers (1). The presence of the target protein in the purified fractions was verified by its size on a denaturing polyacrylamide gel (SDS-PAGE) at different stages of purification. A 25kDa polypeptide was consistently observed on the denaturing gel following gel filtration and ion exchange chromatographic steps of purification. This partially purified protein exhibited ribonuclease activity when incubated with RNA. The digestion products were analyzed further by liquid chromatography coupled with mass spectrometry (LC-MS) to understand the specificity of RNA cleavage. The data related to nucleoside specificity and the ability of this enzyme to map nucleoside modifications in RNA will be presented. This base-specific ribonuclease can be a useful tool for mapping nucleoside modifications in RNA such as tRNA and rRNA.

References:
Rojo MA, et al. (1994) Planta 194:328-338.

Keywords: ribonuclease, cusativin, LC-MS

140. Structure, Conformations, and Thermodynamics of the Three-way Junction (3WJ) of Phi29 pRNA

Mario Vieweger (Nanobiotechnology Center, Markey Cancer Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA), Hui Zhang (Nanobiotechnology Center, Markey Cancer Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA), Dan Shu (Nanobiotechnology Center, Markey Cancer Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA), Peixuan Guo (Nanobiotechnology Center, Markey Cancer Center, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA)

Abstract not available online - please check the printed booklet.

141. Translation rate evaluation with ribosome profiling data

Hao Wang (Lane center for computational biology, Carnegie Mellon University), Joel McManus (Department of Biological Science, Carnegie Mellon Univeristy), Carl Kingsford (Lane center for computational biology, Carnegie Mellon University)

Abstract:
Ribosome profiling is a recently developed sequencing technique that provides approximately 30-base sequences that are protected by ribosomes translating a transcript. Because it measures activity at translation, we can use it to more directly measure protein abundance and understand buffering or amplification of differences in transcription. The challenge with this data is mapping short sequences to isoforms to get an isoform-level estimation of ribosome positions. We are developing a method that combines a model of ribosome motion with short-read mapping in an expectation-maximization framework to estimate isoform-specific ribosome positions. Using this approach, we will be able to estimate sequence-specific rates of ribosome motion and to relate transcript abundance with translational efficiency.

References:
1. Ingolia, N. T. et al. (2009). science, 324(5924), 218-223.
2. Guo, H. et al.(2010). Nature,466(7308), 835-840.
3. Reuveni, S. et al. (2011). PLoS comp. bio., 7(9), e1002127.

Keywords: ribosome profiling, translation rate, translation model

142. Probing the effect of different ligands on the conformational dynamics of a transcriptionally acting preQ1 riboswitch using single molecule FRET microscopy

Jiarui Wang (Department of Chemistry, University of Michigan), Krishna Chaitanya Suddala (Department of Biophysics, University of Michigan)

Abstract:
Non-coding RNAs play crucial roles in a multitude of biological processes such as translation, messenger RNA (mRNA) splicing and regulation of gene expression. Riboswitches are regulatory elements found mainly in the 5’ untranslated regions of numerous bacterial mRNAs capable of modulating gene expression in response to the binding of cellular metabolites. Riboswitches
comprise two functional components: a ligand binding aptamer domain and an expression platform whose conformation decides the fate of gene expression. Despite its smallest size among all reported aptamers, the Bacillus subtilis (Bsu) preQ1 (pre-queuosine) riboswitch boasts precise control of the expression of genes involved in the biosynthesis of queuosine. The co-crystal structure of the Bsu aptamer domain bound to its ligand preQ1 showed a pseudoknot structure. However, the nature of folding pathway remained elusive, until recently. We have recently used single molecule fluorescence resonance energy transfer (smFRET) and computational simulations to show that the ligand-free Bsu aptamer adopts a pre-folded conformation in which the single stranded A-rich tail interacts with stem-loop P1-L1. Previous studies indicated that the Bsu riboswitch binds closely related ligands preQ0 and guanine with similarly favorable affinities. The effects of these ligands on the conformational dynamics, however, were not previously probed and can reveal to what extent near-cognate ligands stabilize the folded state to affect gene regulation. Here, we use smFRET to study the effects of preQ0 and guanine in comparison to preQ1 on the kinetics of Bsu aptamer folding. Our preliminary data show that at saturating concentration and in the presence of Mg2+, guanine stabilizes the folded state as potently as preQ0. However, surprisingly, in the absence of Mg2+, guanine tends to stabilize the folded state more efficiently than preQ0. We anticipate this comparative study to elucidate the ligand-mediated folding pathway of the Bsu aptamer.

