Rustbelt RNA Meeting 2017

Poster abstracts

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

Hiba Al-Ashtal (Biological Sciences, University of Pittsburgh), Courtney Rubottom (Biological Sciences, University of Pittsburgh), Dr. Andrea J. Berman (Biological Sciences, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

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

Lama AlAbdi (Biochemistry department, Purdue University), Ming He (Biochemistry department, Purdue University), Allison B. Norvil (Biochemistry department, Purdue University), Christopher J. Petell (Biochemistry department, Purdue University), Humaira Gowher (Biochemistry department, Purdue University)

Abstract not available online - please check the printed booklet.

3. Polyamine modulation of T-box riboswitch function for antibacterial drug discovery

Ali H. Aldhumani (Department of Chemistry & Biochemistry, Ohio University, Athens Ohio), Hannah Cherry (Department of Chemistry & Biochemistry, Ohio University, Athens Ohio), Jennifer V. Hines (Department of Chemistry & Biochemistry, Ohio University, Athens Ohio)

Abstract:
Aliphatic amine and polyamine cations are known to be bioavailable in high concentrations in the presence of both DNA and RNA implying a nonspecific interaction. However, there is growing evidence that biogenic amines may serve a specific structural role by stabilizing RNA through the formation of nucleotide- and structure-specific interactions. The effect of amines and polyamines on the function of the T-box riboswitch will be presented. The T-box riboswitch is typically found in the 5′-noncoding region of Gram-positive bacterial mRNAs where it regulates gene expression in response to cognate tRNA aminoacylation ratios. Previous studies indicate that biologically unfeasible levels of Mg2+ are required for the T-box riboswitch to optimally function in vitro. Our results show that in the absence of Mg2+, biogenic amines spermidine, spermine, and cadaverine can compensate to facilitate binding of the tRNA to the T-box riboswitch antiterminator element. There is a reduced dependency on Mg2+ with the tRNA-antiterminator complex forming at even 0 mM Mg2+ when in the presence of these polyamines.

Identification and characterization of amines as potential co-factors or modulators of the T-box riboswitch will assist in the development of a novel potential drug pharmacophore as well as a more thorough understanding of the druggability of this riboswitch. Due to the increased resistance to antibiotics, it has become imperative to investigate novel drug targets such as the T-box riboswitch. Based on the observed agonist activity with the biogenic amines, we have designed a series of cationic peptides to potentially identify an inhibitor of the riboswitch. The peptides were designed based on molecular docking studies and one peptide has been identified which modulates tRNA binding to the antiterminator and also in vitro transcription function of the T-box riboswitch.

Keywords: Polyamine, Peptide, T-box Riboswitch

4. Novel AD risk genes identified through a tissue-specific transcriptome-wide differential expression and co-expression analysis

Prabhakar Sairam Andhey (Department of BioHealth, IUPUI), Jingwen Yan (Department of BioHealth, IUPUI)

Abstract:
Alzheimer’s disease (AD) is the most type of brain dementia characterized by gradual impairment of cognitive functions and it usually starts in the hippocampus, deep within and part of the temporal lobe of the brain. Understanding the biological alterations inside these brain regions are essential for more insights of disease mechanism. In this study, we analyzed the gene expression profiles collected in temporal cortex of both AD and health controls to investigate the tissue-specific transcriptional changes associated with AD. By adjusting for age, gender, RIN and post mortem interval (PMI) effect, 39 genes ( >2 fold changes) were identified to be differentially expressed between AD patients and normal controls with FDR corrected p value smaller than 0.05. Top ones include NPNT (p=3.1e-17), VGF (p=2.28e-09), SLC7A2 (p=4.51e-9), AC026691.1 (p=1.51e-8) and METTL7B (2.63e-8). When adding APOE group as an extra covariate, 24 out of 39 genes remain to be statistically significant. We further map these genes to Reactome functional interaction network and found that many of them are located very closely to known AD-risk genes, e.g. APOE, CLU, BIN1, CD40 and PICALM. This study paves the way for investigating the tissue-specific biological activities underlying the progression of AD.

References:
1.Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics. 2009, 25(8):1091-3.

Keywords: Alzheimer disease, differential expression, network analysis

5. Choosing representatives for Equivalence Classes of RNA structures and development of new visualization features for RNA 3D structure annotation and analysis

Sri D. Appasamy (Department of Biological Sciences, Bowling Green State University), Blake Sweeney (European Bioinformatics Institute), Jamie J. Cannone (Center for Computational Biology and Bioinformatics, University of Texas at Austin), Craig L. Zirbel (Department of Mathematics and Statistics, Bowling Green State University), Neocles B. Leontis (Department of Chemistry, Bowling Green State University)

Abstract:
The number and diversity of new RNA structures deposited in the Protein Data Bank (PDB) is rapidly increasing in response to interest generated by discoveries of new functional roles for RNA molecules. Mining the new structures for novel RNA folds, 3D motifs, and protein interactions requires identifying the best structures for each distinct RNA, but presently PDB provides no filtering mechanism to help users. In collaboration with Nucleic Acid Database (NDB), we have developed an automated, weekly pipeline to compare all RNA chains from each PDB file, group them by sequence and geometry in “Equivalence Classes” (EC), and select high-quality representatives for each EC to populate reduced-redundancy “representative” sets of RNA 3D structures (http://rna.bgsu.edu/rna3dhub/nrlist), useful for searching and RNA 3D motif extraction. We have improved the selection methods to provide greater stability and take advantage of multiple quality data, including structure completeness, resolution, clash scores, and Real Space Refinement (RSR) statistics.
We are integrating new visualizations on our website (http://rna.bgsu.edu). Heat maps have been added to display structural similarity within each EC group. Annotated pairwise interactions and RSR values can be visualized on 2D structures for selected molecules. Several new features will be made available to the interactive molecule viewer in our web applications; i) displaying steric clashes in the 3D motifs, ii) displaying variable extent of neighboring regions based on distance entered by the user, and iii) coloring 3D motifs by different criteria (e.g. chain, CPK, structure quality indicators such as Real Space Refinement statistics). Specific examples will be given to show the utility of the different visualization features for facilitating the annotation and analysis of RNA 3D motifs.

Keywords: Equivalence classes, RNA 3D motifs

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

Waqar Arif (University of Illinois at Urbana Champaign), Raiya Sarwar (Carle Foundation Hospital), Andrew Batey (Carle Foundation Hospital), Suthat Liangpunsakul (Indiana University School of Medicine), Auinash Kalsotra (University of Illinois at Urbana Champaign)

Abstract not available online - please check the printed booklet.

7. Determination of the roles of Trm7, Trm732, and Trm734 orthologs in 2’-O-methylation of human and fruit fly tRNA

Regan O. Bales (Chemistry, Northern Kentucky University), Justen B. Mamaril (Chemistry, Northern Kentucky University), Michael P. Guy (Chemistry, Northern Kentucky University)

Abstract not available online - please check the printed booklet.

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

Zahra Batool (Department of Biological Sciences, University of Illinois at Chicago), Yury Polikanov (Department of Biological Sciences, University of Illinois at Chicago)

Abstract not available online - please check the printed booklet.

9. Identification and characterization of S-box riboswitches that regulate at the level of translation initiation

Divyaa Bhagdikar (Department of Microbiology and Center for RNA biology , The Ohio State University, Columbus, OH 43210 ), Frank J. Grundy (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus, OH 43210 ), Tina M. Henkin (Department of Microbiology and Center for RNA biology , The Ohio State University, Columbus, OH 43210 )

Abstract not available online - please check the printed booklet.

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

Abigail Bieker (Biology, Lewis University), Elizabeth Przekwas (Biology, Lewis University), Mallory Havens (Biology, Lewis University)

Abstract not available online - please check the printed booklet.

11. A computational framework for classifying RNA-binding proteins based on their preference for RNA structural motifs

Arun Kumar Boddapati (BioHealth Informatics and Indiana University-Purdue University), Sarath Chandra Janga (BioHealth Informatics and Indiana University-Purdue University)

Abstract:
RNA molecules are composed of conserved sequences or structural components. RNA molecules with structural components form 3D structures and contain secondary structures which are recognized as structural motifs. For instance, the secondary structures can be hairpin loops, helices, internal loops, bulges and junction loops. RNA Binding Proteins (RBPs) predominantly recognizing these “structural elements” can be categorized as structure recognizing RBPs while those exhibiting a preference for sequences can be classified as sequence binding RBPs.
RNA binding proteins are components of the post-transcriptional mechanism of the cell which binds to RNA by recognizing motifs on RNA. Although several algorithms have been developed to identify the RNA recognition motifs of an RBP, most approaches assume either a sequence or structural preference of an RBP. In this study, we adopt an unbiased approach to classify 172 RBPs based on their experimentally known CLIP-sequencing binding site profiles into sequence, structure or their preference to recognize both. Our approach employs BEAM [1] (BEAr Motif finder) tool together with BEAR[2] encoding of RNA motifs and RNAfold[3] tools to find conserved structural motifs across known RNA binding sites from various CLIP and eCLIP datasets in the public domain.
The RBPs were categorized into sequence recognizing and structure recognizing proteins according to a newly proposed Motif Structure Score (MSS). The score considers the coverage of the conserved sequences across the top ten most significant motifs recognized from the output of BEAM and assigns a value from 0 to 1 to determine the sequence to structure recognition preference of an RBP. Based on the distribution of Motif Structure Score, we classified the RBPs above a threshold of 0.6 as structure recognizing and while those below 0.4 as sequence recognizing.
Such an unbiased classification of RBPs based on available CLIP profiles can provide an ensemble of recognition profiles recognized by RBPs to enable a deeper understanding of their sequence/structural binding space along with their combinatorial regulatory mechanisms.

References:
1. Pietrosanto, M., et al., A novel method for the identification of conserved structural patterns in RNA: From small scale to high-throughput applications. Nucleic acids research, 2016. 44(18): p. 8600-8609.
2. Mattei, E., et al., A novel approach to represent and compare RNA secondary structures. Nucleic acids research, 2014. 42(10): p. 6146-6157.
3. Hofacker, I.L., RNA secondary structure analysis using the Vienna RNA package. Current protocols in bioinformatics, 2009: p. 12.2. 1-12.2. 16.

Keywords: RBP, Structure, Motif Structure Score

12. Characterization of U5NU Binding by Vaccinia Capping Enzyme

Rachel Bowman (Department of Biological Sciences, SUNY University at Buffalo), Dr. Paul Gollnick (Department of Biological Sciences, SUNY University at Buffalo)

Abstract not available online - please check the printed booklet.

13. The effect of tRNA modifications on pseudohyphal growth in yeast

Jennifer H. Brooks (Chemistry, Northern Kentucky University), Daisy J. DiVita (Chemistry, Northern Kentucky University), Kory Stuessel (Biological Sciences, Northern Kentucky University), John C. Carmen (Biological Sciences, Northern Kentucky University), Michael P. Guy (Chemistry, Northern Kentucky University)

Abstract not available online - please check the printed booklet.

14. Molecular and functional characterization of stem cell messenger ribonucleoprotein complexes (mRNPs)

Kasun Buddika (Department of Biology, Indiana University, Bloomington IN 47405), Nicholas Sokol (Department of Biology, Indiana University, Bloomington IN 47405)

Abstract not available online - please check the printed booklet.

15. Role of Unique C-terminal Domain in an Aminoacyl-tRNA Trans-editing Protein from Arabidopsis thaliana

Jun-Kyu Byun (The Ohio State University ), Marina Bakhtina (The Ohio State University), Karin Musier-Forsyth (The Ohio State University)

Abstract:
Some aminoacyl-tRNA synthetases (aaRSs) are error-prone, mispairing non-cognate amino acids with cognate tRNAs. To prevent mistranslation, many aaRSs have evolved quality control mechanisms. Bacterial prolyl-tRNA synthetases (ProRSs) mischarge noncognate Ala onto tRNA^Pro and possess an insertion (INS) domain that can deacylate Ala-tRNA^Pro. However, some bacteria and all eukaryotes lack this editing domain and instead, encode a free-standing editing domain homolog, ProXp-ala, that edits Ala-tRNA^Pro in trans. Here, we report that ProXp-ala from plants has a unique C-terminal extension domain (CTD) of unknown structure that is missing from some bacterial and all eukaryotic ProXp-ala domains. This domain is predicted to encode a long helical segment connected to the catalytic domain via a random coil. Ancient aaRSs are believed to have existed as single-domain enzymes and additional domains reflect selective evolutionary adaptations to enhance specificity and efficiency of aminoacylation, as well as to confer new functions. Similarly, additional domains fused to ProXp-ala may be indicative of enhanced specificity or new functions. To determine the function of the CTD in plant ProXp-ala, we prepared a truncated Arabidopsis thaliana (At) ProXp-ala variant (ΔC-ProXp-ala). Circular dichroism spectroscopy showed that the secondary structure of ProXp-ala is not perturbed upon truncation. In vitro deacylation of At Ala-tRNA^Pro showed that the truncation resulted in a 16-fold decrease in the deacylation rate relative to wild-type ProXp-ala, primarily due to a tRNA binding defect. We hypothesize that this extra domain may confer strong tRNA binding via specific RNA interactions. Ongoing studies will investigate this hypothesis, as well as the In vivofunction of At ProXp-ala, using available At knockdown strains. Understanding the functional significance of the ProXp-ala CTD will reveal new insights into how plants have uniquely evolved to avoid mistranslation.

References:

1. Das M, Vargas-Rodriguez O, Goto Y, Suga H, Musier-Forsyth K. Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation. Nucleic Acids Research. 2014;42(6):3943-3953.
2. Vargas-Rodriguez O, Musier-Forsyth K. Exclusive Use of trans-Editing Domains Prevents Proline Mistranslation. The Journal of Biological Chemistry. 2013;288(20):14391-14399.

Keywords: aminoacyl tRNA synthetase, Trans editing protein, ProXp-ala

16. Regulation of eIF2 kinase GCN2 in translational control

Kenneth Carlson (Department of Biochemistry and Molecular Biology Indiana University School of Medicine, Indianapolis, IN), Andy Hudmon (Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN), Millie Georgiadis (Department of Biochemistry and Molecular Biology Indiana University School of Medicine, Indianapolis, IN), Ronald C Wek (Department of Biochemistry and Molecular Biology Indiana University School of Medicine, Indianapolis, IN)

Abstract not available online - please check the printed booklet.

17. De novo Design of Transcriptional and Translational RNA Repressors

Paul D. Carlson (Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University)

Abstract not available online - please check the printed booklet.

18. Genome-wide atlas of alternative polyadenylation in the forage legume, red clover

Manohar Chakrabarti (Department of Plant and Soil Sciences, University, Lexington, KY, USA. 40546-0312), Randy D. Dinkins (USDA-ARS, Forage-Animal Production Research Unit, Lexington, Kentucky, USA. 40546), Arthur G. Hunt (Department of Plant and Soil Sciences, University, Lexington, KY, USA. 40546-0312)

Abstract not available online - please check the printed booklet.

19. Temporally controlled expression of ESRP2 licenses fetal-to-adult switch in alternative splicing required for hepatocyte maturation

Sushant Bangru (Department of Biochemistry, University of Illinois at Urbana-Champaign), Jackie Chen (Department of Biochemistry, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, College of Medicine, Carl R. Woese Institute of Genomic Biology, University of Illinois at Urbana-Champaign)

Abstract:
Postnatal period of development is critical for mammalian tissues and is coordinated through precise activation of genetic programs that govern differentiation, growth and maturation of individual cell lineages. Here, we describe a cell type- and developmental stage-specific program of alternative splicing that drives sequential replacement of fetal-to-adult protein isoforms in the mouse liver. Deep transcriptome analysis of purified fetal and adult mouse hepatocytes coupled with RNA-binding motif enrichment analysis identified Epithelial Splicing regulatory protein 2 (ESRP2) as the major regulator for these developmental splicing decisions. Targeted deletion of ESRP2 in mice resulted in the failure of fetal-to-adult switch in splicing for hundreds of RNA transcripts that encode proteins involved in terminal differentiation and functional competence of hepatocytes. To delineate its exact role in activation of adult splicing program, we generated transgenic mice with tetracycline-inducible and hepatocyte-specific expression of ESRP2. Remarkably, premature expression of ESRP2 in the livers of newborn pups forced an earlier-than-normal onset of adult splicing program. Collectively, these findings highlight the essentiality and temporal requirement for ESRP2 in allowing fetal-to-adult splicing transitions within maturing hepatocytes and broaden our understanding of regulatory mechanisms needed for postnatal liver development and maturation.

Keywords: Liver, splicing, development

20. Modeling RNA-ligand Structures using chemical shifts

Sahil Chhabra (University of Michigan), Aaron Frank (University of Michigan)

Abstract:
Over the last couple of decades, it has become clear that RNAs can regulate many processes within the cell. Also, it has been discovered that RNA dysregulation is associated with many humans diseases. Acquiring accurate structural information of disease-associated RNAs in complex with small drug-like molecules is crucial in the rational design and discovery of potential therapeutics. Here we examine whether, starting from the sequence of an RNA, computational methods can be used to sample the correct 3D structure of RNA-ligand complex, and whether assigned chemical shifts could be used to discriminate between the correct structures and non-native decoy structures. Using the influenza A virus promoter RNA complex with a small-molecule (PDBID 2LWK, BMRBID 18633) that inhibits viral replication as a model system, we have discovered that not only can computational methods sample native-like conformations of the holo RNA, but more importantly, the modeled RNA structure that best agree with holo chemical shift data are within 2.5 Å of the NMR structure. Moreover, when the ligands are docked onto this structure, we can sample models of the full complex that are within 2.5 Å of the correct structure. To better assess the resolving-power of holo chemical shifts in the context of modeling RNA-ligand complexes, we will carry out similar analysis on additional RNA-ligand complexes for which both chemical shifts and NMR structures are available.

Keywords: RNA, Chemical Shifts, Modeling

21. Analyzing the role of G·U base pairs on the melting behavior of the S. enterica htrA RNA thermometer

Edric K. Choi (Department of Chemistry and Biochemistry, Denison University), Rachel M. Mitton-Fry (Department of Chemistry and Biochemistry, Denison University)

Abstract not available online - please check the printed booklet.

22. Developing a high resolution genome-wide R-loop mapping protocol in yeast

Sara Cloutier (Biochemistry Department, Purdue University), Elizabeth Tran (Biochemistry Department, Purdue University)

Abstract not available online - please check the printed booklet.

23. Structural implications of Gag-mediated assembly during bovine leukemia virus replication

Sarah E. Cooper (Berry College Chemistry Department), Dr. Dominic F. Qualley (Berry College Chemistry Department)

Abstract:
Like most retroviruses, including human immunodeficiency virus type 1 (HIV-1), bovine leukemia virus (BLV) utilizes the structural polyprotein Gag in its replication process. Gag is composed of three different domains: the matrix domain, the capsid domain, and the nucleocapsid domain. The function of these three domains is essential in the viral assembly process, but how they work is still poorly understood. Specifically, it is unknown which domains play an active role in facilitating assembly by packaging the genomic RNA of the virus. Through computational structure modeling and gel-shift annealing assays, the interaction of the Gag domains with viral nucleic acids in BLV was studied. Our results indicate that, unlike HIV-1 Gag, BLV Gag adopts a rigid conformation with little flexibility. Similar to HIV-1 Gag, however, the MA domain is important for membrane binding while the NC domain is responsible for RNA binding and packaging. By better understanding this interaction, the domains responsible for genome packaging can potentially be inhibited in order to disrupt viral assembly and prevent BLV from replicating. Because BLV is an agriculturally significant pathogen that infects domestic cattle, a potential treatment would have an important impact on agricultural production. Additionally, BLV has been proposed as an animal model for human T-cell leukemia virus type 1 (HTLV-1), so treatments for BLV could potentially be translated into human medical therapies.