Keywords: preQ1 riboswitch, Kinetics

143. An interaction with Nanos enhances repression by Pumilio and broadens mRNA target specificity

Chase Weidmann (Biological Chemistry, University of Michigan), Aaron Goldstrohm (Biological Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

144. Dissecting the functions of RNA helicases in splicing by single-molecule FRET

Julia R. Widom (Department of Chemistry, University of Michigan), Matthew L. Kahlscheuer (Department of Chemistry, University of Michigan), Nils G. Walter (Department of Chemistry, University of Michigan)

Abstract:
The process of splicing is a critical step in gene expression, and defects in the splicing pathway are associated with many human genetic diseases. The spliceosome is unlike many macromolecular machines in that it lacks a pre-formed catalytic core and is built through sequential steps of assembly and rearrangement on the template of the pre-mRNA. I will present recent and ongoing work in which single-molecule fluorescence resonance energy transfer (smFRET) was applied to the spliceosome, allowing conformational changes in the pre-mRNA to be monitored in real time. I will describe previous experiments that used the technique of single-molecule pulldown FRET (SiMPull-FRET) to isolate complexes stalled before the first chemical reaction of splicing. I will also describe ongoing work investigating the roles of RNA helicases and ATPases in structural rearrangements of the spliceosome. Prp22 is an RNA helicase found in yeast (with a human homologue, HRH1) that is required for the release of the mRNA product after the second chemical reaction of splicing is complete. I will present experiments in which smFRET is used to investigate the unwinding activity of Prp22 on model substrates, with the goal of understanding how this unwinding activity is functionally utilized in the spliceosome. The study of Prp22 will be extended to the spliceosome using SiMPull-FRET, allowing us to isolate from yeast extract spliceosomal complexes that have been stalled after the second step of splicing by a dominant negative mutation in Prp22. This will permit investigation of the effects of Prp22 on mRNA dynamics after both steps of splicing and during mRNA release.

References:
1. Krishnan, R., Blanco, M. R., Kahlscheuer, M. L., Abelson, J., Guthrie, C. & Walter, N. G. Biased Brownian racheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nat. Struct. Mol. Biol. 20, 1450–1457 (2013).
2. Schwer, B. & Gross, C. H. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 17, 2086–2094 (1998).

Keywords: splicing, helicase, single-molecule

145. mRNA-mediated cooperativity between microRNA target sites

Natalie Hanisch (Department of Mathematics, University of Nebraska, Kearney), Michael Widom (Department of Physics, Carnegie Mellon University)

Abstract:
MicroRNAs downregulate expression of targeted genes. Experimental and bioinformatic evidence suggests multiple target sites on a common gene can cooperatively enhance downregulation[1,2], but the precise mechanism of cooperation is not known. Recent theoretical studies suggest target site interactions can be mediated through the RNA sequence of the gene itself[3]. We confirm the presence of both positive and negative cooperativity for mammalian microRNA targets, with a distance-dependent weighting towards positive cooperativity (enhanced binding affinity). We show that positivity of cooperativity is greater for well-conserved target sites than for poorly conserved ones.

References:
[1] A. Grimson, et al., "MicroRNA targeting specificity in mammals: determinants beyond seed pairing", Mol. Cell 27 (2007) 91-105
[2] P. Saetrom, et al., "Distance constraints between microRNA target sites dictate efficacy and cooperativity", Nucl. Acids Res. 35 (2007) 2333-42
[3] Y.-H. Lin and R. Bundschuh, "Interplay between single-stranded binding proteins on RNA secondary structure", Phys. Rev. B 88 (2013) 052707

Keywords: microRNA, cooperativity

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

Tanner P. Brondhaver (Chemistry Department Georgetown University), Russell Williams (Chemistry Department University of Louisville), Eugene G. Mueller (Chemistry Department University of Louisville)

Abstract not available online - please check the printed booklet.

147. 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), Medha Raina (Department of Microbiology and Center for RNA Biology, The Ohio State University), Michael Ibba (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.

148. Structure and mechanism of a riboswitch that selectively responds to transition metals

Arati Ramesh (The University of Maryland), Zhiyuan Zhou (Yale University), Kazuhiro Furukawa (Yale University), Ronald Breaker (Yale University)

Abstract not available online - please check the printed booklet.

149. The significance of quaternary structure for catalysis by the Human Thg1 enzyme

Michael Wolfe (Ohio State University Biochemistry Department), Jikang Wang (Ohio State University Biochemistry Department), Akiko Tanimoto (Ohio State Univeristy Biochemistry Department), Vicki Wysocki (Ohio State University Biochemistry Department), William Eberley (Ohio State University Biochemistry Department), Jane Jackman (Ohio State University Biochemistry Department)

Abstract not available online - please check the printed booklet.

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

Eileen Workman (Department of Molecular and Cellular Biochemistry, Center for RNA Biology, Wexner Medical Center at the Ohio State University), Caitlin Kalda (Department of Molecular and Cellular Biochemistry, Center for RNA Biology, Wexner Medical Center at the Ohio State University), Aalapi Patel (Department of Molecular and Cellular Biochemistry, Center for RNA Biology, Wexner Medical Center at the Ohio State University), Daniel J. Battle (Department of Molecular and Cellular Biochemistry, Center for RNA Biology, Wexner Medical Center at the Ohio State University)

Abstract not available online - please check the printed booklet.

151. The identification of inhibitors against Ribonuclease P to probe tRNA processing in vivo

Nancy Wu (Program in Chemical Biology, University of Michigan), Xin Liu (Department of Chemistry, University of Michigan), Avi Raveh (Life Sciences Institute, University of Michigan), David H. Sherman (Department of Medicinal Chemistry, College of Pharmacy, Life Sciences Institute, University of Michigan), Carol A. Fierke (Department of Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

152. Genomics Research Core: Gene Expression Service Projects

Dr. Janette Lamb (Assistant Director, Genomics Research Core, University of Pittsburgh), Deborah J Hollingshead (Genomics Manager, Genomics Research Core, University of Pittsburgh)

Abstract:
The Genomics Research Core (formerly the GPCL) is one of the five Health Sciences Core Research Facilities and offers cost-effective and quality controlled, high throughput genomics services to University of Pittsburgh researchers. In addition to our role in the Health Sciences Core Research Facilities (HSCRF) we are a resource of the University of Pittsburgh Clinical and Translational Science Institute (ctsi). Although the majority of our investigators are involved in health research, we assist with conservation, environmental, basic science, translational, clinical and educational studies. Here we outline a few of the projects for which we have recently provided research services, using three of the many genomics platforms that are available in our facility.

References:
1. Pociask et al. IL-22 Is Essential for Lung Epithelial Repair following
Influenza Infection. Am J. Pathol. 2013; 182, 4, 1286-1296
2. Blazer et al. Monitoring of Wild Fish Health at Selected Sites in the
Great Lakes Basin Methods and Preliminary Results: U.S.Geological Survey
Open-File Report 20141027 p.31,http://dx.doi.org/10.3133/ofr20141027
3. Ambrose et al. Dysregulation of multiple inflammatory molecules in lymph
node and ileum of macaques during RT-SHIV infection with or without
antiretroviral therapy. J Med. Primatol. 2014; 43, 5, 298-309

Keywords:

153. NusG is a sequence-specific RNA polymerase pause factor

Alexander V. Yakhnin (Dept. of Biochemistry and Molecular Biology, Pennsylvania State University), Paul Babitzke (Dept. of Biochemistry and Molecular Biology, Pennsylvania State University)

Abstract:
NusG, which is referred to as Spt5 in archaeal and eukaryotic organisms, is the only transcription factor conserved in all three domains of life. This transcription elongation factor binds to RNA polymerase (RNAP) and assists in DNA-templated RNA synthesis. Data obtained from studies of E. coli NusG (NusGEc) indicate that this protein is capable of increasing transcription processivity by suppressing RNAP pausing. In contrast to E. coli, B. subtilis NusG (NusGBs) dramatically stimulates pausing at two sites in the untranslated leader of the B. subtilis trp operon. These two regulatory pause sites participate in transcription attenuation and translational control mechanisms, respectively. Transcription elongation complexes reconstituted in vitro with nucleic acid scaffolds revealed that NusGBs makes sequence-specific contacts with a T-rich sequence in the non-template DNA strand within the paused transcription bubble. NusGBs protects T residues of the recognition sequence from permanganate oxidation and the degree of protection correlates with the pause-simulation activity. The amino acid sequences of two short regions within the N-terminal domain of NusGBs are primarily responsible for specific recognition of the trp pause signals. The finding that these amino acid sequences are not conserved in NusGEc explains why the E. coli protein is not capable of stimulating pausing at the B. subtilis trp pause sites. E. coli also contains a NusG paralog called RfaH, which is recruited to the transcription elongation complex through recognition of the ops sequence in the non-template DNA strand. The ops site-recognizing amino acid residues of RfaH (Belogurov et al., 2010) are located in the same regions that we identified for NusGBs. Our results suggest that recognition of specific sequences in non-template DNA and stimulation of RNAP pausing is a conserved function of NusG/Spt5-like transcription factors.