Keywords: Bovine leukemia virus, Gag protein, genome packaging

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

Shiba S. Dandpat (Department of Chemistry, University of Michigan, Ann Arbor), Paul E. Lund (Department of Chemistry, University of Michigan, Ann Arbor), Sujay Ray (Department of Chemistry, University of Michigan, Ann Arbor), Nils G. Walter (Department of Chemistry, University of Michigan, Ann Arbor)

Abstract not available online - please check the printed booklet.

25. Lantern: a semi-automated pipeline and repository for annotating lncRNAs with ontologies

Swapna Vidhur Daulatabad (Department of Biohealth Informatics, Indiana University-Purdue University Indianapolis ), Sarath Chandra Janga (Department of Biohealth Informatics, Indiana University-Purdue University Indianapolis )

Abstract:
With advancements in omics technologies, the range of biological processes long non-coding RNAs (lncRNAs) are involved in, is expanding extensively [1, 2]. The accelerating rate of evidence discovery for lncRNAs’ role in various critical biochemical, cellular and physiological processes is thereby necessitating the need for robust lncRNA annotation resources. Although, there are a plethora of resources for annotating genes, despite extensive corpus of lncRNA literature, available resources with lncRNA ontology annotations are rare. Here, we present a semi-automated pipeline and corresponding lncRNA annotation extractor and repository (Lantern), developed using PubMed’s abstract retrieval engine and NCBO’s recommender annotation system [3]. We extracted between 1-150 abstracts per lncRNA, which were subsequently used for extracting annotations with respect to each ontology by querying NCBO’s recommender system via Application Programming Interface (API). To evaluate the quality of annotations in Lantern, we performed an analysis by deploying our pipeline over 182 lncRNAs from lncRNAdb [4] and compared the extracted annotations against annotations mapped onto the lncRNAdb’s manually curated free text. Benchmarking analysis suggested that Lantern has a recall of 0.62 against lncRNAdb for 182 lncRNAs and precision of 0.8 based on manual verification of ontology annotations for 50 lncRNAs. Lantern’s web-interface currently provides Gene Ontology (GO), Human Phenotype Ontology (HPO) and Human Disease Ontology (DOID) annotations for 182 lncRNAs along with 5 different annotation evaluation scores from NCBO recommender system. The extracted annotations for 182 lncRNAs are available at http://www.iupui.edu/~sysbio/lantern/. Lantern will be expanded to increase the number of annotated lncRNAs and corresponding ontologies, to make it a public resource with high quality controlled annotations for improving the annotation of the noncoding transcriptome.

References:
1. Mohanty, V., et al., Role of lncRNAs in health and disease—size and shape matter. Briefings in functional genomics, 2014. 14(2): p. 115-129.
2. Quinn, J.J. and H.Y. Chang, Unique features of long non-coding RNA biogenesis and function. Nature Reviews. Genetics, 2016. 17(1): p. 47.
3. Martínez-Romero, M., et al., NCBO Ontology Recommender 2.0: an enhanced approach for biomedical ontology recommendation. Journal of biomedical semantics, 2017. 8(1): p. 21.
4. Quek, X.C., et al., lncRNAdb v2. 0: expanding the reference database for functional long noncoding RNAs. Nucleic acids research, 2014. 43(D1): p. D168-D173.

Keywords: lncRNA, ontology, automated annotation

26. Molecular basis for mismatch-sensitive cleavage modulation of bilobed Argonaute proteins

Daniel M. Dayeh (Center for RNA Biology, The Ohio State University ), Bradley C. Kruithoff (Center for RNA Biology, The Ohio State University ), Kotaro Nakanishi (Center for RNA Biology, The Ohio State University )

Abstract not available online - please check the printed booklet.

27. The interplay between alternative polyadenylation and RNA quality control in Arabidopsis

Laura de Lorenzo (Department of Plant and Soil Sciences. University of Kentucky, Lexington, KY ), Julia Bailey-Serres (Center for Plant Cell Biology, University of California Riverside, Riverside CA), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY )

Abstract:
In plants, alternative polyadenylation (APA) is a widespread process, affecting a large majority of genes. APA may occur at any position during the transcription of a gene and determine protein length or influence mRNA stability, translation, and/or transport (1). The selection of polyadenylation signals (PAS) from distal to proximal in the 3’-UTR of premRNAs can quantitatively affect protein synthesis, and the PAS selection in upstream regions (5’-UTRs, introns, or protein coding regions; called noncanonical isoforms) is expected to yield RNAs that may be subject to any of RNA quality-control (RQC) processes. The roles of noncanonical isoforms have not been explored and possible connections with negative regulatory mechanisms have been suggested. In this context, the stabilities and translatabilities of Arabidopsis RNA isoforms derived from proximal PAS usage were studied. A genome-wide approach using libraries of 3’-end-directed cDNA tags were generated from RNA isolated from plants treated with a transcriptional inhibitor, or from polysomes purified, and sequenced. The results suggest that each isoform has different properties (2). RNAs with 3’-ends within protein-coding regions and introns were less stable than 3’-UTR PAS and were under-represented in polysomes, suggesting that these RNA isoforms may be subject to quality-control processes. In contrast, 5’-UTR mRNA isoforms were over-represented in polysomes, and as stable as canonical isoforms. Likewise, different RQC mutants were evaluated using 3’-end tags libraries to elucidate an interplay between APA and RQC. Thus, coding-region isoforms are accumulated in decapping mutants, and intronic and 5’-UTR isoforms are accumulated in upf3 mutant. These results show that APA may re-direct transcriptional output into products that are subject to surveillance processes, and thus may constitute a form of negative regulation.

References:
(1) Lutz, C.S., and Moreira, A. (2011). Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression. Wiley Interdiscip. Rev. RNA 2: 22–31.
(2) de Lorenzo, L., Sorenson, R., Bailey-Serres, J., and Hunt A.G. (2017). Noncanonical alternative polyadenylation contributes to gene regulation in response to hypoxia. The Plant Cell 29: 1262–1277.

Keywords: stability-translatability, RNA quality-control, Arabidopsis

28. A new approach to NGS identification of capped 5’ ends

Daniel del Valle-Morales (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, USA), Guramrit Singh (Center for RNA Biology and Department of Molecular Genetics, The Ohio State University , Columbus, Ohio, USA), Ralf Bundschuh (Center for RNA Biology, Department of Physics, Department of Chemistry and Biochemistry, and Division of Hematology, The Ohio State University, Columbus, Ohio, USA), Daniel R. Schoenberg (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, USA)

Abstract:
The 5’ cap is an essential modification in mRNAs that is needed for its translation, and decapping leads to translational suppression and mRNA decay. Capping was originally thought to occur exclusively in the nucleus, however the identification of capped nonsense mediated decay intermediates of β–globin mRNA and of a cytoplasmic pool of Capping Enzyme (CE) led to the discovery of the cytoplasmic capping complex. We previously identified cytoplasmic capping targets by a combination of in vitro degradation with Xrn1 and position-dependent analysis of transcript loss using human exon arrays. These findings contributed to the hypothesis of cap homeostasis where translation of some mRNAs is controlled by cyclical decapping and recapping. Because uncapped mRNAs are susceptible to degradation and/or 5’ end trimming, it is difficult to map the exact location of recapped 5’ ends. To address this we developed a new approach based on use of the SMART reverse transcriptase and next generation sequencing to identify recapped ends. This approach takes advantage of the template switching of the SMART enzyme. The first step in the process involves ligation of a STOP oligo to uncapped mRNAs. The STOP oligo consists of an 18mer RNA oligonucleotide with 3 isomeric nucleotides (iGiCiG) at the 5’ end that prevent template switching. In the next step, SMART enzyme reverse transcribes capped mRNAs and template switches to the SMART oligo. The SMART oligo contains an eight nucleotide barcode and a 5’ adaptor. A second round of PCR incorporates a biotin tagged 5’ adaptor which is selected on streptavidin beads to enrich for 5’ ends. The last step involves incorporating the Illumina sequencing adaptors to bead recovered cDNAs with a final round of PCR. I will present results for individual steps in this process and their validation.

References:
C. Mukherjee, D. Patil, B. Kennedy, B. Bakthavachalu, R. Bundschuh, D. R. Schoenberg; Identification of Cytoplasmic Capping Targets Reveals a Role for Cap Homeostasis in Translation and mRNA Stability, Cell Reports (2012).
R. Machida, Y. Lin; Four Methods of Preparing mRNA 5′ End Libraries Using the Illumina Sequencing Platform, PLOS one (2014).
C. Mukherjee, B. Bakthavachalu, D.R. Schoenberg; The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme, PLOS Biology, (2014).

Keywords: Cytoplasmic Capping, Sequencing

29. Characterization of a putative HSV-like ribozyme in an R2 retrotransposon in the vertebrates Lampetra aepyptera and Lethenteron appendix.

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

Abstract:
R2 retroelements have been extensively characterized in arthropods, but there has been almost no description of R2 elements in vertebrate genomes. We have putatively identified an R2 retrotransposon in the genomes of the lamprey species Lampetra aepyptera and Lethenteron appendix. R2 retrotransposons are a unique class of transposon that move via an RNA intermediate through eukaryotic genomes. These elements insert themselves in the middle of the 28S rDNA gene, and the R2 gene is transcribed as part of the larger rDNA cassette and cleaves itself from the pre-rRNA transcript via a ribozyme encoded in its 5’UTR. We have characterized these putative transposons via sequencing, locus quantification via qPCR, and a measure of transposition activity via 5’ end profiling. We are currently conducting in vitro transcription/cleavage assays on the putative ribozyme to investigate the mechanisms by which the R2 transposition occurs.

Keywords: Retrotransposon, ribozyme

30. Functional and genetic studies of the tRNA modification protein Trm732 in yeast

Daisy J. DiVita (Chemistry, Northern Kentucky University), Hannah Sizemore, Julia K. Ziebro (Chemistry, Northern Kentucky University), Katherine L. Weiner, Gregory OConnor (Biochemistry and Biophysics, University of Rochester), Eric M. Phizicky (Biochemistry and Biophysics, University of Rochester), Michael P. Guy (Chemistry, Northern Kentucky University)

Abstract not available online - please check the printed booklet.

31. Investigation of a Noncoding RNA Handyman in Dictyostelium discoideum

Samantha Dodbele (Ohio State Biochemistry Program, The Ohio State University OSU; Center for RNA Biology, OSU), Blythe Moreland (Center for RNA Biology, OSU; Department of Physics, OSU), Yicheng Long (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU), Ralf Bundschuh (Center for RNA Biology, OSU; Department of Physics, OSU; Division of Hematology and Department of Internal Medicine, OSU), Jane Jackman (Ohio State Biochemistry Program, OSU; Center for RNA Biology, OSU; Department of Chemistry and Biochemistry, OSU)

Abstract not available online - please check the printed booklet.

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

Yilin Dong (Department of Chemistry, Carnegie Mellon University), Subha Das (Department of Chemistry, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

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

Dana Driver (Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Yi Shu (Nanobiotechnology Center, Markey Cancer Center, and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

34. Do allelic differences in Candida albicans Pseudouridine Synthase 4 lead to distinct functions?

Caleb Embree (Department of Biology, Ball State University), Douglas Bernstein (Department of Biology, Ball State University)

Abstract:
The human fungal pathogen Candida albicans genome is diploid and for many genes the A and B alleles have different sequences. Recent technological advances have enabled us to begin to investigate if C. albicans A an B allele products have distinct functions. Pseudouridine synthases modify uridine to pseudouridine in tRNAs, rRNA, snRNA, mRNA, and snoRNAs. C. albicans’ complement of pseudouridine synthases differs significantly from S. cerevisiae, but the functions of the C. albicans pseudouridine synthases have not been investigated. C. albicans pseudouridine synthase 4 (CaPus4) has 13 differences between A and B alleles. In S. cerevisiae, Pus4 modifies uridine to pseudouridine in cytoplasmic and mitochondrial tRNAs at residue 55 and mRNAs. We aim to identify the functions of C. albicans Pus4, and test if A and B alleles have distinct functions.

Keywords: Candida albicans, Pseudouridine, PUS4

35. Performing a genetic screen to identify factors that promote lncRNA-dependent gene repression

Chrishan Fernando (Department of Biochemistry, Purdue University), Cecilia Yiu (Department of Biochemistry, Purdue University), Sara Cloutier (Department of Biochemistry, Purdue University), Siwen Wang (Department of Biochemistry, Purdue University), Elizabeth Tran (Department of Biochemistry, Purdue University)

Abstract:
Long non-coding RNAs (lncRNAs) were once thought not to have useful functions in organisms but rather to be products of aberrant transcription. However, roles are being found for lncRNAs in beneficial processes such as controlling gene expression. In some of these cases, lncRNAs form R-loops in vivo. R-loops are nucleic acid structures consisting of hybridized strands of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) as well as the displaced strand of ssDNA. Formation of these R-loops is important for gene regulation by the lncRNAs. However, factors that promote formation of lncRNA R-loops are not known. The gene PHO84 is being used to study this matter. PHO84 encodes a phosphate transporter in Saccharomyces cerevisiae. Two lncRNAs are transcribed in the antisense direction across PHO84 and repress PHO84 gene expression. This phenomenon may be due to the formation of R-loops. A genetic screen has been designed in S. cerevisiae using a HIS3 reporter to identify genes that suppress gene expression on the PHO84 locus when overexpressed. The HIS3 reporter is currently being evaluated for ability to identify factors promoting R-loop formation.

Keywords: R-loop, RNA-DNA hybrid, lncRNA

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

Tanja Florin (Center for Biomolecular Sciences, University of Illinois at Chicago), Dorota Klepacki (Center for Biomolecular Sciences, University of Illinois at Chicago), Amira Kefi (Center for Biomolecular Sciences, University of Illinois at Chicago; Department of Bioengineering, University of Illinois at Chicago), Nora Vazquez-Laslop (Center for Biomolecular Sciences, University of Illinois at Chicago), Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago)

Abstract not available online - please check the printed booklet.

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

Anna V. Sherwood (Molecular Cellular and Developmental Biology, Center for RNA Biology, The Ohio State University), Jane K. Frandsen (Ohio State Biochemistry Program, Cellular, Molecular & Biochemical Sciences Program, Center for RNA Biology, The Ohio State University), Frank J. Grundy (Department of Microbiology, Center for RNA Biology, The Ohio State University), Tina M. Henkin (Department of Microbiology, Ohio State Biochemistry Program, Molecular Cellular and Developmental Biology, Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

38. RsmC shows RNA chaperone activity during ribosome biogenesis

Keshav GC (Department of Chemistry and Biochemistry, Kent State University), Davidnhan To (Department of Chemistry and Biochemistry, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)

Abstract:
Synthesizing new ribosomes is a critical process in all forms of life. Ribosome biogenesis is a complex process, where the transcription and the folding of ribosomal RNA (rRNA), assembly of ribosomal proteins, introduction of post-transcriptional nucleotide modifications, and rRNA processing occur simultaneously. Protein RsmC is a methyltransferase enzyme that is responsible for the m2G modification located at helix 34 of 16S rRNA. Protein RsmC contains two homologous domains that show tandem duplication. The goal of our research is to understand the importance of protein RsmC in ribosome biogenesis. Biochemical and biophysical experiments carried out in our laboratory suggest that RsmC enzyme also functions as an RNA chaperone that facilitates annealing of 5’- and 3’- RNA strands of 16S helix 34, in addition to its methyltransferase activity. Similarly, our data suggest that protein RsmC can prevent premature binding of tertiary assembly proteins, such as protein S3.

Keywords: Ribosome biogenesis , Modified nucleotides , Methyltransferase

39. Using Cell-SELEX to Identify Modified DNA Aptamers that Bind E. coli

Nicolette Geron (Department of Chemistry, SIUE), Allie Watts (Department of Chemistry, SIUE), Kamran Shavezipur (Department of Mechanical Engineering, SIUE), Mina Sumita (Department of Chemistry, SIUE)

Abstract:
The process of Cell – SELEX is the Systematic Evolution of Ligands by EXponential enrichment. DNA aptamers are selected using a specific target cell. Cell – SELEX will be used to identify modified DNA aptamers that bind with a high affinity to our target cell, Escherichia Coli K12. The modified DNA aptamers will contain deoxyuridine instead of thymidine. It is hypothesized that the modified DNA aptamer will have the stability of traditional DNA and the conformational variety of RNA. The 2’-H in deoxyribose gives DNA more chemical stability than the 2’-OH present in the RNA structure. However, RNA is able to form more conformations due to this structural difference with DNA. Therefore, the aptamers that are identified will have the stability of DNA and the structural variety of RNA providing for a broader selection of aptamers and a higher specificity when binding to E. coli. When we identify the aptamer sequence, we will study the affinity between the aptamer and E. coli.

Keywords: Aptamer, SELEX, Modification

40. The dichotomy of MDM2 splicing in response to genotoxic stress

Andrew K. Goodwin (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio), Dawn S. Chandler (Molecular, Cellular and Developmental Biology Graduate Program and The Center for RNA Biology, The Ohio State University, Columbus, Ohio)

Abstract not available online - please check the printed booklet.

41. Plasmodium Transmission is Critically Regulated by General and Transmission-Specific Deadenylases of the CAF1/CCR4/NOT Complex

Kevin J. Hart (Department of Biochemistry and Molecular Biology, Center for Malaria Research, Pennsylvania State University, University Park, Pennsylvania 16802), Jenna Oberstaller (Center for Global Health and Infectious Diseases Research, Department of Global Health, University of South Florida, 3720 Spectrum Blvd, Suite 404, Tampa, Florida 33612 ), Michael P. Walker, Mark F. Kennedy (Department of Biochemistry and Molecular Biology, Center for Malaria Research, Pennsylvania State University, University Park, Pennsylvania 16802), Ian Padykula (Center for Global Health and Infectious Diseases Research, Department of Global Health, University of South Florida, 3720 Spectrum Blvd, Suite 404, Tampa, Florida 33612 ), John H. Adams (Center for Global Health and Infectious Diseases Research, Department of Global Health, University of South Florida, 3720 Spectrum Blvd, Suite 404, Tampa, Florida 33612 ), Scott E. Lindner (Department of Biochemistry and Molecular Biology, Center for Malaria Research, Pennsylvania State University, University Park, Pennsylvania 16802)

Abstract:
With relatively few known specific transcription factors (ApiAP2 family), Plasmodium parasites regulate the stability and turnover of transcripts to provide more comprehensive gene regulation. However, few proteins that impose translational repression in Plasmodium sexual stages are known, and those that are characterized primarily affect female gametocytes. We have characterized CCR4-1 of the CAF1/CCR4/NOT complex, which we show plays a role in activating male gametocytes, stabilizing transcripts in gametocytes, and regulating host-to-vector transmission. Comparative RNA-seq demonstrates that many transcripts important for these functions are affected. In contrast, genetic deletion of the major deadenylase CAF1 is lethal, yet expression of the N-terminal CAF1 domain is permissive but prevents complex assembly and phenocopies the ccr4-1 deletion. Transgenic P. falciparum parasites expressing a similar CAF1 disruptant supports this data. We conclude that the general and transmission-specialized deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in parasite growth and transmission.

Keywords: CCR4, Translational Repression, Plasmodium

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

Huzaifa Hassan (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University Indianapolis), Sarath C. Janga (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University Indianapolis)

Abstract not available online - please check the printed booklet.

43. Characterization of minimal binding site of modification enzyme RsmG

Caitlin Hawkins (Department of Chemistry and Biochemistry - Kent State University), Kumudie Jayalath (Department of Chemistry and Biochemistry - Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry - Kent State University)

Abstract:
Ribosomes are the molecular machines that carry out protein biosynthesis in all living organisms. They are composed of three different ribosomal RNAs and more than 50 ribosomal proteins. The thermodynamics and kinetics of in vitro ribosomal assembly have been studied extensively. However, during in vivo ribosome biogenesis, ribosome assembly occurs concurrently with transcription, folding, post-transcriptional modification, and processing of rRNA. Unfortunately, the effects post-transcriptional RNA modification enzymes on ribosomal assembly are understudied. My project in Abey lab is to investigate how RNA modification enzyme RsmG influences 30S bacterial ribosome assembly. I have developed a FRET-based assay to monitor the binding of RsmG to ribosomal RNA, which will allow us to determine the binding affinity of RsmG to RNA, and thus calculate thermodynamic cooperativity between the RsmG enzyme and ribosomal proteins. Our findings give us more insight into how modification enzymes modulate the hierarchy of protein addition during ribosome biogenesis.

Keywords: ribosome assembly, RNA-protein interactions, nucleotide modifications

44. Mapping of R-loops in yeast cells using DNase H

Youssef A. Hegazy (Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA), Sara C. Cloutier (Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA), Zheng Xing (Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA), Elizabeth J. Tran (1. Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA. 2. Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA)

Abstract:
R-loops are cellular structures composed of an RNA–DNA hybrid and a displaced single-stranded DNA. R loops occur more frequently in the genome and have greater physiological importance than was previously predicted. They play vital roles in regulating gene expression (1), DNA replication (2), and DNA and histone modifications (3). Paradoxically, while they do play essential positive functions required for important biological processes, they can also contribute to DNA damage and genome instability (4). Recent evidence suggests that R-loops are involved in molecular mechanisms of a number of human diseases, including neurological disorders (5) and cancer (4). We are developing a new strategy for mapping R-loops genome wide. This strategy is based on the use of Neisseria meningitidis CRISPR associated protein (NmeCas9) that has been recently shown to have a unique “DNase H” activity (6). We will present progress on establishing this technique to map R-loops in Saccharomyces cerevisiae.

References:
1. Ginno et al. (2013) Genome Res 23: 1590–1600.
2. Xu et al. (1996) EMBO J 15: 3135–3143.
3. Castellano-Pozo et al. (2013) Mol Cell 52: 583–590.
4. Stirling et al. (2012) Genes & Dev 26:163-175.
5. Powell et al. (2013) Proc Natl Acad Sci U S A 110:13938–13943.
6. Zhang et al. (2015) Mol Cell 60:242–255.

Keywords: R-loop , DNase H, S cerevisiae

45. The utility of NMR relaxation-dispersion data in characterizing invisible states of RNA

Charles G. Hoogstraten (Biochemistry & Molecular Biology, Michigan State University), Samantha L. Alexander (Biochemistry & Molecular Biology, Michigan State University), Alison M. Gjidoda (Biochemistry & Molecular Biology, Michigan State University), Neil A. White (Biochemistry & Molecular Biology, Michigan State University), Patrick O. Ochieng (Biochemistry & Molecular Biology, Michigan State University), Robert Fidis (Biochemistry & Molecular Biology, Michigan State University)

Abstract:
NMR spin-relaxation dispersion measurements have the ability to report on the chemical shift changes between the major structure of a protein or RNA and a lightly-populated “invisible state” that cannot be analyzed via the usual techniques of structural biology. Our group and others have introduced and applied techniques to use uniformly- and specifically-labeled ¹³C nuclei to detect such invisible states in RNA. In many cases, the question of interest is whether the observed minor conformer is a functionally relevant form or simply an unstructured state. For example, the sampling of a conformation resembling a ligand-bound form can be taken as evidence for a conformational-capture binding mechanism.

In this work, we examine the feasibility of detecting such sampling using NMR. In the docking transition of the hairpin ribozyme, both loops A and B undergo dramatic conformational changes between the free and bound form. We use contemporary chemical-shift calculation algorithms to predict the spectral shifts expected upon the transition of loop A from its free form to a hypothetical “docked-like” conformation. In parallel, we report progress toward the derivation of a reference set of ¹H and ¹³C chemical shifts for nucleotide residues in unstructured RNA regions. Comparison of the predictions of chemical shift changes for loop A unfolding vs. docked-state sampling allows the feasibility of distinguishing these two possibilities using relaxation-dispersion measurements to be assessed.

Keywords: Ribozyme, NMR Spectroscopy, Chemical Shift

46. The evolutionary path of the mammalian mitochondrial ribosome: How to fold a functional RNA with fewer Guanines

Maryam Hosseini (Chemistry, Bowling Green State University), Neocles B. Leontis (Chemistry, Bowling Green State University)

Abstract not available online - please check the printed booklet.

47. Assessment of candidate factors required for RNA editing in Physarum polycephalum mitochondria

Jillian Houtz (Department of Biochemistry and the Center for RNA Science & Therapeutics, CWRU), Jonatha Gott (The Center for RNA Science & Therapeutics, CWRU)

Abstract not available online - please check the printed booklet.

48. Bacterial cell can live without free 5S rRNA

Shijie Huang (Center for Biomolecular Sciences, University of Illinois, Chicago), Dorota Klepacki (Center for Biomolecular Sciences, University of Illinois, Chicago), Luc Jaeger (University of California, Santa Barbara), Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois, Chicago)

Abstract:
The 120 nucleotide-long 5S rRNA is a universal component of cytoplasmic ribosomes of all living organisms. Together with the 23S rRNA and a handful of ribosomal proteins it assembles into the bacterial large ribosomal subunit. 5S rRNA has been suggested to be critical for ribosome assembly, to play a role in the catalysis of peptide bond formation, participate in signal transmission within the ribosome and be involved in other activities. However, even after decades of research, the true function of 5S rRNA remains unclear. The lack of new tools and methodologies hinders the progress towards the understanding the role of 5S rRNA in translation.
To develop a novel system for analyzing the functions of 5S rRNA in the living cell and to implement new approaches to enable ribosome engineering, we succeeded in fusing 5S with 23S rRNA into a single molecule. This was achieved by inserting circularly-permuted 5S rRNA at two different 23S rRNA locations. The resulting 23S-5S rRNA hybrid molecule assembles into a functional large ribosomal subunit. Furthermore, we were able to engineer E. coli cells in which all the ribosomes carry the 23S-5S hybrid rRNA and, therefore, we have created a cell which completely lacks free 5S rRNA. These results unequivocally show that free 5S rRNA is dispensable for the ribosome assembly or function.
We have also introduced 5S rRNA into Ribo-T, the ribosome with tethered small and large subunits, which has been previously built upon a hybrid 16S-23S rRNA molecule. The preliminary result shows resulting single-RNA ribosome is capable of catalyzing polypeptide formation. We envision that the 23S-5S hybrid or the single-RNA ribosome could be powerful new tools for studying 5S rRNA functions, biogenesis and evolution. The single-RNA ribosome makes it possible to create an orthogonal translation system in the cell which does not exchange any of its RNA components with the ‘housekeeping’ ribosome.

Keywords: Ribosome, 5S rRNA, Orthogonal translation

49. Quantitative Analysis of the Effect of RTR1 Knockout on the Global Proteome in Saccharomyces cerevisiae

Katlyn D Hughes (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana ), Guihong Qi (Proteomics Core, Indiana University School of Medicine, Indianapolis, Indiana), Whitney Smith-Kinnaman (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana), Amber L Mosley (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana / Proteomics Core, Indiana University School of Medicine, Indianapolis, Indiana)

Abstract not available online - please check the printed booklet.

50. Using Surface Plasmon Resonance (SPR) to Determine DNA Aptamers that Binds to E. coli for Biosensor Development

Sun Jeong Im (Department of Chemistry, SIUE), Hailey Bowen (Department of Chemistry, SIUE), Mina Sumita (Department of Chemistry, SIUE)

Abstract:
Pathogenic Escherichia coli (E. coli) strains can cause food poisoning. One possible way to prevent food poisoning is by detecting the harmful E. coli via biosensor with DNA aptamer. The DNA aptamer is selected using a method called systematic evolution of ligands by exponential enrichment (SELEX). However, the SELEX method is time consuming when identifying the aptamer. Therefore, we are developing SELEX with surface plasmon resonance (SPR-SELEX). SPR technique can select and detect the affinity to the target molecule in the same time to simplify the SELEX process. The aptamer selection against E. coli with SPR-SELEX will proceed for the future biosensor.

Keywords: Aptamer, SELEX, SPR

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

Mahdieh Jadaliha (Cell and Developmental Biology, UIUC), K.V. Prasanth (Cell and Developmental Biology, UIUC)

Abstract not available online - please check the printed booklet.

52. NMR studies to observe transiently populated conformational states in the RNA binding protein TRAP

Cameron Jamshidi (Department of Chemistry and Biochemistry, The Ohio State University), Ian R. Kleckner (Department of Chemistry and Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

53. Seten: a tool for systematic identification and comparison of processes, phenotypes, and diseases associated with RNA-binding proteins from condition-specific CLIP-seq profiles

Gungor Budak (Biohealth Informatics, IU School of Informatics and Computing, IUPUI), Rajneesh Srivastava (Biohealth Informatics, IU School of Informatics and Computing, IUPUI), Sarath Chandra Janga (Biohealth Informatics, IU School of Informatics and Computing, IUPUI)

Abstract:
RNA-binding proteins (RBPs) control the regulation of gene expression in eukaryotic genomes at post-transcriptional level by binding to their cognate RNAs. Although several variants of CLIP (crosslinking and immunoprecipitation) protocols are currently available to study the global protein-RNA interaction landscape at single-nucleotide resolution in a cell, currently there are very few tools that can facilitate understanding and dissecting the functional associations of RBPs from the resulting binding maps. Here, we present Seten, a web-based and command line tool, which can identify and compare processes, phenotypes, and diseases associated with RBPs from condition-specific CLIP-seq profiles. Seten uses BED files resulting from most peak calling algorithms, which include scores reflecting the extent of binding of an RBP on the target transcript, to provide both traditional functional enrichment as well as gene set enrichment results for a number of gene set collections including BioCarta, KEGG, Reactome, Gene Ontology (GO), Human Phenotype Ontology (HPO), and MalaCards Disease Ontology for several organisms including fruit fly, human, mouse, rat, worm, and yeast. It also provides an option to dynamically compare the associated gene sets across data sets as bubble charts, to facilitate comparative analysis. Benchmarking of Seten using eCLIP data for IGF2BP1, SRSF7, and PTBP1 against their corresponding CRISPR RNA-seq in K562 cells as well as randomized negative controls, demonstrated that its gene set enrichment method outperforms functional enrichment, with scores significantly contributing to the discovery of true annotations. Comparative performance analysis using these CRISPR control data sets revealed significantly higher precision and comparable recall to that observed using ChIP-Enrich. Seten's web interface currently provides precomputed results for about 200 CLIP-seq data sets and both command line as well as web interfaces can be used to analyze CLIP-seq data sets. We highlight several examples to show the utility of Seten for rapid profiling of various CLIP-seq data sets. Seten is available on http://www.iupui.edu/∼sysbio/seten/.

References:
The ENCODE Project. 2017. RNA-seq profiling of CRISPR/Cas9 based knockouts of RNA-binding proteins in human cell line K562.
Janga SC. 2012. From specific to global analysis of posttranscriptional regulation in eukaryotes: posttranscriptional regulatory networks. Brief Funct Genomics 11: 505–521.
Chen B, Yun J, Kim MS, Mendell JT, Xie Y. 2014. PIPE-CLIP: a comprehensive online tool for CLIP-seq data analysis. Genome Biol 15: R18.

Keywords: CLIP (crosslinking and immunoprecipitation), RNA-binding proteins, gene set enrichment

54. Pseudouridylation enzyme RsuA influence 30S ribosome assembly

Kumudie Jayalath (Chemistry & Biochemistry, Kent State University), Sanjaya Abeysirigunawardena (Chemistry & Biochemistry, Kent State University)

Abstract:
The ribosome is responsible for protein biosynthesis in all living organisms. Bacterial ribosome biogenesis is a complex process that requires synchronization of various cellular events including ribosomal RNA (rRNA) transcription, ribosome assembly, RNA processing and post-transcriptional modification of rRNA. Ribosomal RNA nucleotide modifications and their respective modification enzymes can modulate rRNA folding and ribosome assembly. The only pseudouridine modification found in E. coli 16S ribosomal RNA is located at position 516 of 16S helix 18 which forms the central pseudoknot of the 30S ribosomal subunit. Our circular dichroism spectroscopic data suggest that the helix 18 model RNA undergoes Mg2+-dependent structural changes only in the presence of the pseudouridine modification at position 516. A FRET-based RsuA binding assay developed in our lab shows thermodynamic anti-cooperativity between ribosomal protein S4 and RsuA that catalyzes pseudouridylation of U516. Our data suggest that the RsuA enzyme binds preferably to nonpseudoknoted (extended) helix 18.

Keywords: Pseudouridine, Ribosome assembly

55. Interaction between Human Glutamyl-Prolyl tRNA synthetase Linker domain and HIV-1 Matrix: Implications for viral infection

Danni Jin, Nathan P. Titkemeier, Alice A. Duchon (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Yiping Zhu (Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY, 10032), Corine St. Gelais (Center for RNA Biology, Center for Retrovirus Research, and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Li Wu (Center for RNA Biology, Center for Retrovirus Research, and Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210), Stephen P. Goff (Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, NY, 10032), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)

Abstract:
Aminoacyl-tRNA synthetases (AARSs) are part of the cellular translational machinery and primarily function to ligate specific amino acids to cognate tRNAs. Mammalian Glutamyl-Prolyl tRNA Synthetase (EPRS) is a bifunctional AARS with a unique Linker domain between two synthetase domains, which has been shown to be involved in several non-canonical functions. EPRS is a component of the multi-aminoacyl-tRNA synthetase complex (MSC) together with seven other AARSs and three scaffold proteins. The genome of Human Immunodeficiency Virus Type 1 (HIV-1) encodes 15 viral proteins, which interact with host factors that affect viral replication. Based on published studies, many MSC components are included in the interactome of HIV-1 Matrix (MA) protein. We hypothesize that this virus-host interaction contributes to efficient viral infection. Our new data suggest that the expression of EPRS is downregulated in HIV-1 infected cells, and HIV-1 infectivity decreases in 293T cells stably overexpressing the Linker. Preliminary data also show that MA interacts with the MSC through the Linker domain in an RNA-dependent manner, but the specific RNA mediating the interaction is unknown. We are unable to detect an interaction between MA and the purified EPRS Linker in vitro in the absence of RNA, and both proteins interact with many tRNA species with similar affinity. MA mutants that fail to interact with the MSC in cells were identified by alanine-scanning mutagenesis, and viruses expressing these MA mutants are less infectious. Based on these observations, we propose that the Linker domain of EPRS restricts HIV-1 infection, and that the virus has evolved to downregulate this host cell protein.

Keywords: HIV-1, EPRS

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

Elizabeth A. Jolley (Department of Chemistry and Center for RNA Molecular Biology, Pennsylvania State University), David C. Tack (Department of Biology and Center for RNA Molecular BIology, Pennsylvania State University), Sarah M. Assmann (Department of Biology and Center for RNA Molecular BIology, Pennsylvania State University), Paul Babitzke (Department of Biochemistry and Molecular Biology and Center for RNA Molecular Biology, Pennsylvania State University), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry, and Molecular Biology and Center for RNA Molecular Biology, Pennsylvania State University)

Abstract not available online - please check the printed booklet.

57. Global Profiling of the Effects of Oxidative Stress on RNA Modifications by Liquid Chromatography-Mass Spectrometry

Manasses Jora (Department of Chemistry, University of Cincinnati), Andrew Burns (Department of Chemistry, University of Cincinnati), Patrick A. Limback (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati)

Abstract:
The goal of this work was to profile by liquid chromatography-tandem mass spectrometry (LC-MS/MS) the oxidative damage exerted by H₂O₂ on RNA modifications¹ using E. coli tRNA as a model system. Oxidative damage to RNA impacts biological systems due to its association with metabolic, mitochondrial, neurological diseases and cancer.^2,3 Oxidation pathways of canonical nucleosides are well documented.⁴ However, the effect of reactive oxygen species (ROS) on modified ribonucleosides is not well known.⁵ Hydrogen peroxide generates ROS (e.g., HOO^•, HO^•) under physiological conditions. Formation of hydroxyl radicals is accelerated via Fenton reactions in the presence of catalysts (e.g., Fe²⁺, Cu²⁺). Apart from the known oxidative products arising from canonical nucleosides, we have documented a list of modified nucleosides that are adversely affected by oxidative stress. These results indicated some modifications (e.g., 2-methylthio-N⁶-isopentenyladenosine, epoxyqueuosine, 2-thiocytidine) are more susceptible to oxidation than others. It was also observed that oxidation damage is more predominant with a copper catalyst. In addition, a new oxidation product with m/z 260.0877 was noticed to increase in abundance upon increasing ROS. Even though this m/z value is identical to that observed for 5-hydroxycytidine, it differs in retention time and MS/MS fragmentation data. The exact molecular structure of this unknown oxidation product is currently under investigation.

References:
1. modomics.genesilico.pl. A Database of RNA Modification Pathways.
2. Sarin, P.; et al.. RNA Biology 2015, 11, 1555.
3. Gu, C.; et al.. FEBS lett. 2014, 588, 4287.
4. Alshykhly, O, R.; et al.. J. Org. Chem. 2016, 80, 6996.
5. Nawrot, B.; et al.. Cell. Mol. Life Sci. 2011, 68, 4023.

Keywords: Oxidative Stress, RNA Damage, Nucleoside Modifications

58. Identification of novel transcripts in brain tissue using third generation sequencing: ISO-seq

Amira Kefi (Department of bioengineering, University of Illinois at Chicago), Lingling Huang (The State Key Lab of Medical Genetics, Central South University, Changsha, China), Qingtuan Meng (The State Key Lab of Medical Genetics, Central South University, Changsha, China), Chunyu Liu (Department of Psychiatry, University of Illinois at Chicago)

Abstract not available online - please check the printed booklet.

59. The effects of peroxide exposure on the RNA of radiotrophic C. neoformans

Melissa Kelley (Chemistry; University of Cincinnati), Lauren Schultz (Chemistry; University of Cincinnati), Ryan Myers (Biology; University of Cincinnati), Patrick A. Limbach (Chemistry; University of Cincinnati)

Abstract:
Cryptococcus neoformans is a radiotrophic fungi capable at surviving in extreme environments. Understanding post-transcriptional RNA modifications may lead to insight on translational regulation and protein synthesis. Here, the impact of peroxide exposure on C. neoformans transfer RNA is investigated through liquid chromatography coupled with mass spectrometry (LC-MS). Peroxide dosage that causes cytotoxicity in S. cerevisiae does not appear to induce changes in C. neoformans modifications or oxidation products in tRNA. However, cytotoxic concentrations in this organism and the tRNA effects will be investigated.

References:
Chan, C et al. A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress response. PLoS Gen. 6. 2010.
Davachova, E. et al. Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi. PLoS ONE. 2(5): e457. 2007
Gu, C et al. tRNA modifications regulate translation during cellular stress. FEBS letters. 588(23): 4287-4296. 2015.
Robinson, K et al. Adaptation of the black yeast Wangiella dermatitidis to ionizing radiation. PLoS ONE. 7(11): e48674. 2012.