References:
Belogurov, G.A., Sevostyanova, A., Svetlov, V., and Artsimovitch, I. (2010) Functional regions of the N-terminal domain of the antiterminator RfaH. Mol. Microbiol. 76:286-301.

Keywords: transcription factor, RNAP pausing, non-template DNA

154. The Molecular Etiology of PRP Mutations in Autosomal Dominant Retinitis Pigmentosis (RP) Mutations

Kevin Yang (University of Chicago), Klaus Nielsen (University of Chicago), Jon Staley (University of Chicago)

Abstract not available online - please check the printed booklet.

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

Yueqin Yang (University of Pennsylvania, Genetics, Philadelphia, PA, 19104), Thomas Bebee (University of Pennsylvania, Genetics, Philadelphia, PA, 19104), Juw Won Park (University of California, Los Angeles, Microbiology, Immunology, and Molecular Genetics, Los Angeles, CA, 90095), Shihao Shen (University of California, Los Angeles, Microbiology, Immunology, and Molecular Genetics, Los Angeles, CA, 90095), Yi Xing (University of California, Los Angeles, Microbiology, Immunology, and Molecular Genetics, Los Angeles, CA, 90095), Russ Carstens (University of Pennsylvania, Genetics, Philadelphia, PA, 19104)

Abstract not available online - please check the printed booklet.

156. Characterization of 3’ to 5’ exonuclease activity of Pop2p from Saccharomyces cerevisiae

Xuan Ye (Center for RNA Molecular Biology & Department of Biochemistry,Case Western Reserve University), Eckhard Jankowsky (Center for RNA Molecular Biology & Department of Biochemistry,Case Western Reserve University)

Abstract not available online - please check the printed booklet.

157. Molecular modulation of T box riboswitch function

Chunxi Zeng (Ohio University), Shu Zhou, Jennifer V. Hines (Ohio University)

Abstract:
The T box riboswitch is a transcription regulation mechanism that controls expression of essential genes in many bacteria. Uncharged cognate tRNA induces read-through (antitermination) of the T box riboswitch and allows expression of downstream genes. Two fluorescently labeled probes were designed to study the riboswitch in real time during multi-round in vitro transcription of the leader region of the glyQS gene. The read-through probe showed good photostability, integrity and target sensitivity. Specific riboswitch response to uncharged cognate tRNAgly was detected with a low nanomolar EC50. Using this validated, moderate throughput riboswitch functional assay, the effect of molecular modulators of T box riboswitch function was then determined.

References:
Green, N.J., et al. (2010) The T box mechanism: tRNA as a regulatory molecule. FEBS Lett., 584, 318–24.
Grundy, F.J., et al. (2002) tRNA-mediated transcription antitermination in vitro: codon-anticodon pairing independent of the ribosome. PNAS, 99, 11121–6.
Putzer, H., et al. (2002) Transfer RNA-mediated antitermination in vitro. Nucleic Acids Res., 30, 3026–33.
Tsourkas, A., et al. (2002) Hybridization of 2’-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets. Nucleic Acids Res., 30, 5168–5174.
Nakano, S., et al. (2009) Facilitation of RNA enzyme activity in the molecular crowding media of cosolutes. J. Am. Chem. Soc., 131, 16881–8.

Keywords: T box riboswitch, molecular beacon, in vitro transcription

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

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

Abstract not available online - please check the printed booklet.

159. Functional characterization of derivatives of the aminoglycoside tobramycin

Hongkun Zhu (Department of Microbiology, The Ohio State University, Columbus, OH, 43210, USA), Keith D. Green (Department of Pharmaceutical Sciences, University of Kentucky, Lexingon, KY, 40536, USA), Sylvie Garneau-Tsodikova (Department of Pharmaceutical Sciences, University of Kentucky, Lexingon, KY, 40536, USA), Kurt Fredrick (Department of Microbiology, The Ohio State University, Columbus, OH, 43210, USA)

Abstract:
Emergence of resistance to antibiotics has become a serious problem in health care. In efforts to find ways to evade the resistance mechanisms, numerous derivatives of the aminoglycoside tobramycin (TOB), substituted at the 6” position of ring III, were made. Here, these TOB variants are characterized with respect to their mode of action, by employing 16S rRNA mutation A1408G, which lies at the primary aminoglycoside binding site in helix h44 of the small subunit. We tested the ability of each to inhibit (i) growth of E. coli strain Δ7 prrn (WT), (ii) growth of aminoglycoside- resistant E. coli strain Δ7 prrn (A1408G), (iii) translocation in WT ribosomes, and (iv) translocation in A1408G ribosomes. Based on the data obtained, the variants fall into different functional classes. Those in class A, like the parental TOB, act through the primary h44 site of ribosome. Some of them are potent inhibitors and may be potential candidates for clinical use. Other derivatives gain novel properties compared to parental tobramycin. For example, compounds 3,4,5,11,16,17 inhibit AMV reverse transcriptase, while compound 6 can promote mRNA secondary structure formation in the absence of ribosomes. A subset of compounds cause cell lysis (1), implicating the membrane as a cellular target in these cases. These data provide insight into the inhibition of translation and growth by aminoglycosides antibiotics.

References:
I. M. Herzog, K. D. Green, Y. Berkov-Zrihen, M. Feldman, R. R. Vidavski, A. Eldar-Boock, R. Satchi-Fainaro, A. Eldar, S. Garneau-Tsodikova, and M. Fridman, Angew. Chem. Int. Ed.2012, 51, 5652.

Keywords: aminoglycoside, ribosome, h44

160. The C-termini of Symplekin, CPSF73 and CPSF100 interact in the pre-mRNA 3' end processing core cleavage factor

Daniel Michalski (Department of Biology, University of Missouri-St. Louis), Mindy Steiniger (Department of Biology, University of Missouri-St. Louis)

Abstract:
A core cleavage complex (CCC) consisting of CPSF73, CPSF100 and Symplekin is required for 3’ end processing of all metazoan pre-mRNAs. We are characterizing in vivo molecular interactions in the CCC required for both assembly and catalysis in Drosophila. To understand which regions of the CCC components are required for complex assembly, we created stably transfected Dmel-2 cell lines expressing full-length (FL), N- and C-terminally truncated CCC factor proteins. IPs show that FL proteins are successfully incorporated into CCC containing endogenous binding partners. IP of N- and C-terminal deletion mutants reveals that the C-termini of Symplekin and CPSF73/100 are required for CCC formation. Analysis of mutant CCC activity is also being investigated. First, endogenous proteins are RNAi-depleted and mutant complexes are created with RNAi-resistant proteins. The 3’ ends of endogenous H2A mRNA from these cells are then mapped. Results indicate that the activity of Symplekin C-terminal mutant complexes correlates with their ability to form CCC. Interestingly, when only the N-terminal 271 aa of Symplekin are deleted, H2A 3’ end misprocessing is observed even though this mutant recruits CPSF73/100 more efficiently than FL Symplekin. We hypothesize that removal of the N-terminal region of Symplekin inhibits binding to the RNA pol II CTD. This reduces the local CCC concentration and causes misprocessing. We also observed partial rescue of the 3’ end processing defect when CCC include only the 200 C-terminal aa of CPSF73. This CPSF73 mutant recruits Symplekin and CPSF100 as well as FL CPSF73, but these CCC lack the CPSF73 active site. We hypothesize that including the C-terminal 200 aa of CPSF73 reduces co-depletion of endogenous Symplekin and CPSF100. Therefore, the small amount of FL CPSF73 unaffected by RNAi-depletion can form functional CCC. While these experiments are ongoing, we have already gained significant insight into CCC molecular interactions and activity.