Keywords: fungi, tRNA, radiotrophism

60. Reactivity and impact of amino-acid-linked platinum(II) analogues in RNA and DNA

Bett Kimutai (Department of Chemistry, Wayne State University, Detroit, MI 48202), Chenchen He (Department of Chemistry, Wayne State University, Detroit, MI 48202), M. T. Rodgers (Department of Chemistry, Wayne State University, Detroit, MI 48202), Christine S. Chow (Department of Chemistry, Wayne State University, Detroit, MI 48202)

Abstract:
RNA is a competitive target of cisplatin and is able to accumulate inert platinum adducts. Aquated cisplatin may have preferential target residues in RNA as observed with DNA. Cisplatin can also be modified in a number of ways with ligands of varied sizes, polarities, and charges. These features can affect the kinetics of interaction with RNA, the types of adducts formed, and the preferred sites of coordination. In this study, amino acids were reacted with platinum to form a series of cisplatin analogues. Further, kinetic studies showed that ornithine-linked platinum (OrnPt) and arginine-linked platinum (ArgPt) are reactive towards both RNA and DNA nucleosides but with different selectivities. The two amino acid complexes have selectivity towards A/dA residues, in contrast to cisplatin which targets dG. Inductively coupled plasma mass spectrometry (ICPMS) quantification reveals higher accumulation of OrnPt and ArgPt on RNA over DNA. ArgPt also forms platinum adducts with poly(A) RNA. Platination by amino-acid-linked platinum analogues at particular sites on the ribonucleosides (i.e., N1, N3, or N7) is found to have varying impacts on their glycosidic bond stabilities. Weak glycosidic bonds accelerate depurination and could ultimately contribute to cell cytotoxicity. Such adducts may affect the biological roles of RNA such as mRNA maturation and protein synthesis.

Keywords:

61. Analysis of Alternative Splicing in Disease Associated Exons

Stephanie Nguyen (Biology, Lewis University ), Jessica King (Biology, Lewis University )

Abstract:
The process of pre-mRNA splicing involves the removal of introns, the noncoding regions within an RNA transcript, and the ligation of coding regions, or exons. Alternative splicing maintains gene regulation through a variation in the specific patterns of exon inclusion and intron exclusion in the final mRNA transcript. This also allows for individual genes to code for multiple proteins, which contributes to diversity. Approximately 33% of genetic mutations are thought to be the result of an improper regulation in splicing causing a disease. It is known that RNA structure can have an influence on alternative splicing patterns. Additionally, the RNA binding protein, Drosha, generally identifies and excises RNA hairpins, but it was recently shown to also act as a splicing factor in a cleavage-independent manner. Therefore, it is thought that Drosha can alter the inclusion rates of exons that form hairpins or the inclusion of exons near a hairpin structure. Drosha expression is known to be altered in some cancers and therefore there may be changes in alternative splicing in disease states. HeLa and HEK-293T cell lines were used in order to determine the alternative splicing patterns of hairpin-structured exons, or those near known miRNAs in cancerous and non-cancerous cells. The spliced ratios were compared to identify genes in which splicing may become altered in disease states where Drosha expression is also altered. The role of Drosha in alternative splicing of these structured exons will be investigated.

References:

1. Kahn, D.H., Jahan, S., Davie, J.R. (2012) Pre-mRNA splicing: Role of epigenetics and implications in disease. Advances in Biological Regulation. 52:3, 377-388.
2. Lim KH, Ferraris L, Filloux ME, Raphael BJ, Fairbrother WG. Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:11093–11098.
3. McManus CJ, Graveley BR. RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev. 2011 Aug;21(4):373-9.

Keywords: Splicing , Drosha

62. Probing the structure of the HIV-1 5´UTR with varying transcription start sites

Jonathan Kitzrow (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Luke Lamorelle (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)

Abstract:
The full-length HIV-1 transcript can act as either mRNA, encoding the Gag and Gag-Pol polyproteins, or as genomic RNA (gRNA) that is selected by Gag during virion assembly. The HIV-1 5′UTR, which is part of all full-length HIV-1 transcripts, is known to regulate many important processes during the viral lifecycle, including RNA splicing, translation, dimerization, and packaging. These processes are regulated, in part, by interactions between host/viral proteins and conserved structural elements within the 5′UTR. Recently, the number of guanosines at the 5′ end of the HIV-1 RNA transcript has been shown to vary due to heterogeneous transcriptional start-site (TSS) selection.¹ 5´-cap-1G transcripts are primarily dimeric and enriched in virions, whereas 5´-cap-2G/3G transcripts are primarily monomeric and enriched on polysomes.^1,2 Mutations designed to destabilize the transactivation response element (TAR) and polyadenylation (PolyA) stem loops of the 5′UTR impair gRNA dimerization.^2,3 Taken together, these results suggest that different TSSs in HIV-1 transcripts influence downstream structures within the 5´UTR, as well as 5´UTR function. To establish the effects of 5´ end heterogeneity on 5´UTR structure, we performed differential selective 2′-hydroxylation analyzed by primer extension (SHAPE) on 5´UTR RNAs with varying numbers of 5´G nucleotides. Results to date suggest nucleotides within TAR/PolyA are more reactive to SHAPE reagents (i.e., less structured) in G3 constructs, whereas nucleotides within the HIV-1 psi packaging element are more reactive in G1 constructs. Thus, preliminary probing studies are consistent with the hypothesis that 5’UTR structure varies depending on TSS selection.

References:

1. Masuda T, Sato Y, Huang YL, Koi S, Takahata T, Hasegawa A, Kawai G, Kannagi M. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA. Sci Rep. 2015;5:17680.
2. Kharytonchyk S, Monti S, Smaldino PJ, Van V, Bolden NC, Brown JD, Russo E, Swanson C, Shuey A, Telesnitsky A, Summers MF. Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome. Proc Natl Acad Sci U S A. 2016;113(47):13378-83.
3. Das AT, Vrolijk MM, Harwig A, Berkhout B. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging. Retrovirology. 2012;9:59.

Keywords: HIV-1, SHAPE, RNA Structure

63. The Evf2 enhancer lncRNA regulates hundreds of enhancer-chromosomal interactions across the extent of chr6 (~150Mb) in mouse developing brain

Ivelisse Cajigas (Department of Pediatrics, Northwestern University), Abhijit Chakraborty (Division of Vaccine Discovery, La Jolla Institute for Allergy & Immunology), Monique Bastidas (Department of Pediatrics, Northwestern University), Kelsey R. Swyter, Sara J. Kohtz (Department of Pediatrics, Northwestern University), Ferhat Ay (Division of Vaccine Discovery, La Jolla Institute for Allergy & Immunology), Jhumku D. Kohtz (Department of Pediatrics, Northwestern University)

Abstract:
Enhancers are defined as DNA sequences capable of regulating genes at a distance, independent of orientation. While validated enhancer regulatory landscapes in vertebrates typically span ~1 megabase (Mb) and facilitate tissue-specific and/or developmentally programmed gene expression, chromosome conformation capture studies (HiCseq and 4Cseq) predict that enhancer interactions can span multi-megabase distances (Dekker, 2016). A major question in genome biology is how DNA regulatory enhancers are selectively targeted to specific genes across megabase (Mb) distances. Here, we show that Evf2, a Dlx5/6 ultraconserved enhancer (Dlx5/6UCE) lncRNA (Feng et al., 2006), regulates genes that are asymmetrically-positioned across 27Mb. The Evf2 RNA cloud localizes to both activated (~1.6Mb distant) and repressed (~27Mb distant) target genes in mouse developing forebrain, controlling distances between Dlx5/6UCE and transcriptional targets. Through both short-range (Dlx6 anti-sense) and long-range (Akr1b8) repression, the Evf2-5’UCE region regulates multiple interneuron subtype genes, identifying a novel signaling pathway in interneuron specification. Surprisingly, Evf2 regulates the number, density, and position of hundreds of Dlx5/6UCE-chr6 interaction sites across chr6 (~150Mb), without affecting transcription. Active histone lysine modifications distinguish Evf2 positively- and negatively-regulated Dlx5/6UCE-chr6 sites, revealing that many sites are marked before Evf2 regulates enhancer chromosomal interactions. These studies reveal that an autosomal cloud-forming enhancer lncRNA regulates genes through antisense and chromosome topological mechanisms, controlling the 3-D architecture of an entire chromosome.

References:
Dekker, J. (2016). Mapping the 3D genome: Aiming for consilience. Nat Rev Mol Cell Biol 17, 741-742.

Feng, J., Bi, C., Clark, B.S., Mady, R., Shah, P., and Kohtz, J.D. (2006). The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator. Genes Dev 20, 1470-1484.

Keywords: Long non-coding RNA, chromosome topology, enhancers

64. tRNA processing in human mitochondria

Agnes Karasik (Department of Biological Chemistry, USU), Markos Koutmos (Departments of Chemistry and Biophysics, University of Michigan)

Abstract:
Mitochondrial diseases affect 1 in every 4000 children born in the US. Despite their frequency, the underlying mechanisms of most mitochondrial diseases are unknown. The majority (>50 %) of mitochondrial diseases are linked to mutations in mitochondrial tRNA (mt-tRNA) genes, which make up only ~10% of the human mitochondrial genome. tRNAs are extensively processed and these maturation steps can be critical for physiological function. The first step in mt-tRNA processing is thought to be the 5’ end maturation of tRNAs by the three-component mitochondrial RNase P protein complex (MRPP-complex). The MRPP complex is comprised of MRPP1 (methyltransferase), MRPP2 (dehydrogenase), and MRPP3 (catalytic nuclease subunit, also referred to as Protein Only RNase P, PRORP). While there is an emerging picture of the importance of the human MRPP-complex to human health, little is known about how the MRPP-complex functions to process precursor mt-tRNAs (pre-mt-tRNAs). Our work demonstrates how patient mutations in pre-mt-tRNAs alter their structure and impact the ability of mitochondrial tRNA maturation enzymes to properly mature mt-tRNAs.
We investigated the binding and processing of 11 human pre-mt-tRNAs with disease-causing mutations by the MRPP-complex using a combination of binding and single turnover assays. We find that a subset of mutations reduces substrate binding to the MRPP-complex. Interestingly, all of the mutations that we investigated are inefficiently processed by the MRPP-complex. We posit that this may be due to alterations in pre-mt-tRNA folding and structure because most of the mutant tRNAs showed altered UV melting profiles. Additionally, our data indicate that some pre-tRNAs require further processing/modification(s) and/or an additional as of yet unidentified protein partner to be processed by the MRPP-complex. We find that pre-mt-tRNAVal is bound ~100 times tighter and processed ~10 times more efficiently by the MRPP-complex when the methyl donor and MRPP1 substrate (SAM) is present in our assays, suggesting that methylation of pre-tRNAVal m1A9 is required for efficient 5’ end cleavage. Taken together, our work reveals the first direct evidence for the role of MRPP1 in the MRPP-complex, and also points to the role of MRPP-complex in human disease.

Keywords: tRNA, processing, mitochondria

65. Mechanisms of ribosome stalling during the translation of positively charged peptides

Daniel Eyler (Department of Chemistry, University of Michigan), Alexandra Rizo (Program in Chemical Biology, University of Michigan), Frances Acevedo, Tyler Smith (Department of Chemistry, University of Michigan), Sarah Keane (Department of Chemistry, Department of Biophysics, University of Michigan), Daniel Southworth (University of California San Francisco), Kristin Koutmou (Department of Chemistry, University of Michigan)

Abstract:
The rate that ribosomes catalyze the addition of amino acids as they translate along an mRNA template is highly variable. Changes in translation speed can have far-reaching consequences, affecting mRNA stability, protein folding, and protein subcellular localization. Both biochemical reporter and genome wide ribosome profiling studies indicate that proteins containing iterated positively charged residues are translated slowly, producing transiently arrested ribosome species. While these stalling events are generally thought to occur because positively charged nascent peptide chains form strong ionic interactions with the negatively charged ribosome exit tunnel, there is little direct evidence for this hypothesis. We previously reported that lysine codon usage (AAA vs AAG codons) is a key determinant for how the ribosome translates poly-lysine containing proteins, suggesting that ribosome stalling relies on more than just peptide-ribosome interactions during the translation of some poly-cationic peptides. Here, we take a high-resolution look at the translation of iterated lysine and arginine containing peptides to directly observe if cationic peptide mediated ribosome stalling occurs, and to characterize the mechanism by which the translation of cationic peptides causes translational stalling. Our combination of structural (electron microscopy and NMR) and kinetic data will provide the first measurements of ribosome elongation rates during the synthesis of poly-arginine peptides, and identify contributions of mRNA and ribosome structure to stalling during the translation of positively charged peptides.

Keywords: ribosome, cationic peptide, stalling

66. Distinct mechanistic features of the atypical SPOUT methyltransferase, Trm10

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

Abstract not available online - please check the printed booklet.

67. Quality control by trans-editing factor prevents global mistranslation of non-protein amino acid α-aminobutyrate

Alexandra B Kuzmishin (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.), Jo Marie Bacusmo, William A Cantara (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.), Yuki Goto, Hiroaki Suga (Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA.)

Abstract:
Accuracy in protein biosynthesis is maintained through multiple pathways, with a critical checkpoint occurring at the tRNA aminoacylation step catalyzed by aminoacyl-tRNA synthetases (AARSs). In addition to Ala-tRNA^Pro editing mediated by the insertion (INS) domain present in most bacterial prolyl-tRNA synthetases (ProRS), single-domain trans-editing factors structurally homologous to INS are present in some organisms. To date, INS-like editing proteins have been shown to act on specific tRNAs that are mischarged with genetically encoded amino acids. However, structurally related non-protein amino acids are ubiquitous in cells and threaten the proteome. Here, we show that Rhodopseudomonas palustris ProXp-x, a previously uncharacterized INS homolog, edits a known ProRS aminoacylation error, Ala-tRNA^Pro, but displays even more robust editing of tRNAs misaminoacylated with the non-protein amino acid α-aminobutyrate (Abu) in vitro and in vivo. Abu is the product of the transamination of oxobutyrate, a metabolite in Ile biosynthesis, and a precursor metabolite in Thr biosynthesis. Abu is mischarged by E. coli ValRS and ProRS in vitro, and in vivo experiments showed that Abu is toxic to an E. coli strain encoding an editing-defective ValRS (1). However, expression of R. palustris ProXp-x rescues cell growth, presumably by removing Abu that has been mischarged onto tRNA^Val by the editing-deficient ValRS. Our results indicate that editing by trans-editing domains such as ProXp-x studied here may offer advantages to cells, especially under environmental conditions where concentrations of non-protein amino acids may challenge the substrate specificity of AARSs.

References:
1. Döring V, Mootz HD, Nangle LA, Hendrickson TL, de Crécy-Lagard V, Schimmel P, Marlière P. Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway. Science. 2001 April 20; 292:501-4.

Keywords: aminoacyl-tRNA synthetase, trans-editing, Rhodopseudomonas palustris

68. Restoration of MYC-repressed miR-29 in pancreatic cancer cells leads to increased reactive oxygen species and gemcitabine sensitization

Jason J Kwon (Medical and Molecular Genetics Dept, Indiana University School of Medicine), Kayla Quirin (Medical and Molecular Genetics Dept, Indiana University School of Medicine), Arafat Alijoufi (Medical and Molecular Genetics Dept, Indiana University School of Medicine), Amira Nafiseh (Medical and Molecular Genetics Dept, Indiana University School of Medicine), Janaiah Kota (Medical and Molecular Genetics Dept, Indiana University School of Medicine)

Abstract not available online - please check the printed booklet.

69. Phenylalanyl-tRNA synthetase regulated quality control is linked to stress responses in Saccharomyces cerevisiae

Amanda Kyle (Department of Microbiology, The Ohio State University), Rebecca Mann (Department of Microbiology, The Ohio State University), Kyle Mohler (Department of Microbiology, The Ohio State University), Michael Ibba (Department of Microbiology, The Ohio State University)

Abstract:
Compared to other essential cellular processes, translation is more error-prone, with an error rate of approximately 1 in 10⁴ codons¹. In translation, aminoacyl-tRNA synthetases (aaRSs) are enzymes which bind amino acids to cognate tRNAs¹. AaRSs have editing processes to prevent the misincorporation of structurally similar amino acids into proteins¹. Recently, it has been shown that phenylalanyl-tRNA synthetase (PheRS) quality control is important in the regulation of the general amino acid control (GAAC) pathway in Saccharomyces cerevisiae². To further explore how quality control affects S. cerevisiae, the growth of a PheRS editing deficient strain was compared to a wild-type (WT) strain in many conditions using phenotypic microarrays. Conditions where differences in growth were observed were assigned to genes with previously established relationships to those chemicals. Many of these genes were associated with the target of rapamycin (TOR) and GAAC pathways³. When grown in caffeine supplemented media, the PheRS editing deficient strain was more tolerant to caffeine than the WT strain³. There is evidence linking mutations in the TOR pathway to differential growth in caffeine³. To explore the relationship between PheRS quality control and the TOR pathway, growth of ∆TOR WT and ∆TOR PheRS editing deficient strains were compared in a chronological lifespan assay. It has been previously documented that ∆TOR yeast mutants have prolonged chronological lifespans³. However, we observed that the ∆TOR mutation in the PheRS editing deficient strain had a decreased effect on chronological lifespan compared to the ∆TOR WT strain. This evidence suggests a relationship between PheRS regulated quality control and stress responses in S. cerevisiae. Since translation is a comparatively error-prone cellular process and there is evidence that mistranslation may be advantageous¹, it is important to understand how lacking quality control affects the cell.

References:
1. Reynolds, N.M., Lazazzera, B.A., and Ibba, M. Cellular mechanisms that control mistranslation. Nature Reviews Microbiology. 2010, 8, 849-856.
2. Mohler, K., Mann, R., Bullwinkle, T.J., Hopkins, K., Hwang, L., Reynolds, N.M., Gassaway, B., Hans-Rudolf, A., Rinehart, J., Polymenis, M., Faull, K. and Ibba, M. Editing of misaminoacylated tRNA controls the sensitivity of amino acid stress responses in Saccharomyces cerevisiae. Nucleic Acids Research. 2017, 45, 3985-3996.
3. Mohler, K., Mann, R., Kyle, A., Reynolds, N. and Ibba, M. Aminoacyl-tRNA Quality Control is Required for Efficient Activation of the TOR Pathway Regulator Gln3p. RNA Biology, In press.

Keywords: aminoacyl-tRNA, TOR pathway, Saccharomyces cerevisiae

70. In-silico design and experimental validation of Boolean logic gates based on a fluorogenic RNA aptamer

Geneva R. LaForce (Department of Chemistry, Ball State University), Victoria R. Goldsworthy (Department of Chemistry, Ball State University), Dayna A. M. Arnett (Department of Chemistry, Ball State University), Emil F. Khisamutdinov (Department of Chemistry, Ball State University)

Abstract:
RNA aptamers that bind a non-fluorescent dye and activate its fluorescence are highly sensitive, non-perturbing, and convenient probes in the field of synthetic biology. These artificial aptamers operate as molecular nanoswitches that experience altered folding and function in response to ligand binding. We demonstrate a computational approach for designing smart RNA nanodevices based on a malachite green (MG) binding RNA aptamer in which fluorescent output is controlled by the binding of DNA oligonucleotide inputs. Four types of RNA switches, possessing AND, OR, XOR, and NAND Boolean logic functions, were created in modular form, allowing MG dye binding affinity to be changed without altering the RNA aptamer core sequence. All computationally designed RNA devices were synthesized and experimentally tested in vitro. The ability to design smart nanodevices based on RNA binding aptamers offers new ways to engineer ligand-sensing regulatory circuits, nucleic acid detection systems, gene control elements, and a promising complement to silicon technology.

Keywords: Boolean logic, RNA aptamers, nanodevice

71. Characterization of K-turns in RNase P RNAs from Halobacteriales

Stella M. Lai (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Lien B. Lai (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Mengxuan Jia, Andrew Norris (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Charles J. Daniels (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

72. Genome-wide Identification of Enzymatic Targets of a DEAD-box RNA Helicase Reveals Roles for RNA Secondary Structure Remodeling in Termination of Transcription

Yu-Hsuan Lai (Department of Biochemistry, Purdue University), Krishna Choudhary (Department of Biomedical Engineering, UC Davis), Sharon Aviran (Department of Biomedical Engineering, UC Davis), Siwen Wang (Department of Biochemistry, Purdue University), Elizabeth Tran (Department of Biochemistry, Purdue University)

Abstract not available online - please check the printed booklet.

73. Identifying novel inhibitors of RNA riboswitches by targeting transient binding pockets

Jessica D. Leuchter (University of Michigan Chemistry Department ), Aaron T. Frank (University of Michigan Chemistry and Biophysics Departments )

Abstract not available online - please check the printed booklet.

74. Validating and Optimizing Mechanistic Model of Cooperative Ligand Binding to the Oligomeric Trp- and RNA-binding protein TRAP

Weicheng Li (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210), Elihu C. Ihms (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210), Ian R. Kleckner (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210), Melody L. Holmquist (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY14260), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210)

Abstract:
Biological networks are frequently regulated via the cooperative binding of multiple ligands. Cooperativity serves to fine tune regulatory circuits by amplifying or dampening a response to a cellular signal. Cooperativity can be synergistic (positive), or interfering (negative), depending on whether the affinity of subsequent ligands binding is increased or decreased by the effect of previous binding event. Despite its ubiquity and importance, our understanding of mechanisms of cooperativity remains limited. TRAP (trp RNA-binding Attenuation Protein) is a homo-undecamer whose RNA binding activity is regulated by binding of L-tryptophan (Trp) to its eleven identical binding sites. The Trp binding events are thought to stabilize the RNA-binding state of TRAP and favor RNA binding. Trp binding events show cooperativity in Bsu TRAP, Bst TRAP, and Bst TRAP A28I via isothermal titration calorimetry (ITC) experiments. By fitting temperature-dependent ITC data using a nearest-neighbor (NN) statistical thermodynamic model, Trp binding to Bst TRAP A28I we quantify the microscopic contributions of Trp binding to the affinity at neighboring sites. The NN model assumes that binding of Trp to one of its 11 sites will energetically affect binding at the adjacent sites (nearest-neighbor) in TRAP ring. This model is able to reproduce thermodgrams of Trp-binding to Bst TRAP A28I over a range of temperatures. We continue to apply this NN model to test its ability to predict Trp and RNA binding to wild-type Bst TRAP and Bsu TRAP.

References:
1.Ihms, E. C., Kleckner, I. R., Gollnick, P., & Foster, M. P. (2017). Mechanistic Models Fit to Variable Temperature Calorimetric Data Provide Insights into Cooperativity. Biophysical journal, 112(7), 1328-1338.

Keywords: Cooperativity, ITC, Statistical thermodynamic model

75. The Hydrophobic Effect from Conjugated Chemicals or Drugs on in Vivo Biodistribution of RNA Nanoparticles

Zhefeng Li (Ohio State University, College of Pharmacy, Columbus, Ohio, United States), Danny Jasinski (Ohio State University, College of Pharmacy, Columbus, Ohio, United States), Hongran Yin (Ohio State University, College of Pharmacy, Columbus, Ohio, United States), Peixuan Guo (Ohio State University, College of Pharmacy, Columbus, Ohio, United States)

Abstract not available online - please check the printed booklet.

76. Polyadenylation in the Apicomplexa

Ashley T. Stevens (Department of Plant and Soil Sciences, University of Kentucky), Josiah Liew (Department of Plant and Soil Sciences, University of Kentucky), Daniel K. Howe (Department of Veterinary Science, University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky)

Abstract:
The process by which the 3’-ends of precursor mRNAs are processed and polyadenylated is an essential step for gene expression in eukaryotes and one at which gene expression may be regulated. In the best characterized of systems, mRNA polyadenylation is mediated by a sizeable complex of proteins that recognizes the polyadenylation signal AAUAAA (and similar A-rich counterparts in yeast and plants), motifs present 5’ (or upstream) of the polyadenylation signal, motifs situated downstream of the poly(A) site, and the actual cleavage/polyadenylation site itself (1). To date, the consensus picture of polyadenylation comes largely from studies conducted in animals and yeast. However, considerable variety in poly(A) signals exists in other organisms (2-4).
With these ideas in mind, a bioinformatics and high throughput sequencing study of polyadenylation in members of the phylum Apicomplexa has been conducted. Analysis of the published genomes for S. neurona, T. gondii, and P. falciparum suggest that the apicomplexan polyadenylation complex is either highly reduced, or consists of several (as yet unidentified) novel subunits. Antibodies raised against the most highly-conserved poly(A) complex subunit (CPSF73) have been used to confirm the presence of the expected polypeptide in one of these organisms, S. neurona. Experiments intended to clarify further the nature of the complex – reduced or vastly different - are in progress.
The nature of the apicomplexan poly(A) signal is unclear. To gain further insight into this, a high throughput sequencing approach was taken, focusing on three apicomplexans - S. neurona, T. gondii, and N. caninum. The results indicate that all three organisms possess a similar, distinctive polyadenylation signal that is reminiscent of that seen in higher plants. These results seem at odds with the bioinformatics study, since the subunits that recognize the analogous polyadenylation signal in mammals are not seen in the bioinformatics study. The resolution of this paradox is the focus of current research.

References:
1. Shi, Y. and Manley, J.L. (2015) The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site. Genes Dev, 29, 889-897.
2. Franzen, O., Jerlstrom-Hultqvist, J., Einarsson, E., Ankarklev, J., Ferella, M., Andersson, B. and Svard, S.G. (2013) Transcriptome profiling of Giardia intestinalis using strand-specific RNA-seq. PLoS Comput Biol, 9, e1003000.
3. Fuentes, V., Barrera, G., Sanchez, J., Hernandez, R. and Lopez-Villasenor, I. (2012) Functional analysis of sequence motifs involved in the polyadenylation of Trichomonas vaginalis mRNAs. Eukaryot Cell, 11, 725-734.
4. Wodniok, S., Simon, A., Glockner, G. and Becker, B. (2007) Gain and loss of polyadenylation signals during evolution of green algae. BMC Evol Biol, 7, 65.

Keywords: polyadenylation, RNA processing, evolutionary conservation1

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

Kefei Liu (Department of Radiology and Imaging Sciences, Indiana University School of Medicine), Li Shen (Department of Radiology and Imaging Sciences, Indiana University School of Medicine), Hui Jiang (Department of Biostatistics, University of Michigan)

Abstract not available online - please check the printed booklet.

78. SHAPE Analysis of the RSV Genomic RNA 5'-leader Secondary Structure and Gag Interactions

Shuohui Liu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Tiffiny Rye-McCurdy (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Christiana Binkley (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH)

Abstract not available online - please check the printed booklet.

79. Selectivity mechanisms of aminoacyl-tRNA trans-editing factor

Xiao Ma (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Eric M. Danhart, Marina Bakhtina, William A. Cantara, (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Alexandra B. Kuzmishin, Brianne L. Sanford (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Marija Kosutic, Ronald Micura (Institute of Organic Chemistry, Center for Molecular Biosciences, Leopold Franzens University), Yuki Goto, Hiroaki Suga (Department of Chemistry, Graduate School of Science, The University of Tokyo), Kotaro Nakanishi, Mark P. Foster, Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for charging amino acids onto their cognate tRNAs. The structural features of the aaRSs catalytic domains provide a high degree of selectivity for attachment of the correct amino acid; however, given the similar stereochemical properties of many of the amino acids, errors in tRNA charging can occur, especially for the smaller and isometric amino acids. Many organisms possess editing enzymes that function in trans to hydrolyze mischarged tRNAs to maintain high fidelity translation. ProXp-ala, a structural homolog of the editing domain from ProRS, can deacylate mischarged Ala-tRNA^Pro. To determine how ProXp-ala selectively deacylates Ala-tRNA^Pro over the cognate Pro-tRNA^Pro, or Ala-tRNA^Ala, we performed NMR, site-directed mutagenesis mapping and MD simulations of ProXp-ala with an uncharged microhelix^Pro and a non-hydrolyzable, amide-linked Ala-microhelix^Pro substrate analog. Relaxation experiments showed that helix α2, which exhibits dynamics on the ps-ns timescale, is less mobile when bound to Ala-microhelix^Pro, but remains dynamic when bound to the uncharged microhelix^Pro. Relaxation dispersion experiments also reveal that this helix is dynamic on the μs-ms timescale. Flexibility of helix α2 is also supported by a new crystal structure (PDB: 5VXB) of the protein obtained in a different crystallographic space group than a previously published structure (PDB: 1VJF); the structures differ only at in helix α2. Correspondence between stereochemical properties of amino acids and the deacylation activity of ProXp-ala sheds light on both the role of size and aminoacyl functional groups in the discrimination mechanism. These suggest that chemical selection, size exclusion and conformational selection contribute to allow ProXp-ala to conduct specific deacylation of Ala-tRNA^Pro. We expect the high-resolution structure of ProXp-ala in complex with its substrate analogue to further illuminate its mechanism.

References:
Danhart, E. M., Bakhtina, M., Cantara, W. A., Kuzmishin, A. B., Ma, X., Sanford, B. L., Košutić, M., Goto, Y., Suga, H., Nakanishi, K., et al. (2017) Conformational and chemical selection by a trans-acting editing domain. Proc. Natl. Acad. Sci. 114, 6774–6783.

Keywords: conformational selection, NMR, trans-editing

80. The Effect of Metal Ions on Lariat Debranching Enzyme Cleavage Activity

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

Abstract:
Lariat debranching enzyme (Dbr1p) is a metallophosphoesterase that specifically cleaves the 2'-5' phosphate linkage in lariats introns formed during splicing. It is a crucial enzyme and deletion is lethal in most organisms. The homology of the Dbr1p conserved active site residues to those of the DNA repair enzyme MRE11 as well as early in vitro experiments on Saccharomyces cerevisiae Dbr1p suggested that the active site harbors two Mn²⁺ ions for proper function. However, recent crystal structures of Entamoeba histolytica Dbr1p suggest Fe²⁺ and Zn²⁺ or Mn²⁺ and Zn²⁺ as the active site metal ions needed for full activity. We have found, intriguingly, while S. cerevisiae Dbr1p is inactivated by EDTA, E. histolytica Dbr1p retains significant activity. Here we present our assays on the debranching activity of E. histolytica Dbr1p with different metal ions following EDTA treatment. We report the effect of ions of Group II metals as well as transition metals on the observed rate constant for cleavage of branched RNA substrates.

Keywords: Lariat debranching enzyme, Branched RNA, Metallophosphoesterase

81. A study of aminoglycoside binding to 16S rRNA

Prabuddha Madubashitha (Department of Chemistry, Wayne State University, Detroit, MI 48202), Evan Jones (Department of Chemistry, Wayne State University, Detroit, MI 48202), Christine S. Chow (Department of Chemistry, Wayne State University, Detroit, MI 48202)

Abstract:
Despite the potency of aminoglycosides (AG), development of bacterial resistance and adverse side effects on human health including ototoxicity are the major challenges of using AGs as antibiotics. Modifying currently available AGs is a potential strategy in developing better antibiotics that maintain activity but with reduced anti-mitoribosomal binding and associated ototoxicity. Towards this end, knowledge of the binding of a particular AG to its target at the molecular level is important. The objective of our study was to determine the binding affinity and examine drug-induced structural changes of a modified AG, 4'-O-ethyl paromomycin, and helix 44 (h44) of the aminoacyl-tRNA site (A site) of 16S rRNA. By using both chemical footprinting and a fluorescence-based assay, 4'-O-ethylparomomycin, was shown to have affinity for both the bacterial ribosome and bacterial h44 construct. More importantly, 4'-O-ethyl paromomycin displays improved selectivity towards the bacterial A site compared to the corresponding mitochondrial construct in the fluorescence-based assay. This result supports the observed reduction of ototoxicity for 4'-O-ethyl paromomycin.1 NMR data suggest that 4'-O-ethyl paromomycin retains a similar binding mode to that of 4, 5-disubstituted AGs to the bacterial A-site rRNA. These results will lead to a better understanding of drug-rRNA interactions at the molecular level.

References:
Reference
1. Duscha, S., Boukari, H., Shcherbakov, D., Salian, S., Silva, S., Kendall, A., Kato, T., Akbergenov, R., Perez-fernandez, D., Bernet, B., Vaddi, S., Thommes, P., Schacht, J., Crich, D., Vasella, A., Böttger, E. C. MBio. 2014, 5, e01827-14

Keywords:

82. Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo

Rebecca Mann (Department of Microbiology Ohio State University ), Kyle Mohler (Department of Microbiology Ohio State University), Michael Ibba (Department of Microbiology Ohio State University )

Abstract:
Amino acid misincorporation during protein synthesis occurs due to misacylation of tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids has been observed in a variety of contexts, less work has been done to directly assess the extent to which specific tRNAs are misacylated in vivo, and the identity of the misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation profiling (ISAP), a method to identify and quantify the amino acids attached to a tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific isoacceptor tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNA^Phe and tRNA^Tyr species were isolated from total aminoacyl-tRNA extracted from both yeast and Escherichia coli. Isolated aminoacyl-tRNAs were washed until free of detectable unbound amino acid and subsequently deacylated. Free amino acids from the deacylated fraction were then identified and quantified by mass spectrometry. Using ISAP allowed quantification of the effects of quality control on the accumulation of misacylated tRNA species under different growth conditions.

References:
Reynolds, N.M., Lazazzera, B.A., and Ibba, M. (2010) Cellular mechanisms that control mistranslation. Nature Reviews 2010, 8, 849-856.

Mohler, K., Mann, R. and Ibba, M. (2017) Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo. Methods, 113:127-131.

Keywords: tRNA, aaRS

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

Aidan C. Manning (Medical Sciences Program, Indiana University, Bloomington, Indiana), Sarah N. Deffit, Pranathi Vadlamani, Heather A. Hundley (Medical Sciences Program, Indiana University, Bloomington, Indiana), Brian A. Yee, Emily C. Wheeler, Alain Domissy, Gene W. Yeo (Department of Cellular and Molecular Medicine, Stem Cell Program and Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA), Suba Rajendren, Michael C. Washburn (Department of Biology, Indiana University, Bloomington, Indiana)

Abstract not available online - please check the printed booklet.

84. Use of SHAPE-Seq and FRET to elucidate structure-function relationships in archaeal RNase P, a multi-subunit catalytic ribonucleoprotein

Ila A. Marathe (Department of Chemistry and Biochemistry, The Ohio State University), Paul D. Carlson (Department of Chemical and Biological Engineering, Northwestern University), Ben Donovan, Michael G. Poirier (Department of Physics, The Ohio State University), Shiqin Miao, Jie Mao, Dennis Bong (Department of Chemistry and Biochemistry, The Ohio State University), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

85. tRNA recognition and catalysis by eukaryoticThg1 3’-5’ polymerase

Ashanti Matlock (Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

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

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

Abstract not available online - please check the printed booklet.

87. Widespread presence of internal translation initiation sites in Escherichia coli

Sezen Meydan (Center for Biomolecular Sciences, University of Illinois at Chicago, USA), James Marks (Center for Biomolecular Sciences, University of Illinois at Chicago, USA), Dorota Klepacki, Amira Kefi (Center for Biomolecular Sciences, University of Illinois at Chicago, USA), Tonu Margus (Department of Molecular Biology, Umea University, Sweden), Nora Vazquez-Laslop (Center for Biomolecular Sciences, University of Illinois at Chicago, USA), Alexander S. Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago, USA)

Abstract not available online - please check the printed booklet.

88. Post-transcriptional regulation upon tumor-suppressor loss

Wayne Miles (Molecular Genetics, The Ohio State University Comprehensive Cancer Center, The Ohio State University)

Abstract not available online - please check the printed booklet.

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

Vibhor Mishra (Howard Hughes Medical Institute, Department of Biology, Indiana University Bloomington), Jasleen Singh ( Department of Biology, Indiana University Bloomington), Akihito Fukudome ( Department of Biology, Indiana University Bloomington), Craig S Pikaard (Howard Hughes Medical Institute, Department of Biology, Indiana University Bloomington)

Abstract not available online - please check the printed booklet.

90. Glyoxals as in vivo RNA structural probes of guanine base pairing

David Mitchell III (Department of Chemistry, Penn State University), Laura E. Ritchey (Department of Chemistry, Penn State University), Hongmarn Park (Department of Biochemistry and Molecular Biology, Penn State University), Paul Babitzke (Department of Biochemistry and Molecular Biology, Penn State University), Sarah M. Assmann (Department of Biology, Penn State University), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Penn State University)

Abstract:
Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice (Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli, Gram positive and Gram negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues.

References:
1. Staehelin M. 1959. Inactivation of virus nucleic acid with glyoxal derivatives. Biochim Biophys Acta 31: 448-454.

2. Nakaya K, Takenaka O, Horinishi H, Shibata K. 1968. Reactions of glyoxal with nucleic acids. Nucleotides and their component bases. Biochim Biophys Acta 161: 23-31.

3. Aubert M, Bellemare G, Monier R. 1973. Selective reaction of glyoxal with guanine residues in native and denatured Escherichia coli 5S RNA. Biochimie 55: 135-142.

4. Krymkiewicz N. 1973. Reactions of methylglyoxal with nucleic acids. FEBS Lett 29: 51-54.

5. Shapiro R, Cohen BI, Clagett DC. 1970. Specific acylation of the guanine residues of ribonucleic acid. J Biol Chem 245: 2633-2639.

Keywords: Glyoxal, RNA structure, in vivo RNA probing

91. Modulating the insulin receptor splicing in pediatric sarcomas

Matias Montes (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), Brianne Sanford (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital), Thomas Bebee (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital), Frank Rigo (Ionis Pharmaceuticals, Carlsbad CA), Peter Houghton (Greehey Childrens Cancer Research Institute, University of Texas Health Science Center), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital)

Abstract not available online - please check the printed booklet.

92. Investigating the effects of PUS5 deletion on mitochondrial encoded protein expression in Candida albicans and Saccharomyces cerevisiae

Allyson R. Morris (Department of Biology, Ball State University ), Douglas A. Bernstein (Department of Biology, Ball State University )

Abstract:
While RNA is made of only 4 bases, these bases can be modified in over 100 distinct ways. These modifications play critical roles modulating RNA function. Pseudouridine is the most common modified nucleoside, and is found in all kingdoms of life. However, the role of pseudouridylation in translation and RNA function is not well understood. Pseudouridylation is found at dozens of sites in Eukaryotic cytoplasmic rRNA and cytoplasmic ribosomes translate thousands of proteins. As such, it is challenging to study the effects individual sites of pseudouridylation have on translation. In contrast, in fungi, mitochondrial ribosomes translate only eight genes encoded by the mitochondrial genome and mitochondrial rRNA contains only one highly conserved pseudouridine which is made by the pseudouridine synthase Pus5. We find Saccharomyces cerevisiae pus5Δ deletion is more sensitive to drugs that inhibit oxidative phosphorylation such as oligomycin, suggesting they have a defect in mitochondrial function. We will investigate the roles of Pus5 mediated pseudouridylation on mitochondrial protein expression. We will use mass spectrometry to determine if mitochondrial rRNA pseudouridylation is required for wild type mitochondrial gene translation. Investigation of this highly conserved RNA modification will lead to a better understanding of how defects in pseudouridylation lead to human disease and could lead to the identification of novel antifungal drug targets.

Keywords: Candida albicans, Saccharomyces cerevisiae , Mitochondrial protein expression

93. Analysis of alternative splicing in disease associated exons

Stephanie Nguyen (Biology, Lewis University), Jessica King (Biology, Lewis University)

Abstract:
The process of pre-mRNA splicing involves the removal of introns, the noncoding regions within an RNA transcript, and the ligation of coding regions, or exons. Alternative splicing maintains gene regulation through a variation in the specific patterns of exon inclusion and intron exclusion in the final mRNA transcript. This also allows for individual genes to code for multiple proteins, which contributes to diversity. Approximately 33% of genetic mutations are thought to be the result of an improper regulation in splicing causing a disease. It is known that RNA structure can have an influence on alternative splicing patterns. Additionally, the RNA binding protein, Drosha, generally identifies and excises RNA hairpins, but it was recently shown to also act as a splicing factor in a cleavage-independent manner. Therefore, it is thought that Drosha can alter the inclusion rates of exons that form hairpins or the inclusion of exons near a hairpin structure. Drosha expression is known to be altered in some cancers and therefore there may be changes in alternative splicing in disease states. HeLa and HEK-293T cell lines were used in order to determine the alternative splicing patterns of hairpin-structured exons, or those near known miRNAs in cancerous and non-cancerous cells. The spliced ratios were compared to identify genes in which splicing may become altered in disease states where Drosha expression is also altered. The role of Drosha in alternative splicing of these structured exons will be investigated.

References:
Kahn, D.H., Jahan, S., Davie, J.R. (2012) Pre-mRNA splicing: Role of epigenetics and implications in disease. Advances in Biological Regulation. 52:3, 377-388.

Lim KH, Ferraris L, Filloux ME, Raphael BJ, Fairbrother WG. Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:11093–11098.

McManus CJ, Graveley BR. RNA structure and the mechanisms of alternative splicing. Curr Opin Genet Dev. 2011 Aug;21(4):373-9.

Keywords: Alternative Splicing , Drosha

94. Development of a genome-wide screen in S. cerevisiae to identify novel retrograde tRNA nuclear importers and re-exporters

Regina T. Nostramo (Department of Molecular Genetics; The Ohio State University), Anita K. Hopper (Department of Molecular Genetics; The Ohio State University)

Abstract not available online - please check the printed booklet.

95. The Molecular Mechanisms of ADAR3 in Glioblastoma

Eimile Oakes ( Genome, Cell and Developmental Biology Program, Indiana University), Ashley Anderson ( Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Gabrielle McNary ( Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Aaron A. Cohen-Gadol (Department of Neurosurgery, Indiana University School of Medicine, Goodman Campbell Brain and Spine, Indianapolis, IN), Heather A. Hundley (Medical Sciences Program, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine)

Abstract not available online - please check the printed booklet.

96. Programmable Nucleic Acid Based Polygons with Controlled Neuroimmunomodulatory Properties for Predictive QSAR Modeling

M. Brittany Johnson, Ian Marriott (Department of Biological Sciences, University of North Carolina at Charlotte), Justin R Halman, Emily Satterwhite, Kirill Afonin (Nanoscale Science Program, Department of Chemistry), Alexey V. Zakharov (National Center for Advancing Translational Sciences, National Institutes of Health), My Bui, Kheiria Benkato, Victoria Goldsworthy, Emil Khisamutdinov (Department of Chemistry, Ball State University), Taejin Kim (Department of Chemistry, New York University), Enping Hong, Marina A. Dobrovlskaia (Nanotechnology Characterization Lab, Cancer Research Technology Program, Leidos Biomedical Research, Inc, Frederick National Laboratory for Cancer Research)

Abstract:
In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential to understand the immunostimulatory aptitude of such tools and identify potential complications. This paper presents a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. It is demonstrated that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance the current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, quantitative structure-activity relationship modeling is conducted. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predicts the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.

Keywords: QSAR, RNA nanotechnology, immunology

97. Mechanisms and functional consequences of processing and generating the intronic Drosophila RNase P RNA

Geeta Paslule (The Ohio State University, Department of Molecular Genetics), Lien B. Lai (The Ohio state University, Department of Chemistry and Biochemistry ), Venkat Gopalan (The Ohio state University, Department of Chemistry and Biochemistry ), Amanda Simcox (The Ohio State University, Department of Molecular Genetics)

Abstract:
RNase P, an essential and conserved ribonucleoprotein that catalyzes removal of the 5' leader from pre-tRNAs, is comprised of a catalytic RNA (RNase P RNA, RPR) and a variable number of proteins (1). In eukaryotes, RPR was considered a canonical RNA Pol III-regulated transcript, until we discovered that in insects and crustaceans it is embedded in an intron of a Pol II regulated recipient gene, and lacks signals for Pol III transcription. In Drosophila, we showed that RPR is transcribed as part of a Pol II-regulated recipient gene transcript (2). To enable this switch in transcriptional control, which occurred ~500 mya, we hypothesize that nucleases involved in processing other ncRNAs were coopted to trim the 5' and 3' ends of the RPR’s recipient intron and generate the mature RPR. To identify these nucleases, I used RNAi knockdown of candidates in Drosophila cells and examined the impact on RPR biogenesis. My results suggest that XRN2 and the exosome are involved in maturation of RPR. Interestingly, XRN2, a 5' to 3' exonuclease, affects both 5' and 3' processing. I am currently investigating how 5' and 3' end processing are coupled by determining if XRN2 recruits factors to the 3' end and/or resolves a secondary structure in the RNA to reverse a block on 3' processing. To determine whether the mode of transcription has functional consequence(s), I have engineered transgenes regulated by an ancestral-type, Pol III promoter. Purification of RNase P from cells expressing the transgene shows that Pol III-derived RPR co-purifies with the holoenzyme. Preliminary results from pre-tRNA cleavage assays suggest that the Pol III-derived RPR may be active. I am using CRISPR/Cas9 genome editing to generate a fly solely expressing a Pol III-regulated RPR, as this will enable a phenotypic analysis at the molecular, cellular and organismal levels. Collectively, these results will determine whether there is a link between transcriptional control, biogenesis, and function for Drosophila RPR.

References:
1. Lai LB, Vioque A, Kirsebom LA, Gopalan V (2010) Unexpected diversity of RNase P, an ancient tRNA processing enzyme: challenges and prospects. FEBS Lett 584(2):287–296
2. Manivannan SN, Lai LB, Gopalan V, Simcox A (2015) Transcriptional Control of an Essential Ribozyme in Drosophila Reveals an Ancient Evolutionary Divide in Animals. PLoS Genet 11(1): e1004893. https://doi.org/10.1371/journal.pgen.1004893

Keywords: RNase P RNA, Transcriptional control, Splicing

98. Nascent RNA sequencing and capture of minimally disturbed newly divided cells

Luke S. Parks (Department of Radiation Oncology, University of Michigan), Michelle Paulsen (Department of Radiation Oncology, University of Michigan), Mats Ljungman (Departments of Radiation Oncology, Environmental Health Sciences, University of Michigan)

Abstract not available online - please check the printed booklet.

99. Competition between the RNA exosome and TFIIS for backtracked RNA Polymerase II

Sarah A. Peck (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), M.J. Fox, W.R. Smith-Kinnaman, J. F. Victorino, G.O. Hunter (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Hongyu Gao, Yunlong Liu (Center for Computational Biology and Bioinformatics, Indiana University School of Medicine), Amber L. Mosley (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine)

Abstract not available online - please check the printed booklet.

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

Justin Peterson (Molecular and Cellular Biochemistry Indiana University Bloomington ), Heather A. Hundley (Medical Sciences Program, and Department of Biochemistry and Molecular Biology, Indiana )

Abstract not available online - please check the printed booklet.

101. Structural and functional characterization of human Argonaute4

Hong-Duc Phan (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210), Miseul Park (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210), James A. Brackbill (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210), Kotaro Nakanishi (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, 43210)

Abstract:
Human Argonaute4 (AGO4) is a slicer-independent Argonaute protein that uses the loaded microRNA to bind to target mRNAs and triggers translational repression and deadenylation. Recent studies reported that AGO4 plays specialized roles in regulating spermatogenesis and internal ribosome entry site-driven translation although those molecular mechanisms remain unknown. To understand how AGO4 treats the bound miRNA differently from other paralogues, we determined the crystal structure of AGO4 in the complex with guide RNA at 1.9 Å. Unlike the previously reported model, our structure showed that a different glutamate was rearranged to complete the pseudo catalytic tetrad. The consensus of the rearrangement with other paralogues imply another role of the corresponding glutamate in addition to the catalytic reaction. An AGO4-specific insertion loop, previously proposed to be poised on the PIWI domain, actually protruded towards the nucleic acid-binding channel. Our target-strand docking model suggested that the loop interfered with the propagation of guide-target duplex. In contrast, AGO2 possessed a kink-turn at the corresponding position and did not seem to interact with the duplex. Supporting this, an implant of the AGO4-specific insertion loop into AGO2 completely disrupted the target cleavage. These data demonstrate that the specific loop confers a different target specificity on AGO4.

Keywords: AGO4, crystal structure, insertion loop

102. Identification of functions of pseudouridine synthase 7 in Candida albicans

Ethan Pickerill (Ball State University Biology Department), Dr. Douglas Bernstein (Ball State University Biology Department)

Abstract:
Candida albicans is the most prevalent human fungal pathogen. To identify new antifungal targets we must first better understand what differentiates fungi from humans at the molecular level. We are interested in RNA modification and in particular how the process of RNA modification differs between fungal taxa and humans. We study pseudouridine, the most common RNA modification. Pseudouridylation occurs in all organisms and can be present in tRNA, rRNA, mRNA, snoRNA, and snRNA. This study investigates the effects of CRISPR mediated deletion of Pseudouridine synthase 7 in C. albicans. The PUS7 knockout has defects in filamentation and grows slower than wild type C. albicans. The PUS7 knockout also has lesser virulence than wild type C. albicans in the Galleria mellonella in vivo infection model. Additionally, northern blot and high throughput sequencing indicates the PUS7 knockout has defects in rRNA processing. In the future, we will identify C. albicans Pus7 substrates and explore the possibility of a Pus7 consensus sequence.

References:
Binkley J, Arnaud M, Inglis D, Skrzypek M, Shah P, Wymore F, Binkley G, Miyasato S, Simison M, Sherlock G (2014). The Candida Genome Database. Nucleic Acids Res 42 (Database issue); D711-6
Charette, M. & Gray, M. in IUBMB Life, Vol. 49 341-351 (2000).
Delarze, E., Ischer, F., Sanglard, D., & Coste, A. (2015). Adaptation of a gaussia princeps luciferase reporter system in candida albicans for in vivo detection in the galleria mellonella infection model. Virulence, 6(7), 684-693
Hamma, T. & Ferré-D'Amaré, A.R. Pseudouridine Synthases. Chemistry & Biology 13, 1125-1135 (2006).
Mayer, F., Wilson, D., & Hube, B. (2013). Candida albicans pathogenicity mechanisms. Virulence, 4(2), 119-128. doi:10.4161/viru.22913
Vyas, V.K., Barrasa, M.I. & Fink, G.R. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv 1, e1500248 (2015).

Keywords: Pseudouridine, Pseudouridine Synthase 7, Candida Albicans

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

Abigail Bieker (Biology, Lewis University), Elizabeth Przekwas (Biology, Lewis University), Mallory Havens (Biology, Lewis University)

Abstract not available online - please check the printed booklet.

104. tRNA modification profile changes during growth phase transitions in Bacillus subtilis

Christina Psihountas (Chemistry Department, University of Cincinnati), Manasses Jora (Chemistry Department, University of Cincinnati), Patrick A. Limbach (Chemistry Department, University of Cincinnati)

Abstract:
Transfer Ribonucleic Acids (tRNAs) have more modified bases than the other species of RNA. Temperature, culture media, stress, and cell development are suspected to influence tRNA nucleoside modifications in a qualitative and/or quantitative nature. Current databases do not reflect modification changes or detail the culturing conditions of obtained data. Changes in tRNA modifications are being studied in the gram positive and spore forming bacteria, Bacillus subtilis. Here we show that there is a change in the tRNA profile as the bacteria progresses through its life cycle. This method will also be used to monitor tRNA profiles during sporulation. We hypothesize that we will see a change in nucleoside abundance when vegetative cell and spore tRNA profiles are compared. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we can detect and characterize modifications from total tRNA samples obtained at different phases of growth and during sporulation.

References:
El Yacoubi, Basma, Marc Bailly, and Valérie de Crécy-Lagard. 2012. Annual Review of Genetics 46(1): 69-95.
Xiao Wang and Chuan He. 2014. Molecular Cell 56: 5-12.
K. Okamoto and B.S. Vold. 1992. Biochimie 74: 613-618.
Yun-Hua Jeng and Roy H. Doi. 1975. Journal of Bacteriology, 121(3): 950-958
Meyer, F., et al. 2008. BMC Bioinformatics 9(1): 386.

Keywords: growth phase, tRNA modifications, LCMS

105. Time-dependent differential alternative splicing in mammalian tissues

Krithika R Subramanian (Department of Pharmacology and System Physiology, University of Cincinnati and Department of Pediatrics, Division of Biomedical Informatics, Cincinnati Childrens Hospital Medical Research Center), Nathan Salomonis (Department of Pediatrics, Division of Biomedical Informatics, Cincinnati Childrens Hospital Medical Research Center and Department of Biomedical Informatics, University of Cincinnati), Christian I Hong (Department of Pharmacology and System Physiology, University of Cincinnati )

Abstract:
Abstract:

Recent data suggests that alternative splicing (AS) may play a profound role in the temporal regulation of RNA transcripts to maintain circadian rhythm (CR). Mammalian CR is controlled by a core clock molecular complex in the hypothalamus and downstream by clock controlled genes (CCGs). Dysfunctional CR is implicated in sleep, behavioral, neurological disorders and even in cancer. Though AS is proven to add a regulatory layer of control in addition to transcription regulation, its exact role in establishing or maintaining the core clock and other peripheral clocks in organ systems are not well studied. To establish a critical link between AS and CR across multiple mammalian tissues and develop automated bioinformatics workflows for these analyses, we performed a deep analysis of RNA-Seq data from 12 tissues in mice, sampled every 6 hrs for a 48hr time course.
Using non-parametric periodicity detection algorithms we identified thousands of rhythmic splicing events, with varying frequencies among the different tissues examined. Comparison of these splicing events and genes across circadian RNA-Seq datasets by-and-large confirmed the validity of these predictions, with improved accuracy relative to exon microarray circadian splicing predictions. From these data, we observe a disproportionate number of splicing events relative to circadian gene expression changes across the same tissues, particularly in the brain, which has the highest proportion of splicing events (17% of all events detected). While a high percentage of circadian transcriptional regulation occurred in the Heart, Brown adipose, Skeletal Muscle and Liver, these same tissues showed some of the least frequent circadian splicing (~5% of all events in each). We also observe that the large majority of the rhythmic splicing events are tissue specific whereas, a small proportion is (n=49) consistently regulated across multiple tissues (n≥5). These data suggest that alternative splicing differentially tunes circadian expression in specific tissues, likely regulated by tissue specific splicing regulators. Further understanding of the role of these splicing regulators and splicing events may yield prospective chrono-therapeutic targets, which we aim to evaluate experimentally.

Keywords: Alternative splicing, Circadian rhythm

106. Dimerization of ADARs contributes to substrate specificity of A-to-I editing in vivo

Suba Rajendren (Genome, Cell and Developmental Biology Program, Indiana University Bloomington), Justin Peterson (Molecular and Cellular Biochemistry Indiana University Bloomington), Heather A. Hundley (Medical Sciences Program, and Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Bloomington)

Abstract not available online - please check the printed booklet.

107. Lariat Debranching Enzyme Cleavage of Backbone Branched RNAs With Non-Canonical Branch-Point Residues

Timothy Ratterman (Department of Chemistry, Carnegie Mellon University), Stephanie Mack (Department of Chemistry, Carnegie Mellon University), Subha Das (Department of Chemistry, Carnegie Mellon University)

Abstract:
In splicing, lariat introns are formed typically with a conserved branch-point adenosine. Lariat debranching enzyme (Dbr1p) that specifically cleaves the 2',5'-phosphodiester linkage in lariat RNA thus encounters the branch-point adenosine in natural substrates. Preliminary data from our lab suggests that S. cerevisiae Dbr1p cleaves backbone branched RNAs (bbRNAs) in which the branch-point residue is not adenosine. Recent crystal structures of the active site of Dbr1p from E. histolytica show how the branch-point residue is accommodated in the active site. Here we describe our synthesis of dual fluorescently labeled bbRNAs with C, G and U besides A as branch-point residues. These dual-labeled bbRNAs enable real time kinetics assay of the Dbr1p cleavage reaction. From these assays we analyze the binding and cleavage activity of E. histolytica Dbr1p towards these bbRNAs with non-canonical branch-point residues.

Keywords: Dbr1p, bbRNA

108. Regulatory elements in the HIV-1 5´UTR modulate Gag binding specificity

Joshua-Paolo C. Reyes (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Erik D. Olson (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)

Abstract:
The 5ʹUTR of the HIV-1 genomic RNA (gRNA) contains a structured RNA element (termed Psi) that is specifically recognized by the HIV-1 Gag polyprotein, ensuring that two copies of gRNA are packaged into newly assembled virions. However, the mechanism by which Gag recognizes gRNA over other cellular RNAs and spliced viral RNAs is not well understood. A recent study suggested that a negative regulatory element upstream of Psi reduces high-affinity Gag binding, and a positive downstream regulatory element counteracts the upstream element and restores high-affinity binding^1,2. These elements are proposed to form a long-range interaction that promotes packaging of only full-length gRNA and excludes packaging of spliced RNAs. Using a fluorescence anisotropy-based salt-titration binding assay, which measures the electrostatic and nonelectrostatic (i.e., specific) components of protein binding RNA, we have previously shown that Gag interacts with a 109-nt Psi RNA construct with high specificity and relatively few electrostatic interactions³. Using a 356-nt RNA construct that now includes the upstream negative regulatory element in addition to Psi, we observed a loss in Gag binding specificity and an increase in electrostatic interactions. However, a 400-nt construct that additionally contains the positive element, restored highly specific binding and reduced the electrostatic interactions, similar to the 109-nt Psi RNA. Interestingly, Gag binding specificity remained high even in constructs wherein the 44 nt of the downstream positive regulatory element were scrambled. Thus, while the presence of downstream sequences appears to be important for high-specificity Gag binding, their identity is not. Taken together, these data are consistent with a mechanism whereby the negative and positive regulatory elements flanking Psi modulate Gag binding but call into question the long-range interaction model.

References:
¹Abd El-Wahab, E. W., Smyth, R. P., Mailler, E., Bernacchi, S., Vivet-Boudou, V., Hijnen, M. et al. Specific recognition of the HIV-1 genomic RNA by the Gag precursor. (2014) Nat Commun 5, 4304.

²Bernacchi, S., Abd El-Wahab, E. W., Dubois, N., Hijnen, M. E. W., Smyth, R. P. et al. HIV-1 Pr55Gag binds genomic and spliced RNAs with different affinity and stoichiometry. (2017) RNA Biology 14:1, 90-103

³Webb J. A., Jones C. P., Parent L. J., Rouzina I, Musier-Forsyth K. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging. (2013) RNA 19, 1078-88.

Keywords: HIV-1 Gag, HIV-1 Psi, gRNA Packaging

109. Screen for chemical inhibitors of repeat associated non-AUG (RAN) translation

Udit Sheth (Undergraduate Program in Neuroscience, University of Michigan and Department of Neurology, University of Michigan), Katelyn Green (Department of Neurology, University of Michigan and Cellular and Molecular Biology Graduate Program, University of Michigan), Veronica Towianski (Department of Neurology, University of Michigan), Michael Kearse (Department of Neurology, University of Michigan and Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine), Peter Todd (1Department of Neurology, University of Michigan and Veteran Administration Medical Center, Ann Arbor, MI)

Abstract not available online - please check the printed booklet.

110. Transcriptome analysis of developing lens reveals abundance of novel transcripts and extensive splicing alterations

Rajneesh Srivastava (Biohealth Informatics, IU School of Informatics and Computing, IUPUI), Gungor Budak (Biohealth Informatics, IU School of Informatics and Computing, IUPUI), Soma Dash (Biological Sciences, University of Delaware), Salil A. Lachke (Biological Sciences, University of Delaware), Sarath Chandra Janga (Biohealth Informatics, IU School of Informatics and Computing, IUPUI)

Abstract:
Lens development involves a complex and highly orchestrated regulatory program. In this study, we investigate the transcriptomic alterations and splicing events during mouse lens formation using RNA-seq data from multiple developmental stages, and construct a molecular portrait of known and novel transcripts. We present a database “Express” (http://www.iupui.edu/~sysbio/express/) that unifies the processed mouse lens transcriptome across multiple developmental stages. Express provides heatmap and browser view allowing easy navigation of the genomic organization and expression of transcripts or gene loci. It enables the export of both the visualizations and the embedded data to facilitate downstream analysis. We also demonstrate that the extent of novelty (compared to annotated isoforms from reference databases) of expressed transcripts decreases significantly in post-natal lens compared to embryonic stages. Characterization of novel transcripts into partially novel transcripts (PNTs) and completely novel transcripts (CNTs) (novelty score ≥ 70%) revealed that the PNTs are both highly conserved across vertebrates and highly expressed across multiple stages. Functional analysis of PNTs revealed their widespread role in lens developmental processes while hundreds of CNTs were found to be widely expressed and predicted to encode for proteins. We verified the expression of four CNTs across stages. Examination of splice isoforms revealed skipped exon and retained intron to be the most abundant alternative splicing events during lens development. We validated by RT-PCR and Sanger sequencing, the predicted splice isoforms of several genes Banf1, Cdk4, Cryaa, Eif4g2, Pax6, and Rbm5. Finally, we present a splicing browser Eye Splicer (http://www.iupui.edu/~sysbio/eye-splicer/), to facilitate exploration of developmentally altered splicing events and to improve understanding of post-transcriptional regulatory networks during mouse lens development.

References:
Khan SY, Hackett SF, Lee MC, Pourmand N, Talbot CC Jr & Riazuddin SA. Invest Ophthalmol Vis Sci. 2015 Jul;56(8):4919-26.
Dash, S., Siddam, A.D., Barnum, C.E., Janga, S.C. & Lachke, S.A. Wiley Interdiscip Rev RNA. 2016 Jul;7(4):527-57.
Lachke, S.A. & Maas, R.L. Wiley Interdiscip Rev Syst Biol Med. 2010 May-Jun;2(3):305-23.
Srivastava, R., Budak, G., Dash, S., Lachke, S.A. & Janga, S.C. Transcriptome analysis of developing lens reveals abundance of novel transcripts and extensive splicing alterations. Scientific Reports. 2017 (In press).
Budak G., Dash, S., Srivastava, R., Lachke, S.A. & Janga, S.C. Express: A database of transcriptome profiles encompassing known and novel transcripts across multiple development stages in eye tissues. Exp Eye Res. (Accepted)

Keywords: Mouse development, Eye tissue, Alternative splicing

111. Polyadenylation in the Apicomplexa

Ashley T. Stevens (Department of Plant and Soil Sciences, University of Kentucky), Josiah Liew (Department of Plant and Soil Sciences, University of Kentucky), Daniel K. Howe (Department of Veterinary Science, University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky)

Abstract:
The process by which the 3’-ends of precursor mRNAs are processed and polyadenylated is an essential step for gene expression in eukaryotes and one at which gene expression may be regulated. In the best characterized of systems, mRNA polyadenylation is mediated by a sizeable complex of proteins that recognizes the polyadenylation signal AAUAAA (and similar A-rich counterparts in yeast and plants), motifs present 5’ (or upstream) of the polyadenylation signal, motifs situated downstream of the poly(A) site, and the actual cleavage/polyadenylation site itself (1). To date, the consensus picture of polyadenylation comes largely from studies conducted in animals and yeast. However, considerable variety in poly(A) signals exists in other organisms (2-4).

With these ideas in mind, a bioinformatics and high throughput sequencing study of polyadenylation in members of the phylum Apicomplexa has been conducted. Analysis of the published genomes for S. neurona, T. gondii, and P. falciparum suggest that the apicomplexan polyadenylation complex is either highly reduced, or consists of several (as yet unidentified) novel subunits. Antibodies raised against the most highly-conserved poly(A) complex subunit (CPSF73) have been used to confirm the presence of the expected polypeptide in one of these organisms, S. neurona. Experiments intended to clarify further the nature of the complex – reduced or vastly different - are in progress.

The nature of the apicomplexan poly(A) signal is unclear. To gain further insight into this, a high throughput sequencing approach was taken, focusing on three apicomplexans - S. neurona, T. gondii, and N. caninum. The results indicate that all three organisms possess a similar, distinctive polyadenylation signal that is reminiscent of that seen in higher plants. These results seem at odds with the bioinformatics study, since the subunits that recognize the analogous polyadenylation signal in mammals are not seen in the bioinformatics study. The resolution of this paradox is the focus of current research.

References:
1. Shi, Y. and Manley, J.L. (2015) The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site. Genes Dev, 29, 889-897.

2. Franzen, O., Jerlstrom-Hultqvist, J., Einarsson, E., Ankarklev, J., Ferella, M., Andersson, B. and Svard, S.G. (2013) Transcriptome profiling of Giardia intestinalis using strand-specific RNA-seq. PLoS Comput Biol, 9, e1003000.

3. Fuentes, V., Barrera, G., Sanchez, J., Hernandez, R. and Lopez-Villasenor, I. (2012) Functional analysis of sequence motifs involved in the polyadenylation of Trichomonas vaginalis mRNAs. Eukaryot Cell, 11, 725-734.

4. Wodniok, S., Simon, A., Glockner, G. and Becker, B. (2007) Gain and loss of polyadenylation signals during evolution of green algae. BMC Evol Biol, 7, 65.

Keywords: polyadenylation, RNA processing, evolutionary conservation

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

Qinyu Sun,Vidisha Tripathi, Deepak K. Singh,Qinyu Hao (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign), Je-Hyun Yoon,Kyung-Won Min,Sylvia Davila, Richard Zealy (Department of Biochemistry and Molecular Biology, Medical University of South Carolina), Xiao Ling Li, Ashish Lal, (Genetics Branch, Center for Cancer Research, National Cancer Institute), Dawood Dudekula, Elin Lehrmann, Yongqing Zhang, Kevin G. Becker (Laboratory of Genetics, National Institute of Aging-Intramural Research program, NIH), Myriam Gorospe (Laboratory of Genetics, National Institute of Aging-Intramural Research program, NIH), Supriya G. Prasanth, Kannanganattu V. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign)

Abstract not available online - please check the printed booklet.

113. Mitochondrial gene expression responds to lack of nutrients but not to different nutrient sources in Trypanosoma cruzi

Emily K. Susa (Biomedical Sciences, University of Minnesota Medical School, Duluth campus), Sara L. Zimmer (Biomedical Sciences, University of Minnesota Medical School, Duluth campus)

Abstract:
Trypanosoma cruzi is a vector-borne protozoan parasite that causes Chagas’ disease. The parasite has a complex life cycle which involves a variety of different environments and nutrient sources. In the midgut of the insect vector, glucose and amino acids are available to T. cruzi, but these resources dwindle over time and prompt differentiation into a nonreplicative but highly infective life stage. Upon contact with a host, T. cruzi invades mammalian cells and resumes replication, likely utilizing fatty acids for energy. To better understand the complicated relationship between nutrient source, metabolism and life stage differentiation, we are examining changes in the T. cruzi mitochondrial genome within different life stages. We are interested in the mitochondrial genome because of its roles in metabolism and because it exhibits life-stage specific remodeling in related species. Mitochondrial genome regulation must occur post-transcriptionally in the form of RNA editing, translational control, and stability. Previously, we examined mRNA transcript abundance between the insect midgut (epimastigote) stage and the insect hindgut (metacyclic trypomastigote) stage. Significant changes were detected in mature mRNA abundance between these life stages, but the differences appeared to have a stronger connection with the process of starvation than with the process of differentiation. We now examine mitochondrial transcript abundance in the intracellular (amastigote) stage. Expression is similar to that of actively dividing epimastigotes, and different from the starved population used to infect the mammalian cultures, with RNA editing as well as turnover appearing to be the cause of the differences. Our results indicate that environmental nutrient depletion rather than predominant source of energy is the major stimulus for changes to mitochondrial gene expression across the life cycle.

Keywords: mitochondria, editing, Trypanosoma cruzi

114. Kinetics of drug-ribosome interactions defines the cidality of macrolide antibiotics

Maxim Svetlov (University of Illinois at Chicago), Nora-Vazquez Laslop (University of Illinois at Chicago), Alexander Mankin (University of Illinois at Chicago)

Abstract:
Ribosome is a large ribonucleoprotein particle responsible for the synthesis of all proteins in a living cell. Due to its vital role, ribosome is one of the main antibiotic targets in bacteria. It is well known that the interaction of antibiotic with its target molecule can cause dormancy (bacteriostasis) or induce death (cidality) of bacteria. The bactericidal capacity is one of the most critical characteristics of antibacterial agents. However, the current understanding of the fundamental differences in the binding or action of bacteriostatic and bactericidal antibiotics is very rudimentary. Here, by examining the action of several structurally related but chemically distinct ribosome-targeting bacteriostatic and bactericidal macrolide inhibitors we have identified a key difference, which correlates with the ability of the drugs to cause cell death. While bacteriostatic and bactericidal macrolide antibiotics, which bind in the nascent peptide exit tunnel, exhibit comparable affinity to the ribosome, the bactericidal antibiotics are characterized by a significantly slower dissociation rates. The presence and the nature of an extended alkyl-aryl side chain in the macrolide structure, which establishes idiosyncratic interactions with the ribosome, are essential for the characteristically slow rate of dissociation of bactericidal inhibitors. Importantly, low level of residual translation in cells treated with normally bactericidal antibiotics can protect cells from killing even at concentrations of the drugs that significantly exceed those required for cell growth arrest. On the basis of our findings, we propose a new conceptual framework for explaining difference in cidality of antibiotics of the same class.

Keywords: ribosome, macrolides, bactericidal antibiotics

115. Specificities of HTLV-1 Gag RNA-binding domains: implications for genomic RNA dimerization and primer annealing

Yu-Ci Syu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Weixin Wu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Joshua Hatterschide (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Alice Duchon, Yingke Tang (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), William Cantara (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210)

Abstract:
During assembly of retrovirus particles, the Gag polyprotein selectively packages two copies of full-length viral genomic RNA (gRNA) in a process mediated by a specific interaction between Gag and the gRNA packaging signal. In HIV-1, the nucleocapsid (NC) domain of Gag is critical for specific gRNA selection and tRNA primer annealing to the primer binding site (PBS). In contrast, the mechanism of gRNA packaging and primer annealing in deltaretroviruses such as human T-cell lymphotropic virus type 1 (HTLV-1) are less clear. Previous studies suggested that deltaretroviral matrix (MA) plays a more important role than NC in the initial gRNA selection process (Sun, M., et al, J. Virol 2014). To test this hypothesis, UV cross-linking and selective 2'-hydroxyl acylation analyzed by primer extension (XL-SHAPE) were used to determine the secondary structure of the HTLV-1 5' leader for the first time, and to characterize HTLV-1 MA- and NC-gRNA interactions. SHAPE probing of gRNA alone did not support the formation of the previously proposed stem-loop 1 (SL1) and revealed that the PBS is embedded in a highly-structured hairpin. XL-SHAPE identified sites of both MA and NC interaction with the gRNA, as well as conformational changes upon protein binding. Moreover, a new primary dimerization initiation site (DIS) located in the U5 region was identified and confirmed by in vitro dimerization assays. We also demonstrated that the putative primer for HTLV-1 reverse transcriptase (RT), tRNA^Pro, is not efficiently annealed to the complementary PBS by either HTLV-1 NC or MA. Deletion of the gRNA sequence that is base paired to the PBS significantly increases full-length tRNA^Pro annealing, which suggests this sequence may play a role in regulating primer annealing in HTLV-1. Interestingly, a fragment derived from the 3'-18 nt of tRNA^Pro is present in HTLV-1 particles (Ruggero, K., et al, J. Virol 2014) and can be fully annealed to the PBS even in the absence of a chaperone protein. Studies to identify the RT primer in HTLV-1 virions are currently underway.

References:
Ruggero, K., Guffanti, A., Corradin, A., Sharma, V.K., De Bellis, G., Corti, G., Grassi, A., Zanovello, P., Bronte, V., Ciminale, V., & D'Agostino, D.M. Small noncoding RNAs in cells transformed by human T-cell leukemia virus type 1: a role for a tRNA fragment as a primer for reverse transcriptase. J. Virol. 88, 3612-3622 (2014).
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.
88, 1271-1280 (2014).

Keywords: HTLV-1, Genome dimerization, Primer annealing

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

Courtney Szyjka (Department of Biological Sciences, University at Buffalo), Natalie McAdams (Department of Microbiology and Immunology, University at Buffalo), Emily Bonacquisti (Department of Biological Sciences, University at Buffalo), Justin Durland (Department of Biological Sciences, University at Buffalo), Paul Gollnick (Department of Biological Sciences, University at Buffalo)

Abstract:
Transcriptional regulation of the tryptophan (trp) biosynthetic operon in Bacillus subtilis is controlled by the trp RNA-binding attenuator protein (TRAP). Regulation of this operon was initially described as depending on 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 thus halting transcription of the trp genes. In limiting tryptophan conditions, TRAP does not bind the trp leader RNA, the anti-terminator forms and the genes are transcribed and translated to produce the tryptophan biosynthesis enzymes. However, recent work has shown that, 1) formation of the terminator RNA structure isn’t sufficient to induce transcription termination in the absence of TRAP and 2) in vivo TRAP can induce termination in the absence of the terminator RNA structure. Together these observations suggest that TRAP may have a more direct role in transcription attenuation. Consistent with this suggestion, we have recently isolated a TRAP mutant (E60K) that is capable of binding RNA and tryptophan at wild-type (WT) levels, but is deficient in transcription attenuation. We used a suppression screen to identify protein interactors of TRAP that are necessary for efficient transcription attenuation in the trp operon. By placing the toxic mazF gene under control of the trp leader region, we identified B. subtilis proteins that will enhance transcription attenuation of the E60K TRAP mutant in vivo. A candidate gene produced from the screen is yteA, a currently uncharacterized gene that shares homology with E. coli transcription factor dksA. Through use of strains with transcriptional fusions between the trp leader and β-galactosidase, we show that a mutant attenuator (disrupted stem) shows decreased TRAP-mediated regulation when yteA is knocked out.

Keywords: transcription attenuation, transcription termination, suppression screen

117. Investigating the importance of the bulge region for S. enterica htrA RNA thermometer behavior using SHAPE assays.

Yow Yong Tan (Department of Chemistry and Biochemistry, Denison University), Rachel M. Mitton-Fry (Department of Chemistry and Biochemistry, Denison University)

Abstract:
RNA thermometers (RNATs) are structural elements that can be found in the 5′- untranslated region of bacterial mRNAs that encode for heat shock proteins or virulence factors. They regulate the expression of these genes by changing conformation in response to temperature. Such regulation enables immediate response to environmental changes and allows pathogenic bacteria to detect the successful invasion of a warm-blooded host. In Salmonella enterica, an RNA thermometer in the htrA gene regulates the expression of HtrA, an enzyme critical for Salmonella virulence. Our previous work showed that a single-nucleotide bulge plays a significant role in tuning the htrA thermometer melting behavior. To better our understanding of the importance of the bulge region, we studied the melting behavior of a mutant htrA thermometer that switched the position of the base pairs flanking the bulge. We performed Selective 2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) assays on the mutant and wild-type at different temperatures representing the standard growth conditions, warm-blooded body temperatures, and heat-shock conditions. Our results reveal that this mutation seems to have a subtle effect on the melting behavior of the thermometer.

Keywords: SHAPE, RNAT, htrA

118. Improving the sequence coverage of RNA modification mapping by LC-MS

Priti Thakur (Department of Chemistry, University of Cincinnati), Patrick Limbach (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati)

Abstract not available online - please check the printed booklet.

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

Jackson B. Trotman (Center for RNA Biology, The Ohio State University), Bernice Agana (Department of Chemistry and Biochemistry, The Ohio State University), Andrew J. Giltmier (Center for RNA Biology, The Ohio State University), Chandrama Mukherjee (Center for RNA Biology, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Daniel R. Schoenberg (Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

120. Identifying regulators of C9orf72 ALS/FTD associated RAN translation

Yi-Ju Tseng (Molecular, Cellular, and Developmental Biology Graduate Program, University of Michigan, Ann Arbor, MI), Alexander E. Linsalata (Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI), Katelyn M. Green (Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI), Nicholas Santoro (Center for Chemical Genomics, Life Sciences Institute, University of Michigan, Ann Arbor, MI), Martha J. Larson (Center for Chemical Genomics, Life Sciences Institute, University of Michigan, Ann Arbor, MI), Peter K. Todd (Department of Neurology, University of Michigan, Ann Arbor, MI; VA medical Center, Ann Arbor, MI)

Abstract:
A GGGGCC repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently this locus was shown to support Repeat-Associated Non-AUG (RAN) translation, a non-canonical initiation process that generates toxic dipeptide repeat proteins from C9orf72 repeat-containing mRNAs. To better understand the mechanism underlying RAN translation, we performed a genome-wide siRNA screen using luciferase-based reporters for G₄C₂-expanded C9orf72 RAN translation (C9RAN). We identified 642 of genes that exhibited selective inhibition of RAN translation while sparing translation of an AUG initiated control. One candidate gene, the RNA and DNA helicase DHX36, was validated using overexpression and knock-down studies at both the DNA and RNA level. Overexpression of DHX36 strongly suppressed global translation while sparing C9RAN translation across reading frames. In contrast, knockdown of DHX36 caused a greater impact on RAN translation than AUG initiated translation. Identifying factors that selectively modulate RAN translation should lay the groundwork for rational therapeutic design in these currently untreatable neurologic disorders.

Keywords: C9orf72, DHX36, RAN translation

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

Ullas Valiya Chembazhi (Biochemistry, University of Illinois at Urbana-Champaign), JuYeon Lee (Chemistry, University of Illinois at Urbana-Champaign), Sang Ba Ho (Biochemistry, University of Illinois at Urbana-Champaign), Kevin Yum (Biochemistry, University of Illinois at Urbana-Champaign), Steven C. Zimmerman (Chemistry, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Chemistry, University of Illinois at Urbana-Champaign)

Abstract not available online - please check the printed booklet.

122. Use of pulsed EPR to probe protein cofactor-mediated long-range remodeling of the catalytic RNA in archaeal RNase P

Vibhuti Wadhwa (Department of Chemistry and Biochemistry, The Ohio State University), Xiao Ma (Department of Chemistry and Biochemistry, The Ohio State University), Antonia Duran (Ohio State Biochemistry Program, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
In all domains of life, RNase P is an essential enzyme required for processing 5’-leader sequence of precursor tRNAs to yield a mature tRNA. This universally conserved ribonucleoprotein form of RNase P consists of a single RNA subunit (RPR) and variable number of protein subunits (RPPs): one RPP in bacteria, up to five in archaea and up to ten in eukarya.¹ Although the RPR is active on its own in vitro, the RPPs are essential in vivo. Biochemical reconstitution of the archaeal RPR with RPPs significantly improves the enzyme’s substrate affinity and cleavage rate.^2,3 Archaeal RNase P serves as an excellent model to understand the need the for increased protein complexity in higher organisms, due to its tractability and homology of archaeal and eukaryotic RPPs.¹ To probe the structural changes induced in the archaeal RPR upon binding of RPPs, we are using double electron-electron resonance (DEER), a pulsed EPR technique, that allows distance measurements between site-specifically spin labeled RPPs or RPR. Insights into the conformational rearrangement of the RPR as a function of RPPs will provide information about subunit positions and structural changes that are responsible for modulating enzymatic activity.

References:
(1) Lai, L. B., Vioque, A., Kirsebom, L. A., and Gopalan, V. (2010) Unexpected diversity of RNase P, an ancient tRNA processing enzyme: Challenges and prospects. FEBS Lett.
(2) Chen, W. Y., Pulukkunat, D. K., Cho, I. M., Tsai, H. Y., and Gopalan, V. (2010) Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex. Nucleic Acids Res. 38, 8316–8327.
(3) Cho, I.-M., Lai, L. B., Susanti, D., Mukhopadhyay, B., and Gopalan, V. (2010) Ribosomal protein L7Ae is a subunit of archaeal RNase P. Proc. Natl. Acad. Sci. U. S. A. 107, 14573–14578.

Keywords: RNase P, EPR, Spin label

123. XL-SHAPE Analysis of Human Lysyl-tRNA Synthetase Interaction with HIV-1 Genomic RNA

Petra C. Wallenmeyer (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH), Kevin A. Garayalde Batista (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH), Joshua Hatterschide (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH), Karin Musier-Forsyth (Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research,The Ohio State University, Columbus, OH)

Abstract:
Reverse transcription of HIV-1 genomic RNA (gRNA) is initiated from the 3' end of human tRNA^Lys3, which is annealed to the primer binding site (PBS) in the 5'UTR. All tRNALys isoacceptors are specifically packaged into HIV-1 particles, along with human lysyl-tRNA synthetase (LysRS). Moreover, specific interactions between a tRNA-like element (TLE) in the 5'UTR and LysRS are proposed to facilitate targeting of tRNA^Lys3 to the PBS.¹ The TLE mimics the U-rich anticodon loop of tRNA^Lys3 and is located proximal to the PBS. A previous study showed that high-affinity LysRS binding to the 5'UTR required additional stem-loop (SL) elements within the gRNA Psi packaging signal.¹ Previous work also showed that the isoform of LysRS that is packaged into HIV-1 particles is phosphorylated on S207.² To further characterize the interaction between the HIV-1 5’UTR and LysRS and to understand the effect of LysRS phosphorylation on this interaction, cross-linking and selective 2'-hydroxyl acylation analyzed by primer extension (XL-SHAPE) studies were performed using WT LysRS and the phosphomimetic S207D LysRS variant. SHAPE studies revealed that both proteins confer moderate RNA flexibility changes in the TLE of both WT dimeric and monomeric RNA constructs. Both proteins also confer decreases in flexibility in a bulge in SL1 of Psi, consistent with studies showing that SL1 contributes to high-affinity binding of LysRS to gRNA.¹ Thus, a specific SL1:LysRS interaction may contribute to LysRS binding to the 5′UTR and cross-linking studies currently underway will address this question.

References:
1. Jones CP, Saadatmand J, Kleiman L, Musier-Forsyth K. Molecular mimicry of human tRNA^Lys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing. RNA. 2013;19(2):219-229.

2. Duchon AA, St-Gelais C, Titkemeier N, Hatterschide J, Musier-Forsyth K. HIV-1 exploits dynamic multi-aminoacyl-tRNA synthetase complex to enhance viral replication. J. Virol. 2017, in press.

Keywords: HIV, XL-SHAPE, LysRS

124. Evaluating the hybridization of backbone branched and spliceosomal RNAs

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

Abstract:
In the pre-messenger RNA splicing process, introns or non-coding regions of RNA are removed as lariats that form a loop with the 5'-end of the intron attached to an internal branch-point adenosine via a 2',5'- phosphodiester linkage. The highly conserved U6 and U2 small nuclear RNA (snRNA) found in spliceosomes are known to form duplexes along the branch point and recently observed in the yeast cryo-EM structures. We aim to study these interactions in vitro with synthetic backbone branched RNAs (bbRNAs) that can mimic the lariat intron sequences near the branch point. Here, we study the effects of bbRNAs and the effects of the 2',5'- phosphodiester bond on the thermal stability of RNA duplexes that would form with U2 and U6 snRNAs and variants through optical thermal melting measurements. These data will enable us to design RNA-based hybridization and splicing assays.

Keywords: backbone branched RNA, splicing, melting temperature

125. Recent Updates to the RNAstructure Software Package for RNA Secondary Structure Prediction and Analysis

Richard M. Watson (Biochemistry and Biophysics, University of Rochester), Michael Sloma (Biochemistry and Biophysics, University of Rochester), David H. Mathews (Biochemistry and Biophysics, University of Rochester)

Abstract:
    RNAstructure is a suite of software tools for RNA secondary structure prediction, structure analysis, and sequence alignment. It is comprised of command-line programs, a desktop graphical user interface (GUI), and online webservers. RNAstructure has received a number of improvements over the past year. The programs can now predict structures for nucleic acids of any size alphabet, and therefore modified or designed residues can be included in structure prediction. The Java-based GUI has been enhanced with new tools and is now the primary GUI on all platforms (Windows, Mac OS X, and Linux). It now features a workflow-based UI in addition to the previous single-task operations. A new graphical structure editor has been added, which allows users to visually edit CT and dot-bracket structure files and also produce publication-quality RNA structure drawings.
    Several new command-line tools have been added to the package: design generates a sequence to fold with low ensemble defect to a target structure. ProbScan calculates loop probabilities in thermodynamic ensembles. CycleFold predicts non-canonical base pairs. Rsample calculates basepair probabilities for a sequence that has multiple conformations. TurboFold II predicts alignments in addition to conserved RNA structures for a set of input RNA homologs.
    Additional improvements have been made to quality control and distribution of the package; regression testing of the command-line tools has been enhanced and new regression tests of the GUI have been added. Improved installers are available for Windows and Mac operating systems. Parallel versions of most programs are now available to speed predictions on computers with multiple processor cores.  Overall, these changes improve the ease, accuracy, and speed of RNA secondary structure analysis for users from a variety of backgrounds and skill levels.

References:

• Mathews, D.H., Turner, D.H., and Watson, R.M. 2016. RNA secondary structure prediction. Curr. Protoc. Nucleic Acid Chem. 67:11.2.1-11.2.19. doi: 10.1002/cpnc.19
• Spasic, A., Assmann, S.M., Bevilacqua, P.C. and Mathews, D.H. "Modeling RNA secondary structure folding ensemble using SHAPE mapping data." (Manuscript in preparation).
• Sloma, M.F. and Mathews, D.H. "Accurate prediction of RNA non-canonical pairs using probability estimates." (In review).
• Harmanci, A.O., Sharma, G., and Mathews, D.H. BMC Bioinformatics, 12:108. (2011).
• Reuter, J.S. and Mathews, D.H. "RNAstructure: software for RNA secondary structure prediction and analysis." BMC Bioinformatics, 11:129. (2010).

Keywords: RNA, Secondary Structure, Prediction

126. Bru-Seq reveals modulation of mRNA stability by Human Pumilio proteins

Michael Wolfe, Peter Freddolino (Department of Biological Chemistry, University of Michigan, Department of Computational Medicine and Bioinformatics, University of Michigan), Trista Schagat (Department of Biological Chemistry, University of Michigan), Michelle Paulsen, Mats Ljungman (Department of Radiation Oncology, University of Michigan), Brian Magnuson (Department of Radiation Oncology, University of Michigan), Daeyoon Park, Aaron Goldstrohm (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota), Chi Zhang (Department of Biological Sciences, University of Texas at Dallas)

Abstract:
Post-transcriptional gene regulation in eukaryotic systems is widely controlled through the binding of diverse regulators, including miRNAs and proteins, to the 3’ untranslated region of target mRNA molecules.1-3 The RNA binding proteins Human Pumilio1 (PUM1) and Pumilio 2 (PUM2) have been demonstrated to negatively regulate the protein expression of mRNA targets through binding of the consensus sequence 5’-UGUANAUW-3’.3-6 Previous studies in the Goldstrohm lab using RNA-seq have revealed nearly 1000 mRNA targets that either increase or decrease in expression when both PUM1 and PUM2 are knocked down with RNAi.7 However, RNA-seq is only able to assess the steady state levels of mRNA in any given condition and thus PUM effect on mRNA stability cannot be directly assessed. In order to separate the effects of mRNA stability from transcriptional activation, we employ a combination of Bromouridine sequencing (Bru-Seq) and Bromouridine-Chase sequencing (BruChase-Seq), in both WT and PUM knockdown HEK293 cells.8 Here, we reveal a total of 308 genes that have a statistically significant change in mRNA stability upon PUM knockdown. Of these genes, 252 increased in mRNA stability and 56 decreased in mRNA stability. On the contrary, only one gene was shown to have a significant change in transcription rate upon PUM KD. Motif analysis showed high mutual information between the UGUANAUW motif and genes that increased in mRNA stability upon PUM KD, suggesting that the primary mode of PUM mediated repression of gene expression is through the modulation of mRNA stability.


References:
References:
1). Mitchell et al. Nat Struct Mol Biol. Jan 2013; 20(1): 127–133.
2) Lee et al. Science. 2001 Oct 26;294(5543):862- 4.
3) Quenault et. al. Trends Cell Biol. 2011 Feb;21(2):104-12.
4) Wickens et al. Trends Genet. 2002 Mar;18(3):150-7.
5) Spassov et al. Gene. 2002 Oct 16;299(1-2):195-204
6) Galgano et. al. PLoS ONE. 2008; 3(9): e3164.
7) Bohn et al. (submitted, under review)
8) Paulsen, M. T. et al. Methods 67, 45–54 (2014).


Keywords: Bru-seq, mRNA stability , RBPs

127. Alternate exon junction complexes potentiate branched nonsense-mediated mRNA decay pathways

Justin Mabin (Department of Molecular Genetics, the Ohio State University), Lauren Woodward (Department of Molecular Genetics, the Ohio State University), Robert Patton (Department of Physics), Mengxuan Jia (Department of Chemistry and Biochemistry), Vicki Wysocki (Department of Chemistry and Biochemistry), Guramrit Singh (Department of Molecular Genetics, the Ohio State University)

Abstract:
Pre-mRNA splicing deposits the exon junction complex (EJC) 24 nt upstream of most exon-exon junctions in a sequence-independent manner. The stable EJC core consists of eIF4AIII, Y14 and Magoh, which serves as an interaction platform for more dynamic peripheral EJC proteins that direct mRNA export, localization, translation and nonsense-mediated mRNA decay (NMD). To date, the specificity of the peripheral EJC proteins in target RNA selection and the resulting impact on mRNA fate remains largely unknown. We recently reported that MLN51, a protein widely presumed to be an EJC core factor, and several peripheral EJC proteins are sub-stoichiometric in EJCs purified from human cells. We have now discovered that MLN51 and RNPS1, two EJC proteins previously implicated in NMD, exist in two mutually exclusive EJCs in human and mouse cells. Surprisingly, while some mRNAs are preferentially bound by one of the alternate EJC factors, most endogenous mRNAs including many that are targeted for NMD have similar alternate EJC occupancy. Notably, as compared to MLN51-EJCs, RNPS1-EJCs are more enriched in non-canonical sites, which we previously showed to overlap with SR protein binding sites. Indeed, biochemical and proteomic analysis of purified MLN51 and RNPS1 complexes from HEK293 cells shows that while the two alternate EJCs share the three EJC core proteins, only RNPS1-EJCs associate with SR proteins. Intriguingly, RNPS1 depletion causes >2-fold upregulation of most of the endogenous NMD targets tested while MLN51 depletion leads to modest or no increase in their levels. Overall, our data suggests that endogenous EJCs exist in at least two mutually exclusive complexes defined by either RNPS1 or MLN51. While both complexes are likely to be active in NMD, they may represent two distinct NMD branches in which RNPS1-EJCs, in cooperation with SR proteins, promote more efficient RNA turnover as compared to SR protein devoid MLN51-EJCs. We are currently testing this model to understand how EJC composition and its cooperativity with SR protein impacts mRNA fate.

Keywords: EJC, NMD

128. Characterization of the mammalian DEAD-box protein DDX5 reveals functional conservation with S. cerevisiae ortholog Dbp2 in transcriptional control and glucose metabolism

Zheng Xing (Department of Biochemistry, Purdue University), Siwen Wang (Department of Biochemistry, Purdue University), Elizabeth Tran (Department of Biochemistry, Purdue Center for Cancer Research, Purdue University)

Abstract:
DEAD-box proteins are a class of non-processive RNA helicases that dynamically modulate the structure of RNA and ribonucleoprotein complexes (RNPs). However, the precise roles of individual members are not well understood. Work from our lab revealed that the DEAD-box protein Dbp2 in Saccharomyces cerevisiae is an active RNA helicase in vitro that functions in transcription by promoting mRNP assembly, repressing cryptic transcription initiation, and regulating long non-coding RNA activity. Interestingly, Dbp2 is also linked to glucose sensing and hexose transporter gene expression. DDX5 is the mammalian ortholog of Dbp2 that has been implicated in cancer and metabolic syndrome, suggesting that the role of Dbp2 and DDX5 in glucose metabolic regulation are conserved. Herein, we present a refined biochemical and biological comparison of yeast Dbp2 and human DDX5 enzymes. We find that human DDX5 possesses a 10-fold higher unwinding activity than Dbp2, which is partially due to the presence of a mammalian/avian specific C-terminal extension. Interestingly, ectopic expression of DDX5 rescues the cold sensitivity, cryptic initiation defects, and impaired glucose import in dbp2∆ cells, suggesting functional conservation. Consistently, we show that DDX5 promotes glucose uptake and glycolysis in mammalian cells, a process that is upregulated in cancers. We will present evidence that DDX5 may be a novel entry point for therapeutic targeting of cancer-specific metabolism.

Keywords: DEAD-box helicase , Glycolysis

129. Favorable Biodistribution, Specific Targeting and Conditional Endosomal Escape of RNA Nanoparticles in Cancer Therapy

Congcong Xu (College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, Ohio, USA), Farzin Haque (Nanobio Delivery Pharmaceutical Co. Ltd., Columbus, OH, USA), Daniel L. Jasinski (College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, Ohio, USA), Daniel W. Binzel (College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, Ohio, USA), Dan Shu (College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, Ohio, USA), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; College of Medicine, Dorothy M. Davis Heart and Lung Research Institute )

Abstract:
The past decades have witnessed the success transition of several nanotechnologies into the clinic. However, specific delivery of therapeutics to tumors is hindered by several barriers including cancer recognition and tissue penetration; particle heterogeneity and aggregation; unfavorable pharmacokinetic profiles such as fast clearance and organ accumulation. With the advent of RNA nanotechnology, a series of RNA nanoparticles have been successfully constructed to overcome many of the aforementioned challenges for in vivo cancer targeting with favorable biodistribution profiles. Compared to other nanodelivery platforms, the physiochemical properties of RNA nanoparticles can be tuned with relative ease for investigating the in vivo behavior of nanoparticles upon systemic injection. The size, shape, and surface chemistry, especially hydrophobic modifications, exert significant impacts on the in vivo fate of RNA nanoparticles. Rationally designed RNA nanoparticles with defined stoichiometry and high homogeneity have been demonstrated to specifically target tumor cells while avoiding accumulation in healthy vital organs after systemic injection. RNA nanoparticles can deliver therapeutics such as siRNA and anti-miRNA to block tumor growth in several animal models. Although the release of anti-miRNA from the RNA nanoparticles has achieved high efficiency of tumor regression in multiple animal models, the efficiency of endosomal escape for siRNA delivery needs further improvement. This presentation will focus on the advances and perspectives of this promising RNA nanotechnology platform for cancer targeting and therapy.

References:
[1] P. Guo, The emerging field of RNA nanotechnology. Nature Nanotechnology 5 (2010) 833-842.
[2] D. Shu, Y. Shu, F. Haque, S. Abdelmawla, and P. Guo, Thermodynamically stable RNA three-way junctions for constructing multifuntional nanoparticles for delivery of therapeutics. Nature Nanotechnology 6 (2011) 658-667.
[3] D. Jasinski, F. Haque, D.W. Binzel, and P. Guo, Advancement of the Emerging Field of RNA Nanotechnology. ACS Nano 11 (2017) 1142-1164.
[4] Y. Shu, F. Haque, D. Shu, W. Li, Z. Zhu, M. Kotb, Y. Lyubchenko, and P. Guo, Fabrication of 14 Different RNA Nanoparticles for Specific Tumor Targeting without Accumulation in Normal Organs. RNA 19 (2013) 766-777.
[5] H. Li, K. Zhang, F. Pi, S. Guo, L. Shlyakhtenko, W. Chiu, D. Shu, and P. Guo, Controllable Self-Assembly of RNA Tetrahedrons with Precise Shape and Size for Cancer Targeting. Adv. Mater. 28 (2016) 7501-7507.

Keywords: RNA nanotechnology, pRNA-3WJ, Biodistribution

130. Maternal dead-end 1 promotes translation of nanos1 by binding the eIF3 complex

Tristan Aguero (University of Miami), Zhigang Jin (University of Illinois), Sandip Chorghade (University of Illinois), Auinash Kalsotra (University of Illinois), Mary Lou King (University of Miami), Jing Yang (University of Illinois)

Abstract:
In the developing embryo, primordial germ cells (PGCs) represent the exclusive progenitors of the gametes, and their loss results in adult infertility. During early development, PGCs are exposed to numerous signals that specify somatic cell fates. To prevent somatic differentiation, PGCs must transiently silence their genome, an early developmental process that requires Nanos activity. However, it is unclear how Nanos translation is regulated in developing embryos. We report here that translation of nanos1 after fertilization requires Dead-end 1 (Dnd1), a vertebrate-specific germline RNA-binding protein. We provide evidence that Dnd1 protein, expression of which is low in oocytes, but increases dramatically after fertilization, directly interacts with, and relieves the inhibitory function of eukaryotic initiation factor 3f, a repressive component in the 43S preinitiation complex. This work uncovers a novel translational regulatory mechanism that is fundamentally important for germline development.

Keywords: Germline development, Translation regulation, Dnd1

131. Rbfox1 accomplishes extraordinary RNA binding specificity by optimizing discrimination against non-cognate sequences

Xuan Ye (Center for RNA Science and Therapeutics, Case Western Reserve University), Fan Yang (Department of Chemistry, University of Washington), Gabriele Varani (Department of Chemistry, University of Washington), Eckhard Jankowsky (Center for RNA Science and Therapeutics, Case Western Reserve University)

Abstract not available online - please check the printed booklet.

132. Inhibition of HIV-1 reverse transcriptase by modified aminoglycosides

Lanqing Ying (Department of Microbiology and Center for RNA Biology, The Ohio State University), Hongkun Zhu (Department of Microbiology and Center for RNA Biology, The Ohio State University), Nagaraja Tirumuru, Li Wu (Department of Veterinary Biosciences, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus), Marina Y. Fosso (Department of Pharmaceutical Sciences in the College of Pharmacy, University of Kentucky), Sylvie Garneau-Tsodikova (Department of Pharmaceutical Sciences in the College of Pharmacy, University of Kentucky), Kurt Fredrick (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

133. A sensitivity analysis of RNA folding nearest neighbor parameters quantifies the uncertainty in RNA secondary structure prediction and reveals correlations between parameter values

Jeffrey Zuber (Biochemistry and Biophysics, University of Rochester), B. Joseph Cabral (Computational Sciences, Moderna Therapeutics), Iain McFadyen (Computational Sciences, Moderna Therapeutics), David M. Mauger (Computational Sciences, Moderna Therapeutics), David H. Mathews (Biochemistry and Biophysics, University of Rochester)

Abstract:
RNA nearest neighbor thermodynamic parameters are often used to predict RNA secondary structures in order to develop hypotheses about structure-function relationships for RNA sequences, in RNA sequence design, and to identify new functional RNAs. These parameters were derived from optical melting experiments on sets of small model oligonucleotides. To better understand the precision of RNA secondary structure prediction, this work examines how experimental errors in optical melting experiments propagates to errors in the derived nearest neighbor parameter values and hence to uncertainty in structure prediction. This work identifies previously undocumented correlations between nearest neighbor parameters. This work also shows that the precision of RNA secondary structure prediction is more robust than was indicated by previous work that was based on perturbation of the nearest neighbor parameters. Additionally, this work provides an estimate for the precision of RNA secondary structure prediction as well as guidance for future optical melting experiments.

Keywords: RNA Secondary Structure, Nearest Neighbor Parameters, Sensitivity Analysis