Keywords: pre-mRNA 3 end processing, protein-protein interactions, Drosophila

161. Elucidation of the protein-RNA interactions that dictate human T-lymphotropic virus type-1 viral genomic RNA packaging

Weixin Wu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), William A. Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH 43210)

Abstract:
In most retroviruses, viral genomic RNA (vRNA) packaging is mediated by a specific interaction between the nucleocapsid (NC) domain of the viral Gag polyprotein and vRNA psi (Ψ) packaging signals. Recent studies have shown that the matrix (MA) domain of Gag also plays a role in vRNA packaging (1). In HIV-1, NC is critical for selective packaging of vRNA over various other cellular RNAs. By comparison, the mechanisms of vRNA packaging in deltaretroviruses, which include bovine leukemia virus (BLV), human T-lymphotropic virus type-1 and -2 (HTLV-1 and -2), are less clear. Previous studies showed that both BLV Gag NC and MA domains are involved in vRNA packaging (2). Our recent results support an important role of HTLV-2 MA in nucleic acid binding (3). Therefore, we have proposed that deltaretroviruses may use a different vRNA packaging mechanism from that of HIV-1 in which MA plays a larger role in vRNA selection at the initial stage. This project aims to understand the HTLV-1 MA-vRNA interactions that are responsible for selective vRNA interaction and packaging. We have prepared and optimized the purification of recombinant HTLV-1 MA. Fluorescence anisotropy binding assays reveal that HTLV-1 NC bound to a non-specific 20-mer DNA oligonucleotide (Kd = 230 nM), but failed to bind to RNA stem loops SL1 or SL2 derived from the putative vRNA packaging signal. In contrast, HTLV-1 MA binds with moderate affinity to ssDNA, SL2 and SL1-SL2 (Kd = 1.1 μM, 1.9 μM and 1.7 μM, respectively); no binding was detected to SL1 alone. These results suggest that HTLV-1 MA has moderate nucleic acid binding ability and may play a role in specific vRNA interaction and selection. Salt-titration and competition-binding studies are underway to examine binding specificity. We are also currently mapping the interactions between HTLV-1 proteins and larger vRNA fragments encompassing the full-length 5’-UTR (nt 1-448) and upstream gag (nt 449-624), which are the likely locations of Ψ.

References:
1. Parent, L. J.; Gudleski, N., Beyond plasma membrane targeting: role of the MA domain of Gag in retroviral genome encapsidation. J Mol Biol 2011, 410 (4), 553-64.
2. Wang, H.; Norris, K. M.; Mansky, L. M., Involvement of the matrix and nucleocapsid domains of the bovine leukemia virus Gag polyprotein precursor in viral RNA packaging. J Virol 2003, 77 (17), 9431-8.
3. Sun, M.; Grigsby, I. F.; Gorelick, R. J.; Mansky, L. M.; Musier-Forsyth, K., Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging. J Virol 2014, 88 (2), 1271-80.

Keywords: Deltaretrovirus, HTLV-1 , MA

162. Running out of rice?: Effects of Environmental Stresses on Gene Expression in Rice

Marissa L. Gentle (Department of Chemistry, Pennsylvania State University), Laura Richey (Department of Chemistry, Pennsylvania State University), Myasia Cherry (Department of Chemistry, Pennsylvania State University), El-Isha Battle (Department of Chemistry, Pennsylvania State University), Zhao Su (Department of Chemistry, Pennsylvania State University), Sarah M. Assmann (Department of Biology, Pennsylvania State University)

Abstract:
Running out of rice?: Effects of Environmental Stresses on Gene Expression in Rice
Marissa L. Gentle, Laura Richey, Myasia Cherry, El-Isha Battle,
Zhao Su, Sarah M. Assmann, Phillip C. Bevilacqua
SEECoS Mentor/ Undergraduate Research Summer 2014
Department of Chemistry, Pennsylvania State University, University Park, PA 16802

Abstract

We are all affected by the production and yield of rice. Rice feeds over half the world’s population consumption by humans or livestock. With our rising population, the rice yield that farmers are producing will not be enough to sustain humanity in thirty years time. We are studying rice because of changing environmental conditions that are moving us into a worldwide rice shortage. Our research focuses on understanding genes that have various regulation patterns under stress conditions (e.g. heat, cold, salt, drought). This will help us further comprehend which environmental stresses rice plants are most responsive to. Gene expression is best studied by determining RNA levels. To do this, we obtained RNA from rice seedlings, performed reverse-transcription to create cDNA, and ran PCR (polymerase chain reaction) and qPCR (quantitative polymerase chain reaction) to amplify the DNA. qPCR is primarily a technique to amplify the RNA present in a stressed sample and quantify how much RNA is present. Data were recorded for two different genes that encode cold shock proteins. For gene one, there was no obvious regulation under the stressed conditions tested. For gene two, there was an up-regulation under high salt and high temperature conditions. In the future we hope to look at different stresses applied to rice strains and different genes encoding cold shock proteins.


Keywords: