Rustbelt RNA Meeting 2018

Poster abstracts

1. A cancer-associated mutation in a human mitochondrial tRNA induces structural rearrangement

Yehong Qiu (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Edric K. Choi (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)

Abstract:
Human mitochondrial (hmt) tRNA genes comprise only 10% of the hmt genome, yet nearly half the mitochondrial disease-associated mutations reside in these genes¹. With these mutations, there is no safety net in the form of genetic redundancy as the hmt genome has only one locus each for the 22 hmt-tRNAs. In mitochondria, transcription generates long polycistronic RNAs, with tRNAs serving as “punctuation” points to facilitate accurate biogenesis of all RNAs. Thus, mutations in tRNA genes adversely affect not only translation but other mitochondrial processes. However, the molecular basis for various diseases arising from mt tRNA mutations is unclear. Here, we sought to test the hypothesis that mt-tRNAs toggle between native and non-native conformations, and that a hepatocellular carcinoma-associated mutation (C6U) in hmt tRNA^Ala might selectively stabilize a non-native fold and thereby prevent 5′-processing by hmt-RNase P. Thermal denaturation studies revealed unexpectedly that the mutant has a higher melting temperature (T_m) compared to the wild-type, a finding that we attribute to stabilization of a non-native conformation. Additional mutations that were engineered to either stabilize or destabilize the non-native fold behaved as predicted. Results from ongoing selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) experiments are expected to further refine our structural models for the native and non-native folds for hmt tRNA^Ala. Taken together, our studies provide a glimpse into a potential mechanism for mitochondrial disease pathogenesis: impaired biogenesis caused by hmt tRNA mutations, which unduly bias an intrinsic conformational switch.

References:
¹ Schon EA, DiMauro S, and Hirano M. (2012) Human mitochondrial DNA: roles of inherited and somatic mutations. Nature Rev Genet., 13: 878-890.

Keywords: mitochondrial tRNA, thermal denaturation, SHAPE

2. A genome-wide alternative polyadenylation atlas of abiotic stress responses in sorghum

Manohar Chakrabarti (Dept. of Plant and Soil Sciences, University of Kentucky, Lexington, KY), Laura de Lorenzo (Dept. of Plant and Soil Sciences, University of Kentucky, Lexington, KY), Salah E. Abdel-Ghany, Anireddy S.N. Reddy (Department of Biology, and Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO), Arthur G. Hunt (Dept. of Plant and Soil Sciences, University of Kentucky, Lexington, KY)

Abstract not available online - please check the printed booklet.

3. A high-throughput, fluorescence turn-on assay to measure RNase P activity

Edric K. Choi (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Sravya Kovvali (Department of Chemistry and Biochemistry, Department of Microbiology, Center for RNA Biology, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

4. A new approach to identifying cytoplasmic capping targets in cells and tissues

Daniel del Valle-Morales (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University), Jackson Trotman (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University), Daniel R. Schoenberg (Center for RNA Biology and Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract not available online - please check the printed booklet.

5. A screen for chemical inhibitors of repeat associated non-AUG (RAN) translation

Udit Sheth (Department of Neurology, University of Michigan, Ann Arbor, MI), Katelyn Green (Department of Neurology and Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI), Veronica Towianski (Department of Neurology, University of Michigan, Ann Arbor, MI), Michael Kearse (Department of Neurology, University of Michigan, Ann Arbor, MI and Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA ), Peter Todd (Department of Neurology, Cellular and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI and VA medical Center, Ann Arbor, MI)

Abstract not available online - please check the printed booklet.

6. A specific Exon Junction Complex composition may support UPF3B-independent nonsense-mediated mRNA decay

Zhongxia Yi (Department of Molecular Genetics, The Ohio State University), Lauren Woodward (Department of Molecular Genetics, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

7. ADAR3 Inhibits RNA Editing and Promotes Differential Gene Expression in Glioblastoma

Pranathi Vadlamani (Medical Sciences Program, Indiana University School of Medicine), Eimile Oakes (Genome, Cell and Developmental Biology Program, Indiana University), Aidan Manning (Medical Sciences Program, Indiana University School of Medicine), Reshma Kurup (Genome, Cell and Developmental Biology Program, Indiana University), Obi Nwosu (Medical Sciences Program, Indiana University School of Medicine), Heather Hundley (Medical Sciences Program, Indiana University School of Medicine)

Abstract not available online - please check the printed booklet.

8. Alternative splicing profile comparison of differentiating T-helper cells to dissect the splicing signatures of Th1, Th2, Th17 and Treg cells

Deepak Kumar Lakshmipathi (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Mir Quoseena (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Benjamin Ulrich (Department of Microbiology and Immunolgy, Indiana University School of Medicine, Cancer Research Institute, Indianapolis), Mark Kaplan (Department of Microbiology and Immunolgy, Indiana University School of Medicine, Cancer Research Institute, Indianapolis, Indiana), Sarath Chandra Janga (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University)

Abstract:
T cell differentiation is regulated through complex changes in cellular transcription. This study focuses on the contribution of Alternative Splicing (AS) events in the differentiation and post differentiation functions of T-helper cells, specifically in Th1, Th2, Th9, Th17 and Treg cells. T cell RNA-seq data from 72hr and 2week post differentiation time points was analyzed using (r-MATS) for alternative splicing events. We observed majority of the significant events are Skipped Exon (SE) events originating from a total of 1,556 genes and Intron Retention (RI) events were the second most abundant event occurring in 1,254 genes at 72 hours post differentiation. These numbers were significantly lower at 2 weeks post differentiation. Data was further validated using PCR and qPCR techniques. Results showed several skipped exonic events in KTN1, IL4RA IL27, Hnrmpd, CREM and Arid4b show different mRNA isoforms across multiple naïve vs differentiated T cell combinations. Overall, RI event associated genes were more prevalent (3,239 genes) than those exhibiting SE (2810 genes). SE events were associated with 10.8% (Th17), 11.2% (Treg), 12.1% (Th2) and 13.9% (Th1) of the genes, a similar trend was observed with RI events with a prevalence of 12.2% (Th17), 12.5% (Treg), 14.2% (Th1) and 14.4% (Th2) of the genes. Gene ontology results showed most of the genes showing SE and RI events are involved in process like ‘Regulation of mRNA splicing and alternative mRNA splicing via spliceosome’, ‘RNA splicing’, ‘mRNA Processing’, ‘RNA Processing’ and ‘RNA Binding’. It was also observed that Introns consistently favored retention at the 3’ end of the gene than the 5’, with upper bound showing 430 genes with introns retained at the 3’ end and 21 genes showing introns retained at the 5’ for the 72 hour time point. Enriched functional ontologies were consistently seen across all cell types to be exclusive for the genes showing RI in the 5’ end vs the 3’ end.

Keywords: T-helper cells, Alternative Splicing , Next Generation Sequencing

9. Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote liver regeneration

Sushant Bangru (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Waqar Arif (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Joe Seimetz, Amruta Bhate, Jackie Chen, Edrees H Rashan (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Russ P Carstens (Dept. of genetics, University of Pennsylvania), Sayeepriyadarshini Anakk (Dept. of Molecular & Integrative Physiology, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Dept. of Biochemistry, University of Illinois, Urbana-Champaign)

Abstract:
During liver regeneration, most new hepatocytes arise via self-duplication; yet, the underlying mechanisms that drive hepatocyte proliferation following injury remain poorly defined. By combining high-resolution transcriptome and polysome profiling of hepatocytes purified from quiescent and toxin-injured mouse livers, we uncover pervasive alterations in messenger RNA translation of metabolic and RNA-processing factors, which modulate the protein levels of a set of splicing regulators. Specifically, downregulation of the splicing regulator ESRP2 activates a neonatal alternative splicing program that rewires the Hippo signaling pathway in regenerating hepatocytes. We show that production of neonatal splice isoforms attenuates Hippo signaling, enables greater transcriptional activation of downstream target genes, and facilitates liver regeneration. We further demonstrate that ESRP2 deletion in mice causes excessive hepatocyte proliferation upon injury, whereas forced expression of ESRP2 inhibits proliferation by suppressing the expression of neonatal Hippo pathway isoforms. Thus, our findings reveal an alternative splicing axis that supports regeneration following chronic liver injury.

Keywords: alternative splicing, hippo pathway, liver regeneration

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

Jose Victorino (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Whitney Smith-Kinnaman (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Samuel Ogunsanya (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Melanie Fox (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine), Yunlong Liu (Department of Medical Genetics, Indiana University School of Medicine), Amber Mosley (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine)

Abstract not available online - please check the printed booklet.

11. An RNA element in PSTVd determines intercellular movement from epidermal to mesophyll cells

Jian Wu (Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210), Neocles Leontis (Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403), Craig Zirbel (Deparment of Mathematics and Statistics, Bowling Green State University, Bowling Green, OH 43403), David M. Bisaro (Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210), Biao Ding (Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210)

Abstract not available online - please check the printed booklet.

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

Eviania Likos (Biological, Geological, and Environmental Sciences - Cleveland State University), Dr. Girish Shukla (Biological, Geological, and Environmental Sciences - Cleveland State University)

Abstract not available online - please check the printed booklet.

13. Auxiliary domains of the mammalian DEAH/RHA helicase DHX36 work in concert with the helicase core domains to remodel RNA structures.

SUKANYA SRINIVASAN (Center for RNA science and Therapeutics, Case Western Reserve University), Zhonghua Liu (Department of Pathology, Case Western Reserve University), Tsan Sam Xiao (Department of Pathology, Case Western Reserve University), Eckhard Jankowsky (Center for RNA science and Therapeutics, Case Western Reserve University)

Abstract:
The DEAH/RHA helicase DHX36 is linked to DNA and RNA G-quadruplex structures and to AU-rich element (ARE) - mediated mRNA deadenylation and decay. In vitro, DHX36 resolves DNA and RNA G-quadruplex structures and unwinds RNA duplexes. However, the molecular basis for DHX36-mediated remodeling of RNA quadruplex and duplex structures remains poorly understood and important biochemical features of DHX36 substrate specificity remain unclear. To address these questions, we combined structural and biochemical analyses of mouse DHX36. We have solved the crystal structure of DHX36 in complex with ADPNP (a non-hydrolysable ATP analog). The structure provides a conformational snapshot of a “closed” nucleotide-bound form of DHX36. It reveals an overall architecture similar to that previously observed for other DEAH/RHA helicases. Biochemical experiments demonstrate that at saturating DHX36 concentration, unwinding rate constants are 4 fold higher for quadruplexes than for duplex structures. In addition, the functional affinity of DHX36 for quadruplexes is more than 20 fold higher than that for duplex structures. The data thus reveal that DHX36 preferentially binds and remodels RNA quadruplex structures, compared to duplex structures. We further show that the N-terminal DSM domain functions not only as a quadruplex binding adaptor, but also promotes the remodeling of both, duplex and quadruplex structures. In addition, we demonstrate that two previously uncharacterized DHX36 regions, a 5’-β-hairpin in the helicase core, and the C-terminal OB-fold domain are essential for the ability of DHX36 to bind and remodel both, quadruplex and duplex structures. Collectively, our data reveal that the auxiliary domains of DHX36 work in conjunction with the helicase core to remodel RNA structures.

Keywords: DHX36, RNA G-quadruplex, RNA duplex

14. Biological significance of multiple, parallel primary tRNA nuclear export pathways in budding yeast

Kunal Chatterjee (Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, U.S.A, Center for RNA Biology, The Ohio State University, Columbus, Ohio, U.S.A), Shubhra Majumder (Presidency University, Kolkata, West Bengal, India), Anita K.Hopper (Department of Molecular Genetics, The Ohio State University, Columbus, Ohio, U.S.A, Center for RNA Biology, The Ohio State University, Columbus, Ohio, U.S.A)

Abstract:
In eukaryotic cells, newly transcribed tRNAs are escorted out of the nucleus to the cytoplasm to participate in translation by the step termed primary tRNA nuclear export. Our recent work revealed that the mRNA exporter Mex67-Mtr2 heterodimer and possibly the protein exporter Crm1, co-function with the canonical tRNA nuclear exporter Los1 in this nuclear export process. Nuclear tRNA export by Los1 also provides an initial tRNA quality control step to generate functional tRNA, as Los1 preferentially binds to end-processed, appropriately structured tRNA. Thus, we sought to assess the fidelity of Los1-independent tRNA nuclear export pathways in yeast by three different approaches. First, we document that Los1-independent export pathways are error-prone, as enhanced levels of 5’–end unprocessed, spliced, non-functional tRNA accumulate in higher levels in los1Δ cells compared to wild-type cells. Second, Mex67-Mtr2 exhibits erroneous tRNA nuclear export, as 5’-end unprocessed, spliced tRNAs are detected in elevated levels in yeast cells under conditions in which Mex67 and Mtr2 are over-expressed. Third, in cells with mex67 or mtr2 mutations, the levels of aberrant tRNA are lower than wild-type cells after 2 h incubation at the non-permissive temperature, possibly because more tRNA are channeled through the high-fidelity Los1-mediated nuclear export pathway under these conditions. The question thus arises, why do cells employ multiple, parallel, but error-prone tRNA nuclear export pathways? We learned that environmental conditions can affect tRNA fidelity, likely due to the alternate use of various tRNA nuclear export pathways. We observed that elevated levels of aberrant m²₂G₂₆ hypomodified tRNA are detected when yeast cells are grown above 30^oC, but not at 23^oC. Thus, yeast cells seem to employ the Los1-independent, error prone tRNA nuclear export pathways in varying environmental conditions, which may confer a yet unknown selective advantage to the cells in those situations.

Keywords: tRNA nuclear export, budding yeast, tRNA quality control

15. Blocking RAN translation enhances FMRP expression and reduces toxicity in Fragile X stem cells.

Shannon E. Wright (Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI), Caitlin M. Rodriguez, Jill M. Haenfler, Yu Lui (Department of Neurology, University of Michigan, Ann Arbor, MI), Michael A. Sutton (Molecular & Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI), Frank Rigo (Ionis Pharmaceuticals, San Diego, CA), Jack M. Parent, Sami J. Barmada (Department of Neurology, University of Michigan, Ann Arbor, MI), Peter K. Todd (Department of Neurology, University of Michigan, Ann Arbor, MI)

Abstract:
Expansion of a CGG repeat in the 5’ UTR of the FMR1 gene underlies a heterogeneous set of human clinical disorders, including Fragile X Syndrome and Fragile X-Associated Tremor/Ataxia Syndrome. While full mutations (>200 repeat expansions) result in FXS typically lead to methylation and silencing of FMR1 expression, patients often display mosaicism of FMR1 methylation and CGG repeat length in individual cells. Large transcribed CGG repeats are potentially toxic as RNA or by triggering Repeat associated non-AUG initiated translation (CGG RAN). Moreover, large repeats can impede translation of FMRP even when transcription is sufficient. Therefore, effective therapies for Fragile X-associated disorders need to simultaneously block CGG RAN and enhance production of FMRP. To this end, our group recently developed a series of antisense oligonucleotides that selectively target RAN initiation sites (RAN ASOs) on the FMR1 transcript. In reporter systems and human cell lines, these ASOs suppress RAN translation at both normal and expanded repeat sizes. Surprisingly, this loss of CGG RAN is associated with a marked increase in endogenous FMRP expression, suggesting that CGG RAN acts normally to inhibit FMRP synthesis. Using patient-derived induced pluripotent stem cells (iPSCs) from an unmethylated full mutation (UFM) carrier, we generated human neurons that exhibit normal FMR1 mRNA transcription but very low FMRP expression. Application of RAN ASOs on these neurons effectively reduced accumulation of CGG RAN products and enhanced neuronal FMRP expression. These biochemical correction were associated with enhanced UFM neuronal survival. Together, these data suggest a native function for CGG RAN in regulating FMRP synthesis and demonstrate that targeting CGG RAN has the potential to correct multiple disease relevant features in Fragile X-associated disorders.

Keywords: RAN translation, Fragile X, iPSCs

16. bPNA probes of RNA tertiary interactions

Shiqin Miao (Department of Chemistry and Biochemistry, Center for RNA Biology), Ila Marathe (Department of Chemistry and Biochemistry), Venkat Gopalan (Department of Chemistry and Biochemistry), Dennis Bong (Department of Chemistry and Biochemistry, Center for RNA Biology)

Abstract:
We demonstrate herein the use of bifacial peptide nucleic acids (bPNAs) as fluorescent probes of RNA loop-loop and protein interactions. Fluorophore-labeled bifacial peptide nucleic acid can bind to U-rich domains in folded RNA to label loop regions with fluorophores that can serve as probes for monitoring long-range RNA interactions. This is demonstrated with previously reported bPNA scaffolds, as well as a new, branched sidechain bPNA we call bPNA(+). This new scaffold displays two melamines on the lysine sidechain, with each melamine targeting two thymine or uracil bases for base triple formation. First, we accomplished scalable synthesis of bPNA(+) and observed sub-nanomolar K_d. Second, we applied fluorophore-labeled bPNA and bPNA(+) as probes for monitoring long-range RNA interactions. We use a hexapeptide and decapeptide bPNA(+), labeled with a FRET pair, respectively, to label RNA secondary structures to provide FRET reporters of RNA-RNA interactions. Initially, we confirmed the site-selectivity of bPNA(+) as driven by length-matching using U-sites of different sizes and bPNA(+) of the corresponding lengths in the ColE1 kissing loop system by FRET. Notably, dye-labeled bPNA(+) exhibits unexpected strong fluorescence turn on to signal both RNA and protein binding: a 2-fold turn on is observed when forming the ColE1 kissing loop complex and an 8-fold turn on is seen when Rop protein interacts with the kissing complex. We anticipate that this fluorescence turn on could be used in measurements of RNA-RNA and RNA-protein interactions and binding affinity measurements.

References:
1. Mao J., DeSantis C., Bong D. (2017). Small molecule recognition triggers secondary and tertiary interactions in DNA folding and hammerhead ribozyme catalysis.J. Am. Chem. Soc., 139, 9815-9818.
2. Zeng Y., Pratumyot Y., Piao X, and Bong D. (2012). Discrete assembly of synthetic peptide-DNA triplex structures from polyvalent melamine-thymine bifacial recognition. J. Am. Chem. Soc. 134, 832-835

Keywords: structural probe, RNA recognition, fluorescence turn on

17. Characterization of a putative R2 retrotransposon and its HSV-like ribozyme in vertebrates

Kennedy L. Stoll (Biology Department, University of Southern Indiana), Rex Meade Strange (Biology Department, University of Southern Indiana), Kimberly J. Delaney (Biology Department, University of Southern Indiana)

Abstract:

Keywords: Retrotransposon, 28S rDNA, ribozyme

18. Characterization of interactions between HIV-1 integrase, nucleocapsid and genomic RNA using AFM and SHAPE

Shuohui Liu (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retroviral Research, The Ohio State University, Columbus, OH), Pratibha C. Koneru (Division of Infectious Diseases, School of Medicine, University of Colorado, Aurora, CO), Gunjan Agarwal (Department of Biomedical Engineering, The Ohio State University, Columbus, OH), Mamuka Kvaratskhelia (Division of Infectious Diseases, School of Medicine, University of Colorado, Aurora, CO), 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.

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

Andrew K. Goodwin (Center for Childhood Cancer and Blood Diseases, The Research Institute at Nationwide Childrens Hospital, Columbus, Ohio), Safiya Khurshid (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.

20. CLEAR: Coverage-based Limiting-cell Experiment Analysis for RNA-seq

Pearlly Yan (Department of Internal Medicine, The Ohio State University), Michael G Sovic, Xi Chen (OSUCCC - Genomics Shared Resources, The Ohio State University), Chi-Ling Chiang, Eileen Hu (Department of Internal Medicine, The Ohio State University), Jiyeon Denninger, Elizabeth Kirby (Department of Psychology, The Ohio State University), Logan A Walker, Ralf Bundschuh (Department of Physics, The Ohio State University), John C Byrd, Natarajan Muthusamy (Department of Internal Medicine, The Ohio State University)

Abstract:
Direct cDNA preamplification protocols developed for single-cell RNA-seq have enabled transcriptome profiling of small clinical samples and rare cells without sample pooling and RNA extraction. Currently, there is no algorithm to distinguish noisy transcripts from robust transcripts in limiting-cell RNA-seq (lcRNA-seq) data to permit their removal from downstream analyses. Herein, we present CLEAR, a workflow that identifies reliably quantifiable transcripts in lcRNA-seq data for differentially expressed gene (DEG) analysis. Libraries at three input amounts from FACS-derived CD5+ and CD5- cells from a single chronic lymphocytic leukemia patient were used to develop CLEAR. When using CLEAR transcripts vs. using all transcripts, downstream analyses reveal more shared transcripts across different input RNA amounts, and improved Principal Component Analysis separation and more DEGs between cell types. As proof-of-principle, CLEAR was applied to an in-house lcRNA-seq dataset and two publicly available datasets. CLEAR can be used in large clinical studies by imputing signal dropouts.

References:
Bhargava et al. (2014). Technical Variations in Low-Input RNA-Seq Methodologies. Scientific Reports, 3678.
Feng et al. (2015). mRIN for Direct Assessment of Genome-Wide and Gene-Specific mRNA Integrity from Large-Scale RNA-Sequencing Data. Nature Communications, 7816.
Ilicic et al. (2016). Classification of low quality cells from single-cell RNA-seq data. Genome Biology, 17(29).
Lun et al. (2016). Pooling across cells to normalize single-cell RNA sequencing data with many zero counts. Genome Biology, 17(75).

Keywords: rare cells, pre-filtering, differential gene expression analysis

21. Cloning and Overexpression of Pseduouridine Monophosphate Synthase from Escherichia Coli

Andrew Riley (Department of Chemistry, Southern Illinois University Edwardsville), Austin Woodard (Department of Chemistry, Southern Illinois University Edwardsville), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville)

Abstract:
RNA modifications are very prevalent throughout nature and play a variety of roles in protein expression. Among these modifications, the most abundant is the modification of uridine to pseudouridine (Ψ). Pseudouridine has been found in the dynamic region of RNA structures, and it is believed to play an important role in the structural dynamics of these motifs. This is believed to be due to the increased rotational freedom of the C-C glycosidic bond between the base and its ribose. Multistep organic synthesis of Ψ has been proven to be very difficult because of its stereochemistry. Therefore, a semi-enzymatic synthesis has been proposed to synthesize Ψ in an efficient and cost-effective manner. This process involves cloning and overexpressing the yeiN gene from E. coli, which codes for pseudouridine synthase. By using this enzyme, isotope-labeled Ψ can be generated in significant quantities for RNA structural studies. It can then be used to give insight into the structural and functional effects of Ψ has on RNA motifs

Keywords: RNA modification, pseudouridine

22. Codon usage and position alter the functions of upstream open reading frames

Yizhu Lin (Department of Biological Sciences, Carnegie Mellon University), Gemma May (Department of Biological Sciences, Carnegie Mellon University), Pieter Spealman (Department of Biological Sciences, Carnegie Mellon University), Lauren Nazzaro (Department of Biological Sciences, Carnegie Mellon University), Hunter Kready (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Translational regulation plays an important role in eukaryotic gene expression. Upstream Open Reading Frames (uORFs) are important regulatory elements located in 5’ mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frame (mORF), but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the impact of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of uORF coding regions on their functions by using a massively parallel reporter assay (FACS-uORF) to determine the regulatory functions of thousands of variants of a uORF from the yeast YAP1 gene. Varying uORF codons resulted in a wide range of functions, including both repressor and enhancer uORFs. The presence of rare codons resulted in the most inhibitory uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF functions. Our results support a model in which uORF coding sequences impact their regulatory functions by altering the speed of uORF translation.

Keywords: uORF, translation, codon usage

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

Rabiul Islam (Department of Chemistry, Wayne State University, Detroit, MI 48202), Nisansala Muthunayake (Department of Biological Sciences, Wayne State University, Detroit, MI 48202), Wesley D. Colangelo (Department of Biological Sciences, Wayne State University, Detroit, MI 48202), Philip R. Cunningham (Department of Biological Sciences, Wayne State University, Detroit, MI 48202), Christine S. Chow (Department of Chemistry, Wayne State University, Detroit, MI 48202)

Abstract not available online - please check the printed booklet.

24. Coordination of G4R1, RNAPII, and AGO proteins to regulate the transcription of developmental genes and proto-oncogenes

Adam E. Richardson (Ball State University, Department of Biology), Josh D. Tompkins (City of Hope, Department of Diabetes Complications & Metabolism), Arthur D. Riggs (City of Hope, Department of Diabetes Complications & Metabolism), Steven N. Akman (St. Francis Roper Cancer Center), James P. Vaughn (Nanomedica Inc.), Philip J. Smaldino (Ball State University, Department of Biology)

Abstract:
Regulation of developmental gene and proto-oncogene transcription is a complex process involving the participation of many proteins. These proteins tightly regulate developmental gene and proto-oncogene expression to prevent uncontrolled, cancerous cell growth and division. One of the many layers in which the expression of these genes is regulated is through G-quadruplex (G4) structures. G4 structures form within guanine-rich regions of DNA and RNA. The guanines self-associate into vertically-stacked planar tetrads, stabilized via Hoogsteen hydrogen-bonding and coordinate bonding with a monovalent cation. The human genome contains 716,310 putative G4 motifs, which are present in upwards of 40% of gene promoters. The degree of enrichment of promoter G4 structures has been shown to correlate with the function of the gene. For example, proto-oncogenes and developmental genes are especially enriched with G4 structures in contrast to tumor suppressor and housekeeping genes. Promoter G4 structures negatively regulate transcription via impeding the transcriptional machinery. The cell can overcome this barrier by using G4 helicases such as G4 Resolvase1 (G4R1) (aliases DHX36 and RHAU), which unwinds G4 structures. AGO2 and RNAPII are also key proteins involved in transcriptional regulation and are binding partners with G4R1. For these reasons, we performed a chromatin immunoprecipitation on chip (ChIP-chip) using antibodies specific to G4R1, RNAPII, and AGO proteins (AGO). We found that G4R1, RNAPII, and AGO binding sites overlap with genes involved with developmental processes, pattern specification processes, and cell fate commitment. Furthermore, under serum starvation, the three proteins co-localized to the enhancer of C-MYC. These data suggest that G4R1, RNAPII, and AGO function together to regulate the transcription of developmental genes and proto-oncogenes.

References:
1. Bishop, J.M., Molecular themes in oncogenesis. Cell, 1991. 64(2): p. 235-248.
2. Bochman, M.L., K. Paeschke, and V.A. Zakian, DNA secondary structures: stability and function of Gquadruplex structures. Nature Reviews Genetics, 2012. 13(11): p. 770.
3. Stöhr, J.R., Proteomic and functional characterization of human Argonaute complexes. 2011, lmu.
4. Möller, A., et al., Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease. Molecular & Cellular Proteomics, 2011: p. mcp. M111. 011767.
5. Giri, B., et al., G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA. Nucleic acids research, 2011. 39(16): p. 7161-7178.
6. Portnoy, V., et al., saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription. Cell research, 2016. 26(3): p. 320.

Keywords: G4R1, Transcription regulation, Development

25. Dazl regulates germ cell survival through a network of polyA-proximal mRNA interactions

Leah L. Zagore (Center for RNA Science and Therapeutics, Case Western Reserve University), Thomas J. Sweet (Center for RNA Science and Therapeutics, Case Western Reserve University), Molly M. Hannigan (Center for RNA Science and Therapeutics, Case Western Reserve University), Sebastien M. Weyn-Vanhentenryck, Chaolin Zhang (Center for Motor Neuron Biology and Disease, Columbia University,), Donny D. Licatalosi (Center for RNA Science and Therapeutics, Case Western Reserve University)

Abstract:
The RNA binding protein Dazl is essential for gametogenesis, but its direct in vivo functions, RNA targets, and the molecular basis for germ cell loss in DAZL null mice are unknown. Here, we mapped transcriptome-wide Dazl-RNA interactions in vivo, revealing Dazl binding to thousands of mRNAs via polyA-proximal 3’UTR interactions. In parallel, fluorescence activated cell sorting and RNA-Seq identified mRNAs sensitive to Dazl deletion in male germ cells. Despite binding a broad set of mRNAs, integrative analyses indicate that Dazl post- transcriptionally controls only a subset of its mRNA targets, namely those corresponding to a network of genes critical for germ cell proliferation and survival. Additionally, we provide evidence that polyA sequences have key roles in specifying Dazl-RNA interactions across the transcriptome. Altogether, our results reveal a mechanism for Dazl-RNA binding, and illustrate that Dazl functions as a master regulator of a post-transcriptional mRNA program essential for germ cell survival.

Keywords: post-transcriptional gene regulation, RNA binding proteins, RNA networks

26. Deciphering mRNA Pseudouridylation in Eukaryotes

Meredith Purchal (Program in Chemical Biology, University of Michigan), Ryan McNassor (Department of Chemistry, University of Michigan ), Taslima Khan (Program in Chemical Biology, University of Michigan), Kristin Koutmou (Department of Chemistry, Program in Chemical Biology, University of Michigan), Markos Koutmos (Department of Chemistry, Department of Biophysics, Program in Chemical Biology, University of Michigan )

Abstract:
Pseudouridine (Y) is among the most abundant post-transcriptional RNA modifications in cells and has long been known to make important contributions to tRNA structure and function.  While traditionally pseudouridine was only thought to occur in non-coding RNAs, recent efforts in mapping pseudouridylation across the transcriptome have identified a multitude of sites consistently pseudouridylated across mRNA and dynamically modified under stress conditions. As appreciation for the complexity of the role of this modification increases, a deeper understanding of the enzymes that catalyze the incorporation of this modification is necessary. Our goal is to describe the sequence and structural elements that cooperatively determine the specificity of pseudouridine synthase (Pus) enzymes towards their mRNA targets. We solved the first crystal structure of S. cerevisiae Pus7, a pseudouridine synthase that primarily modifies mRNA under cellular stress. Furthermore, we developed binding and kinetic assays to interrogate wild-type and mutant Pus7 pseudouridylation activity. Our biochemical work reveals several residues important to Pus7 function (K61, F307, F67, and D256).  Ultimately, the combination of biophysical and biochemical techniques will allow us to dissect the features determining substrate recognition, and help to lay the foundation for a deeper understanding of RNA post-transcriptional regulation.

Keywords: Pseudouridylation, modification

27. Decoupling the roles of peptide and mRNA in ribosome pausing during the translation of positively charged peptide sequences

Tyler Smith (Department of Chemistry - University of Michigan), Alexandrea Rizo (Program in Chemical Biology - University of Michigan ), Sarah C. Keane (Department of Chemistry and Department of Biophysics - University of Michigan), Daniel Southworth (Department of Biochemistry and Biophysics - University of California, San Francisco), Kristin Smith-Koutmou (Department of Chemistry and Program in Chemical Biology - University of Michigan)

Abstract not available online - please check the printed booklet.

28. Detecting miRNA-miRBP interactions with a cell-based split-enzyme assay

Sydney Rosenblum (Program in Chemical Biology, University of Michigan), Daniel A. Lorenz (Program in Chemical Biology, University of Michigan), Amanda L. Garner (Medicinal Chemistry, University of Michigan)

Abstract:
Greater than one-thousand human miRNAs are believed to control the translation of greater than 60% of all protein-coding genes. Accordingly, miRNA dysregulation is a common hallmark of countless human diseases, including cancers, neurological diseases, and cardiac diseases, making miRNA and miRNA binding proteins (miRBPs) attractive drug targets. Recent advances in high-throughput technology have enabled large-scale screening of small molecules and natural products against RNA targets. However, subsequent characterization of promising chemical scaffolds is hindered by the lack of reliable assays designed to detect target engagement of RNA-targeted compounds or protein-RNA interaction inhibitors. Most existing methods of determining target engagement successfully measure ligand engagement of protein targets, but such systems cannot be easily adapted to detect RNA target engagement or the disruption of protein-RNA interactions. The development of novel methods of detecting RNA-ligand and RNA-protein interactions in cells will not only provide tools for studying miRNA-miRBP interactions, but will also facilitate the discovery of miRNA-targeted ligands and miRNA-miRBP inhibitors. Here I describe a cellular assay that utilizes the split-enzyme NanoBiT® (Promega) system to detect the miR-miRBP interaction between the pre-let7 class of miRNA and Lin28. This strategy is currently being expanded to enable the detection and study of various miR-miRBP interactions as well as measure the cellular activity of small molecule inhibitors of such interactions.

Keywords: microRNA , microRNA binding protein, assay

29. Detection of ribonucleoside modifications by liquid chromatography higher-energy collisional dissociation mass spectrometry (LC-HCD-MS) and spectral matching

Manasses Jora (Department of Chemistry, University of Cincinnati), Peter A Lobue (Department of Chemistry, University of Cincinnati), Ningxi Yu (Department of Chemistry, University of Cincinnati), Robert L Ross (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati), Patrick A Limbach (Department of Chemistry, University of Cincinnati)

Abstract:
The goal of this work is to evaluate the feasibility of higher-energy collisional dissociation (HCD)-based fragment ion fingerprints for detection of modified ribonucleosides through spectral matching. To date, more than 160 ribonucleoside modifications have been identified in different organisms [1]. The most well established approach for detection of modified nucleosides is liquid chromatography-tandem mass spectrometry (LC-MS/MS), where retention time, mass-to-charge (m/z) values of precursor and fragment ions are monitored for the observed nucleoside signal. Data processing is generally done manually, and this process is laborious, time consuming, and error prone, thus requiring well trained analysts.
Our studies show that the HCD-based dissociation of ribonucleosides at higher collisional energies (e.g., HCD 80) can create nucleoside-specific fragment ion spectra (fingerprints) resembling pseudo-MSⁿ situation [2]. Such fingerprints have high reproducibility (intra- and interday), and are insensitive to chromatographic conditions, precursor ion abundance and sample matrix. They also enable differentiation of positional isomers exclusively through mass spectrometric behavior (i.e., precursor ion m/z and HCD-based fingerprint). In the current study, we tested the suitability of HCD fingerprints to use as LC-independent reference spectra lists for in-silico analysis of ribonucleoside modifications in a given sample. We present the observations of these studies involving the detection of modified ribonucleosides from E. coli and S. cerevisiae tRNA through NIST-MS Search-based [3] spectral matching.

References:
[1] Boccaletto, P.; Machnicka, M.A.; Purta, E.; Piatkowski, P.; Baginski, B.; Wirecki, T.W.; de Crecy-Lagard, V.; Ross, R.; Limbach, P.A.; Kotter, A.; Helm, M.; Bujnicki, J.M.. Nucleic Acids Res. 2017, 46, D303.
[2] Jora, M.; Burns, A.P.; Ross, R.L.; Lobue, P.A.; Zhao, R.; Palumbo, C.M.; Beal, P.A.; Addepalli, B.; Limbach, P.A.. J. Am. Soc. Mass Spectrom. 2018, 29, 1745.
[3] Yang, X.; Neta, P.; Stein, S.E.. J. Am. Soc. Mass Spectrom. 2017, 28, 2280.

Keywords: Ribonucleoside Modifications, LC-MSMS, Spectral Matching

30. Determinants of substrate specificity for the Trm10 family of tRNA modification enzymes

Nathan Howell (Ohio State University), Aiswarya Krishnamohan (University of Chicago), Jane Jackman (Ohio State University)

Abstract not available online - please check the printed booklet.

31. Determining the role of tRNA modifications on pseudohyphal growth in yeast

Hannah Sizemore (Chemistry & Biochemistry, Northern Kentucky University), Kaylee Fox, Olivia Gilliam (Biology, Northern Kentucky University), Daisy DiVitia (Chemistry & Biochemistry, Northern Kentucky University), Rachel Morgeson, Jenna Kappes (Biology, Northern Kentucky University), John C. Carmen (Biology, Northern Kentucky University), Michael P. Guy (Chemistry & Biochemistry, Northern Kentucky University)

Abstract:
Fungi have the ability to change their morphology to produce hyphae, branching filaments that allow for increased levels of feeding and locomotion. In some pathogenic fungi, the shift from unicellular to hyphal growth is linked to their ability to cause disease. The model yeast Saccharomyces cerevisiae does not form hyphae, but can form pseudohyphae under certain conditions such as nutrient limitation. Previous studies in this yeast have indicated that the shift to pseudohyphal growth requires elongator complex genes such as ELP2, suggesting that tRNA modifications are involved in the transition. The elongator complex is a group of proteins which work together to add a 5-methoxycarbonylmethyl (mcm5) group or a 5-carbamoylmethyl (ncm5) group to tRNAs that have a uridine at position 34. We are using S. cerevisiae as a model to study the role of elongator complex tRNA modifications in the shift to pseudohyphal growth. We have generated S. cerevisiae mutants lacking genes that code for elongator complex proteins to analyze their role in pseudohyphal growth by investigating which tRNA substrate(s) is involved in the shift. We are also measuring elongator modification levels during the shift to pseudohyphal growth, via UPLC. In addition, we have begun to study the role of elongator complex tRNA modifications in the shift in the pathogenic fungus Candida albicans.

Keywords: tRNA, modifications, yeast

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

Nathan McCann (Chemistry, Ball State University), My N. Bui (Chemistry, Ball State University), Kirill Afonin (Chemistry, UNC Charlotte), Emil Khisamutdinov (Chemistry, Ball State University)

Abstract not available online - please check the printed booklet.

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

Whitney Bergman (Department of Chemistry, Washburn University), Keshav G C (Department of Chemistry and Biochemistry, Kent State University), Dr. Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)

Abstract not available online - please check the printed booklet.

34. Disruption of Minor Intron Splicing in Inherited Developmental Disorders

Elizabeth DeLaney (Cellular and Molecular Medicine, Lerner Research Institute), Rosemary C. Dietrich (Cellular and Molecular Medicine, Lerner Research Institute), James M. Hiznay (Cellular and Molecular Medicine, Lerner Research Institute), David E. Symer (Department of Lymphoma/Myeloma-Research, Division of Cancer Medicine, UT MD Anderson Cancer Center), Richard A. Padgett (Cellular and Molecular Medicine, Lerner Research Institute)

Abstract:
Biallelic mutations in the gene encoding the minor spliceosomal small nuclear RNA (snRNA) U4atac (RNU4ATAC) have been identified in patients with three autosomal recessive developmental disorders: microcephalic osteodysplastic primordial dwarfism type I (MOPDI), Roifman Syndrome, and Lowry-Wood Syndrome. While each disorder is pathologically distinct, common features of patients include microcephaly, skeletal dysplasia, growth retardation, and cognitive delay. MOPDI is the most severe of the three disorders, with patients exhibiting severe neurological defects and early death. Twenty-six unique disease-causing mutations have been identified in RNU4ATAC. Current data suggests that RNU4ATAC mutations disrupt U4atac snRNA function and impair minor splicing through several mechanisms. Using our in vivo orthogonal splicing assay, we have characterized the effects of disease-associated RNU4ATAC mutations on minor splicing. Mutations seen in all three diseases in the 5’ stem-loop region of U4atac and those in the Sm protein binding site reduce splicing to less than 30% of the wild-type U4atac, while mutations found only in Roifman and Lowry Wood Syndromes in the Stem II base pairing region have nearly 50% splicing activity. The mechanism of the splicing defects seen with RNU4ATAC mutations varies depending on the mutation’s location. Previous work revealed that mutations to the 5’ stem-loop impair binding of essential U4atac/U6atac di-snRNP protein components and reduce U4atac/U6atac.U5 tri-snRNP formation. We further show that mutations to the Sm protein binding site and Stem II base pairing region result in reduced U4atac abundance, likely due to the mutations interfering with the recruitment of essential protein factors such as the Sm proteins and Prpf3. The wide range in the nature and severity of phenotypes in patients with RNU4ATAC mutations suggest there are likely multiple mechanisms that impair minor splicing in MOPDI and Roifman and Lowry Wood syndrome patients.

Keywords: Minor splicing, snRNA, MOPDI

35. Diverse long-RNAs are differentially sorted into extracellular vesicles secreted by colorectal cancer cells

Scott Hinger (Vanderbilt University), Dr. James G. Patton (Vanderbilt University)

Abstract:
The regulation and functional roles of secreted coding and long noncoding RNAs (lncRNAs, >200nt) are largely unknown. We previously showed that mutant KRAS colorectal cancer (CRC) cells release EVs containing distinct proteomes, miRNAs, and circular RNAs. Here, we comprehensively identify diverse classes of CRC extracellular long RNAs secreted in EVs and demonstrate differential export of specific RNAs. Distinct noncoding RNAs including antisense transcripts and transcripts derived from pseudogenes are enriched in EVs compared to cellular profiles. We detected strong enrichment of Rab13 in mutant KRAS EVs and demonstrate functional delivery of Rab13 mRNA to recipient cells. To assay functional transfer of lncRNAs, we implemented a CRISPR/Cas9 based RNA-tracking system to monitor delivery to recipient cells. We show that gRNAs containing export signals from secreted RNAs can be transferred from donor to recipient cells. Our data support the existence of cellular mechanisms to selectively export diverse classes of RNA.

Keywords: RNA-seq, extracellular RNA, cell-cell communication

36. DksA-like factor YteA enhances transcription attenuation of the trp operon in Bacillus subtilis

Courtney E. Szyjka (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. In vivo transcriptional fusions between the trp leader and lacZ show that mutant attenuators display decreased TRAP-mediated regulation when yteA is knocked out. We also show that purified YteA enhances transcription attenuation in vitro on the WT trp leader in the absence of TRAP and in the presence of both WT and E60K TRAP.

Keywords: attenuation, termination, transcription

37. Editing of Minimally Edited RNA CYb in Trypanosoma brucei

Brianna L. Tylec (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY), Rachel M. Simpson (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY), Runpu Chen (Department of Computational Science and Engineering, University at Buffalo, Buffalo NY), Yijun Sun (Department of Computational Science and Engineering, University at Buffalo, Buffalo NY), Laurie K. Read (Department of Microbiology & Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY)

Abstract:
Trypanosoma brucei, the causative agent of African sleeping sickness, is an early branching Eukaryote belonging to the Class Kinetoplastida. Members of this group undergo uridine (U) insertion/deletion editing of most mitochondrial RNAs. Trans-acting gRNAs are sequentially utilized as templates, ensuring the general 3' to 5' progression of editing along the mRNA to generate a translatable open reading frame. Editing involves multiple protein subcomplexes including the non-enzymatic RNA Editing Substrate Binding Complex (RESC), composed of the RNA Editing Mediator Complex (REMC) and Guide RNA Binding Complex (GRBC). Nine mitochondrial RNAs are edited throughout the length of the transcript, termed "pan-edited", while three genes only require editing over a small portion of the reading frame. These "minimally edited" RNAs are a good model for studying editing since they require only 1-2 gRNAs to complete the process. We characterized the editing of the minimally edited RNA, CYb, in procyclic form T. brucei using the Trypanosome RNA Editing Alignment Tool (TREAT), which takes advantage of high-throughput sequencing technology as well as a uridine in/del specific alignment algorithm to evaluate the extent of editing in a large pool of pre-, partially, and fully edited transcripts. We then knocked down expression of four proteins that play a role in RNA editing (RESC factors TbRGG2 and GAP1, and non-RESC factors RBP16 and MRP1/2), and assessed the effects of the knockdowns on editing initiation and progression of CYb mRNA. We found that editing intrinsically pauses at the steps of initiation and gRNA exchange, and that RBP16 and MRP1/2 knockdowns result in depletion of edited mRNA by different mechanisms.

Keywords: Trypanosoma brucei, RNA Editing

38. EJC protein composition and position on mRNAs are influenced by translation

Lauren Woodward (Molecular Genetics, The Ohio State University), Justin Mabin, Robert Patton (Physics, The Ohio State University), Ralf Bundschuh (Physics, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University)

Abstract:
The Exon Junction Complex (EJC) is comprised of a trimeric core (EIF4AIII, RBM8A, and MAGOH) which is deposited on RNA during splicing 24nt upstream of exon boundaries. The EJC serves as a molecular memory of the splicing reaction and couples diverse post-transcriptional processes by serving as a binding platform for peripherally interacting proteins. Recently, we demonstrated that EJC’s exist in two mutually exclusive biochemical varieties with respect to their peripheral proteins: either bound to CASC3 or RNPS1(Mabin et al., 2018). RNPS1 binds more to EJCs in the nucleus. In contrast, CASC3-bound EJCs represent a later, primarily cytoplasmic EJC composition and display greater sensitivity to the translational status of mRNA. Current models state that EJCs are removed from mRNA co-translationally through the action of the ribosome-associated EJC disassembly factor, PYM. However, translation results in more EJC footprints in the 3’UTR compared to conditions where translation is inhibited. Additionally, EJC binding in the 3’UTR correlates with the number of upstream introns. Thus, EJC footprints in the 3’ may represent EJCs originally deposited at upstream splicing events. This translation-dependent relocation is also evident in EJCs that do not interact with PYM. Taken together, these results suggest translating ribosomes can relocate EJCs downstream of the canonical (-24nt) position independent of PYM-EJC interaction. Future experiments will elucidate the roles of the ribosome of PYM in EJC disassembly.

References:

Mabin, J.W., Woodward, L.A., Patton, R., Yi, Z., Jia, M., Wysocki, V., Bundschuh, R., and Singh, G. (2018). The exon junction complex undergoes a compositional switch that alters mRNP structure and nonsense-mediated mRNA decay activity. BioRxiv.

Keywords: EJC, PYM, EJC disassembly

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

Patrick Stevens (Bioinformatics, Indiana university Purdue University Indianapolis), Sasank Vemuri (Bio Health Informatics, Indiana university Purdue University Indianapolis), Huzaifa Hassan (Bioinformatics, Indiana university Purdue University Indianapolis), Sarath Chandra Janga (Associate Professor, Bioinformatics, Indiana University Purdue University Indianapolis)

Abstract not available online - please check the printed booklet.

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

Patrick Stevens (Bioinformatics, Indiana university Purdue University Indianapolis), Sasank Vemuri (Bio Health Informatics, Indiana university Purdue University Indianapolis), Huzaifa Hassan (Bioinformatics, Indiana university Purdue University Indianapolis)

Abstract not available online - please check the printed booklet.

41. Estimating RNA structure chemical probing reactivities from reverse transcriptase stops and mutations

Angela M Yu (Tri-Institutional Training Program in Computational Biology and Medicine, Weill Cornell Medicine; Chemical and Biological Engineering, Northwestern University), Molly E. Evans (Chemical and Biological Engineering, Northwestern University), Julius B. Lucks (Chemical and Biological Engineering, Northwestern University)

Abstract not available online - please check the printed booklet.

42. Exploring splicing and retrotransposition in a mobile group IIC intron

Claire Smathers (Biochemistry, West Virginia University)

Abstract:
Group II introns are ribozymes that are the expected ancestors of both the spliceosome and non-long terminal repeat retroelements, offering a streamlined system to mechanistically examine both splicing and retrotransposition. These introns are divided into three structural families: IIA, IIB, and IIC. In group IIA and IIB introns, the 5’ splice site is decoded by base-pairing interactions between the intron and exon. In contrast, group IIC introns use a combination of short intron-exon base pairing along with a structured rho-independent terminator stem to achieve 5’ exon definition. Group IIC introns have reduced RNA secondary structure complexity compared to their organellar counterparts, and use an unusual hydrolytic splicing pathway in vitro. Here we show that addition of an intron-encoded protein (IEP) switches the self-splicing activity from a hydrolytic to a branched lariat forming reaction. This reaction produces RNPs capable of mobility reactions into DNA targets. We examined this DNA mobility reaction using a novel fluorescent retrohoming assay. We further explored the mechanisms of splicing and retrohoming by utilizing mutant intron RNA, IEP, and altered DNA substrates. Understanding the biochemistry of group IIC intron splicing and mobility will lead to valuable structural and mechanistic insight into how splicing is regulated, and assist our efforts to determine structures of the intron and RNP complex through X-ray crystallography.

Keywords: intron, retroelement, splicing

43. Fluorescence turn-on upon annealing of cyanine dye-labeled bPNAs to uridine-tract-containing RNAs

Ila Marathe (Department of Chemistry and Biochemistry, The Ohio State University), Shiqin Miao (Department of Chemistry and Biochemistry, The Ohio State University), Yehong Qiu (Department of Chemistry and Biochemistry, The Ohio State University), Khan Cox, Michael Poirier (Department of Physics, The Ohio State University), Dennis Bong (Department of Chemistry and Biochemistry, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy is a powerful technique for studying the assembly of ribonucleoprotein (RNP) complexes. However, site-specific fluor-labeling of long RNAs remains a major technical challenge, and often requires multiple purification steps to remove unincorporated labels. Thus, facile labeling approaches are needed to broaden the utility of smFRET for uncovering RNP assembly and function. To study assembly of archaeal RNase P (comprised of one RNA and five proteins) using ensemble and single-molecule FRET, we used cyanine dye-labeled bifacial peptide nucleic acids (Cy-bPNAs), which bind with high affinity to uridine-tracts (U-tracts)^{1, 2}. The RNase P RNA (RPR) was modified to contain an internal U-tract, and fluor-labeling of the RPR with Cy-bPNA was achieved through a simple heating and cooling step. During the course of these ensemble FRET experiments, we unexpectedly observed that the fluorescence of Cy-bPNA alone is diminished to near baseline but the addition of a U-tract-containing RPR increases the Cy-bPNA’s fluorescence up to 200-fold, an unprecedented enhancement for Cy dyes. This ‘fluorescence turn-on’ has been observed with multiple RNAs (<100 - 380 nt) bearing U-tracts of different lengths at terminal or internal positions, and under varying salt, pH, and temperature conditions. Moreover, preliminary sm-fluorescence experiments revealed strong fluorescence resulting from the binding of Cy-bPNA to an immobilized U-tract-containing RPR. The bPNA-based method for labeling RNAs has broad utility for fluorescence-based techniques including smFRET, and the turn-on phenomenon has potential for further exploration in bio-sensing applications.

References:
1. Mao J., DeSantis C., Bong D. (2017). Small molecule recognition triggers secondary and tertiary interactions in DNA folding and hammerhead ribozyme catalysis. J. Am. Chem. Soc., 139, 9815-9818.
2. Zeng Y., Pratumyot Y., Piao X, and Bong D. (2012). Discrete assembly of synthetic peptide-DNA triplex structures from polyvalent melamine-thymine bifacial recognition. J. Am. Chem. Soc., 134, 832-835

Keywords: Fluorescence, bPNA, RNA fluor-labeling

44. Folding Complex RNA with Fewer Gs: Comparison of Composition and Distribution of Nucleotides in ribosomal RNA from Avian and Mammalian mitochondria.

Cailyn Davish (Chemistry Department Bowling Green State University), Maryam Hosseini (Chemistry Department Bowling Green State University), Neocles Leontis (Chemistry Department Bowling Green State University)

Abstract:
Mitochondrial ribosomes are derived from those of bacteria, and their SSU and LSU rRNAs show large-scale loss of peripheral elements, increase of ribosomal protein content, and drastic reduction of guanine nucleotides. Guanine is the base most sensitive to oxidative damage. In recent work (1), we showed that the remaining Gs in mmt rRNA are strategically concentrated at solvent inaccessible positions where they stabilize the rRNA structure by forming guanine-specific, RNA–RNA and RNA–protein interactions. Moreover, solvent-exposed guanines, conserved in bacteria, are replaced in mmt rRNA by bases less sensitive to oxidation. Birds and mammals shared a common ancestor over 200 million years ago, rather recently in relation to the origin of eukaryotes, but long ago for divergence in mitochondrial sequences to occur. They share warm-bloodedness and high metabolic rates, characteristics correlated with increased production of reactive oxygen species (ROS) in the mitochondrial matrix, where they can damage mitochondrial DNA and RNA (2,3). Here, we examine whether Avian mitochondrial (amt) rRNA shows the same adaptations to minimize or avoid oxidative damage. We compare the distribution of the conserved Gs in bacterial, mmt and amt SSU rRNA.

References:
References:
(1) Maryam Hosseini, Poorna Roy, Marie Sissler, Craig L Zirbel, Eric Westhof, Neocles Leontis; How to fold and protect mitochondrial ribosomal RNA with fewer guanines, Nucleic Acids Research, gky762, https://doi.org/10.1093/nar/gky762.
(2) López-Torres, M., & Barja, G. (2008). Mitochondrial Free Radical Production and Caloric Restriction: Implications in Vertebrate Longevity and Aging. Oxidative Stress in Aging, 149-162. doi:10.1007/978-1-59745-420-9_9
(3) Willi, J., Küpfer, P., Evéquoz, D., Fernandez, G., Katz, A., Leumann, C., & Polacek, N. (2018). Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center. Nucleic Acids Research, 46(4), 1945-1957. doi:10.1093/nar/gkx1308.

Keywords: ribosomal RNA, mitochondrial, Guanine

45. Function and regulation of alternative splicing to enhance tumor suppressor activity of Neurofibromin 2

Ullas V. Chembazhi (Department of Biochemistry, University of Illinois at Urbana-Champaign), Cole Lewis (Department of Biochemistry, University of Illinois at Urbana-Champaign), Miranda Gurra (Department of Biochemistry, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, University of Illinois at Urbana-Champaign)

Abstract:
Hippo signaling pathway functions as a central regulator of organ size in higher organisms, controlling cell proliferation and apoptosis. Recent studies in drosophila and mammals have shown that Neurofibromin 2 (NF2), a bona fide tumor suppressor, causes hippo pathway activation through direct binding and recruitment of LATS to the plasma membrane, promoting LATS phosphorylation by MST1/2-WW45. Nf2 encodes for two major isoforms that differ in the inclusion of exon-16 (E16) and possess distinct capacities to assume the active ‘open’ conformation. Here, we show that a conserved splicing switch in the Nf2 pre-mRNA occurs during postnatal mouse liver development, a period of extensive remodeling during which hepatocytes switch from a proliferative to quiescent state. A steady increase in the inclusion of E16, produces a structural change in the NF2 protein that causes it to act as a constitutive activator of hippo pathway. Further, using real-time and single-cell resolution biochromatic splicing reporter that monitors the exon 16 inclusion, we show that RBFOX2 (RNA Binding Fox-1 Homolog 2) directly regulates E16 splicing and that it is necessary to produce the adult isoform.

Keywords: Tumor suppressor, Rbfox2, developmentally regulated splicing

46. Functional Definition of Domain Structures and Distribution in RNA Lariat Debranching Enzyme

Arundhati Mohanta (Department of Biological Sciences, University of North Carolina at Charlotte), Kausik Chakrabarti (Department of Biological Sciences, University of North Carolina at Charlotte)

Abstract not available online - please check the printed booklet.

47. Functional dissection and comparative analysis of alternative splicing events from lncRNA perturbation screens across human cell types

Felipe Porto (Department of BioHealth Informatics, IUPUI ), Swampna Vidhur Daulatabad (Department of Medical and Molecular Genetics), Sarath Chandra Janga (Center for Computational Biology and Bioinformatics)

Abstract not available online - please check the printed booklet.

48. Functional synergy between Rrp6p and Rrp44p in the nuclear exosome

Armend Axhemi (The Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Ulf-Peter Guenther (European Molecular Biology Laboratory, Heidelberg, Germany ), Christopher D. Lima (Structural Biology Program & Howard Hughes Medical Institute, Sloan Kettering Institute, New York, NY ), Eckhard Jankowsky (The Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)

Abstract not available online - please check the printed booklet.

49. Gene expression and alternative polyadenylation in an Arabidopsis sr45 mutant

Emily Ranseen (Department of Plant and Soil Sciences, University of Kentucky), Jordan Powers (Biochemistry Program, Department of Biology, St. Bonaventure University), Chong Zhang (Department of Biological Sciences, University of Maryland Baltimore County), Hua Lu (Department of Biological Sciences, University of Maryland Baltimore County), Xiao-Ning Zhang (Biochemistry Program, Department of Biology, St. Bonaventure University), Arthur G. Hunt1 (Department of Plant and Soil Sciences, University of Kentucky)

Abstract:
RNA processing is central to gene expression in eukaryotes. In Arabidopsis, mutants impaired in the expression of a splicing factor, SR45, have been described previously. One such mutant, sr45-1, displays flower and seed phenotypes, and has characteristic differences in gene expression in inflorescences; these differences include altered expression of genes associated with defense responses, and are reflected in a greater resistance of the sr45-1 mutant to infection with a bacterial pathogen. To better understand these characteristics, and also to explore possible connections between splicing and polyadenylation, gene expression and alternative poly(A) site choice was studied in wild-type and sr45-1 mutant Arabidopsis. Almost 1000 genes showed significant differences in expression between the sr45-1 mutant and wild-type. As was seen in inflorescence tissues, the uninfected sr45-1 mutant showed altered expression of genes associated with immunity and defense responses, compared with the wild-type. Nearly 1000 genes also showed some measurable differences in poly(A) site choice. About 100 of these were also altered in their expression, suggestive of a role for APA in determining overall gene expression levels. However, these ca. 100 genes included only a small number associated with defense responses. These results confirm the global gene expression profile of the sr45-1 mutant that was seen in inflorescence tissues and reinforce the conclusion that SR45 plays important roles in plant defense responses. They also provide a tenuous but interesting link between APA and gene expression.

References:
1: Zhang XN, Shi Y, Powers JJ, Gowda NB, Zhang C, Ibrahim HMM, Ball HB, Chen SL,
Lu H, Mount SM. Transcriptome analyses reveal SR45 to be a neutral splicing
regulator and a suppressor of innate immunity in Arabidopsis thaliana. BMC
Genomics. 2017 Oct 11;18(1):772. doi: 10.1186/s12864-017-4183-7.

Keywords: alternative splicing, SR45, alternative polyadenylation

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

Diana Renteria (Department of Biological Sciences, University of North Carolina at Charlotte ), Kausik Chakrabarti (Department of Biological Sciences, University of North Carolina at Charlotte )

Abstract not available online - please check the printed booklet.

51. Genome-wide RNA 3’-end Mapping identifies riboregulators and reveals the effects azithromycin on transcription in Pseudomonas aeruginosa

Salini Konikkat (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh PA, 15213 ), Rory Eutsey (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh PA, 15213 ), N. Luisa Hiller (Department of Biological Sciences, Carnegie Mellon University, Pittsburgh PA, 15213 ), Dan Snyder (Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA, 15219), Vaughn Cooper (Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA, 15219), C. Joel McManus (Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave, Pittsburgh PA, 15213 )

Abstract not available online - please check the printed booklet.

52. High - resolution and cell - type specific polysome profiling from mouse tissues

Waqar Arif (Biochemistry, UIUC), Joeseph Seimetz (Biochemistry, UIUC), Sushant Bangru (Biochemistry, UIUC), Mikel Hernaez (Carl R. Woese Institute for Genomic Biology, UIUC), Auinash Kalsotra (Biochemistry, UIUC)

Abstract not available online - please check the printed booklet.

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

Divyaa Bhagdikar (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus, OH 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.

54. Identification of DNA aptamer with modified nucleotide

Nicolette Geron (Department of Chemistry, Southern Illinois University Edwardsville), Sun-Jeong Im (Department of Chemistry, Southern Illinois University Edwardsville), Kamran Shavezipur (Department of Mechanical Engineering, Southern Illinois University Edwardsville), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville)

Abstract:
Our main research interest is the development of a food safety biosensor that can detect foodborne pathogens by using DNA aptamers. The DNA aptamer that has a high affinity and specificity to the target pathogen is identified by a new SPR – SELEX, Surface Plasmon Resonance – Systematic Evolution of Ligands by EXponential enrichment, method. The target cells used are Excherichia Coli, K12. SPR-SELEX selects a deoxyuridine aptamer (dU-Ap) that utilizes deoxyuridine instead of thymidine as well as the other three common dNTPs. 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 dU-Ap 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. This project compares the sequences, stability, and structure of the two DNA aptamers: deoxythymidine aptamers (dT-Ap) and deoxyuridine aptamers (dU-Ap).

Keywords: SELEX, modification, SPR

55. Identification of the enzyme responsible for the acp3U modification in bacteria

Maggie Thomas (Department of Chemistry and Biochemistry), Adrian R. Guy (Department of Chemistry and Biochemistry), Jamison Burchett (Department of Chemistry and Biochemistry), Michael P. Guy (Department of Chemistry and Biochemistry)

Abstract:
Transfer RNAs (tRNAs) are biomolecules needed to translate mRNA into proteins. tRNAs must receive chemical modifications for proper function, and defects are associated with diseases such as cancer. The 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification is formed on some bacterial, plant, and animal tRNAs by an unknown enzyme(s). This modification occurs on residue 47 of some bacterial tRNAs, and due to its location on the Watson-Crick face, interferes with base pairing. To identify the enzyme responsible for the acp3U modification, two approaches are being used. In the first approach, acp3U modification activity is purified from bacterial extracts and detected using either labelled tRNAs, or using engineered tRNAs and a primer extension assay. The second approach uses a plant tRNA and a plant cDNA library expressed in yeast, followed by acp3U detection by primer extension. Identification of the enzyme responsible for the acp3U modification will allow study of the function of acp3U in tRNA, and may help identify the enzyme in other organisms, including humans.

Keywords: tRNA, modifications, bacteria

56. Identify Rbfox-regulated transcripts important for muscle function

Jinghan Zhao (Molecular Genetics, The Ohio State University), Sharon L. Amacher (Molecular Genetics, The Ohio State University), Thomas L. Gallagher (Molecular Genetics, The Ohio State University)

Abstract:
Rbfox RNA-binding proteins are important regulators of muscle-specific splicing. Rbfox proteins contain an RNA recognition motif that is conserved from flies to human and binds to (U)GCAUG motifs that are enriched in introns next to muscle-specific alternative exons. Many muscle-specific splicing events are conserved among vertebrates, and we use the zebrafish model to understand how Rbfox-mediated splicing regulates muscle development and function. In zebrafish, Rbfox1l and Rbfox2 are both expressed in muscle. Double knockdown of rbfox1l and rbfox2 using antisense morpholinos leads to splicing changes of muscle-specific alternative exons, coupled with skeletal muscle paralysis and reduced heart rate. To confirm whether splicing changes and phenotypic effects in morphants are indeed due to Rbfox depletion and not morpholino-induced side effects, I compared phenotype and alternative splicing changes in rbfox genetic mutants versus morphants. Using our unpublished RNA-seq data, I identified candidate Rbfox-regulated transcripts and focused on transcripts that are highly expressed and show substantial splicing changes between wildtype and double morphant embryos. To enrich for direct targets, I selected only those candidates containing intronic Rbfox motifs for further analysis. The majority of candidates (7 of 9 tested) were similarly affected in double mutants and double morphants, although a few showed differences. These results indicate that splicing is similarly affected in morphants and mutants, consistent with the observation that rbfox double mutants and double morphants also have the same morphological phenotypes. Future experiments will test whether Rbfox-dependent transcript isoforms can fully or partially rescue muscle function when microinjected into double mutants. Transcript isoforms that restore muscle function in mutants are likely targets of Rbfox regulation that are necessary for proper muscle development. Our long-term goal is to identify Rbfox-regulated splicing events that are required for muscle function. Insight gained from this work will improve our understanding of Rbfox-regulated muscle splicing and may provide clues for development of therapies for human muscle diseases.

Keywords: alternative splicing, zebrafish, muscle

57. Importance of conserved elements in the Bacillus subtilis thrS canonical T-box riboswitch

Alexander Runyon (Department of Microbiology and 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), Frank J. Grundy (Department of Microbiology, Center for RNA Biology)

Abstract:
T-box riboswitches are cis-acting RNA structures that modulate expression of downstream amino acid-related genes in Gram-positive bacteria. They are induced in response to a specific uncharged tRNA that corresponds to the amino acid specificity of the downstream gene, which is regulated through transcription antitermination or sequestration of the Shine-Dalgarno sequence. T-box riboswitches with all of the conserved elements found in the majority of T-box genes are known as canonical T-box riboswitches. Current biochemical understanding of T-box riboswitch function comes from models that are missing key conserved elements. The Bacillus subtilis thrS riboswitch is located upstream of the threonyl-tRNA synthetase coding sequence and contains all of the key conserved elements, making it a canonical riboswitch. tRNA^Thr-dependent antitermination in vitro has been demonstrated, which allows comparisons of biochemical data obtained from non-canonical riboswitches to that derived from the more common canonical riboswitch organization. Mutations have been generated in conserved elements in thrS, which are known to disrupt these elements in other T-box RNAs. Initial results from in vitro transcription and tRNA binding assays suggest that the conserved elements in thrS are critical for tRNA-dependent antitermination activity. In addition, some elements of thrS have been modified to resemble the corresponding elements of the non-canonical ileS US riboswitch found in Actinobacteria. Our results have shown that modification of elements in thrS results in lower binding affinity for tRNA^Thr. This system will allow us to gain a better understanding of the function of canonical T-box RNAs.

Keywords: Riboswitch, T-box, thrS

58. Inhibition of lariat debranching enzyme with click-branched RNA: effects of the 2'-branch and the branch-point residue

Megan Van Horn (Chemistry, Carnegie Mellon University ), Timothy Chad Ratterman (Chemistry, Carnegie Mellon University ), Stephanie Mack (Chemistry, Carnegie Mellon University ), Subha Das (Chemistry, Carnegie Mellon University )

Abstract:
During RNA splicing, lariat introns are formed via a 2’,5-phosphodiester bond, with an adenosine branch-point residue. Lariat debranching enzyme (Dbr1p) specifically cleaves the 2’,5’-phosphodiester bond in the branch-point of the lariat intron.

Previous studies in our lab have shown that Dbr1p can bind, but not cleave, branched RNAs containing an unnatural 2ʹ,5ʹ-triazole linkage at the branch-point residue generated through ‘click’ chemistry. These click-branched RNAs are competitive inhibitors of the Dbr1p cleavage reaction. Recently our lab has shown that Dbr1p can cleave backbone-branched RNAs with branch-point residues other than adenosine. Here we describe the use of click-branched RNAs with altered 2'-branch sequences and non-canonical branch-point residues to study the effects of the 2'-branch and the branch-point residue identity on the Dbr1p cleavage reaction.

Keywords: Dbr1p, E histolytica, Inhibitor

59. Interactions with Vaccinia Virus K7 Protein Alter the Biochemical Activity of the DEAD-Box RNA Helicase DDX3X

Sarah Venus (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), McKenzie Clapp (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Andrea Putnam (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Eckhard Jankowsky (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)

Abstract:
The DEAD-box RNA helicase DDX3X, which functions in translation initiation and cellular signaling, is targeted by proteins from diverse viruses, including K7, a protein from the poxvirus vaccinia. K7 binds the N-terminus of DDX3X, a region that facilitates oligomerization of DDX3X as well as association with translation initiation factors and stress granules. The impact of K7 on DDX3X’s biochemical activities and roles in RNA metabolism is unknown. We show that K7 inhibits ATP-dependent RNA duplex unwinding activity of DDX3X, but simultaneously promotes DDX3X-mediated ATP hydrolysis. K7 shows similar effects on the S. cerevisiae DDX3X ortholog Ded1p, which shares the N-terminal K7 binding site. Since monomeric Ded1p exhibits higher ATPase, but lower unwinding activity compared to oligomeric Ded1p, our results suggest that K7 impacts the oligomerization of DDX3X and Ded1p. We further show formation of recombinant DDX3X granules in vitro. This allows direct visualization of K7’s effect on DDX3X oligomerization and granule formation. Ultimately, characterizing the interaction between K7 and DDX3X will provide insight into strategies by which viruses target DDX3X to alter RNA metabolism in the host.

Keywords: DDX3X, Granules, K7

60. Investigating active site mutants of lariat debranching enzyme with backbone branched RNA

Timothy Chad Ratterman (Chemistry, Carnegie Mellon University), Brian Graham (Biological Sciences, University of Pittsburgh), Grace Wang (Biological Sciences, University of Pittsburgh), Andrew VanDemark (Biological Sciences, University of Pittsburgh), Subha R. Das (Chemistry, Carnegie Mellon University)

Abstract:
Lariat debranching enzyme (Dbr1p) belongs to the binuclear metallophosphoesterase (MPE) superfamily and processes lariat introns formed during RNA splicing. In the splicing reactions, the 2ʹ,5ʹ-phosphodiester bond formed in the lariat intron is specifically cleaved while the 3ʹ,5ʹ-phosphodiester bonds remain intact. Sequence homology studies have identified a high level of conservation throughout the metallophosphoesterase (MPE) domain. Mutational studies have demonstrated that mutation of certain residues within this highly conserved region greatly diminishes or abolishes enzymatic activity in yeast and human Dbr1p. Recent crystal structures of the E. histolytica Dbr1p suggest critical amino acid residues as contacts with the metal centers or branchpoint adenosine. Here we investigate four mutants of the E. histolytica Dbr1p enzyme and assess their ability to cleave a backbone branched RNA substrate compared to the wild type enzyme. In these mutant enzymes, the changes are to active site residues that are putative ligands for the metal ions or branchpoint adenosine binding. Our data suggest that the E. histolytica Dbr1p tolerates mutations to residues suggested to contact the branchpoint of the branched RNA.

Keywords: Dbr1p, E histolytica, Active site mutants

61. Investigating how DAZL regulates its RNA targets using BioID and immunostaining

Cydni Akesson (The Center for RNA Science & Therapeutics, Case Western Reserve University), Leah Zagore (The Center for RNA Science & Therapeutics, Case Western Reserve University), Mohammed Alzahrani (Case Western Reserve University), Molly Hannigan (The Center for RNA Science & Therapeutics, Case Western Reserve University), Donny Licatalosi (The Center for RNA Science & Therapeutics, Case Western Reserve University)

Abstract:
Deleted in Azospermia-Like (DAZL) is an RNA-binding protein expressed explicitly in germ cells. DAZL’s absence or malfunction causes infertility in male and female mice, and the molecular basis for this outcome is not well understood. Previous work in our lab indicates that DAZL positively regulates its RNA targets via 3’UTR interactions. To investigate how DAZL regulates its RNA targets, we will identify proteins interacting with DAZL using proximity-dependent biotin identification (BioID). We also use BioID and immunostaining to determine how different mutations in DAZL affect its ability to interact with different proteins and its subcellular localization. Thus far, we have found that DAZL’s protein network consists of many RNA processing factors and, although not exposed to stressful conditions in cell culture, many stress granule markers. These protein interactions are altered when point mutations are introduced into DAZL’s RNA Recognition Motif making it unable to bind RNA. Lastly, immunostaining experiments show a remarkable difference in DAZL’s localization in the cell depending on both its ability to bind RNA as well as the presence of its conserved, 27-amino acid intrinsically disordered region. Our results bring us closer to understanding how DAZL is able to access its target mRNAs in the cell.

Keywords: RNA Processing

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

Jazmin L Marks-Burns (Biology, Ball State University), Ally R. Morris (Biology, Ball State University), Douglas A. Bernstein (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 in 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 proteins encoded by the mitochondrial genome and mitochondrial rRNA contains only one highly conserved pseudouridine, which is made by the pseudouridine synthase Pus5. I 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. I hypothesize Pus5 mediated pseudouridylation is important for mitochondrial protein expression. I will use mass spectrometry to determine if mitochondrial rRNA pseudouridylation is required for wild type mitochondrial gene translation. Furthermore, from this mass spectrometry data, I will determine if C. albicans uses an alternative mitochondrial genetic code. 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.

References:
Ansmant, I., Massenet, S., Grosjean, H., Motorin, Y., & Branlant, C. (2000). Identification of the Saccharomyces cerevisiae RNA: pseudouridine synthase responsible for formation of Ψ2819 in 21S mitochondrial ribosomal RNA. Nucleic acids research, 28(9), 1941-1946.
Banks, C.A., S.E. Kong, and M.P. Washburn, Affinity purification of protein complexes for analysis by multidimensional protein identification technology. Protein expression and purification, 2012. 86(2): p. 105-119.
Miranda, I., et al., A genetic code alteration is a phenotype diversity generator in the human pathogen Candida albicans. PLoS One, 2007. 2(10): p. e996
Spenkuch, F., Y. Motorin, and M. Helm, Pseudouridine: still mysterious, but never a fake (uridine)! RNA biology, 2014. 11(12): p. 1540-1554.

Keywords: pseudouridlyation, Candida albicans, mitochondrial rRNA

63. Investigating the post-transcriptional regulatory roles of HuR and SF2 in interleukin-17 mediated inflammation

Xinrui Zhang (Center for RNA Science and Therapeutics, Case Western Reserve University, School of Medicine, Cleveland, OH 44106), Xing Chen (Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44106), Leah Zagore (Center for RNA Science and Therapeutics, Case Western Reserve University, School of Medicine, Cleveland, OH 44106), Xiaoxia Li (Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44106), Donny Licatalosi (Center for RNA Science and Therapeutics, Case Western Reserve University, School of Medicine, Cleveland, OH 44106)

Abstract not available online - please check the printed booklet.

64. Investigating the Role of DDX41 in pre-messenger RNA Splicing and Leukemogenesis

Noah J. Daniels (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH), James M. Hiznay (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH), Courtney E. Hershberger, William M. DiPasquale, Devlin C. Moyer (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH), Sukanya Srinivasan, Eckhard Jankowsky (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH), Jaroslaw P. Maciejewski (Department of Translational Hematology and Oncology Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH), Richard A. Padgett (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH)

Abstract:
DEAD-box RNA helicases are a highly conserved family of proteins involved in nearly all aspects of RNA metabolism, including several members with crucial roles in pre-messenger RNA splicing. Whole exome sequencing has identified DDX41, a member of this family, as recurrently mutated in myelodysplastic syndrome (MDS) patients. MDS is a bone marrow neoplasm that results from a buildup of precursor cells giving rise to subsequent defects in the myeloid lineages. Germline frameshift DDX41 mutations potentially causing DDX41 hemizygosity throughout life have been identified in numerous families with histories of MDS and leukemia. Nearly half of these individuals will acquire a recurrent, somatic, missense mutation, resulting in the conversion of arginine 525 to histidine (R525H) in the highly conserved helicase domain of the second DDX41 allele. Biochemical assays in vitro with recombinant DDX41R525H displayed slightly tighter binding of double stranded RNA and a 3-fold decrease in RNA duplex unwinding compared with DDX41WT protein; this suggests a hypomorphic function. Proteomic analyses have classified DDX41 as a member of the catalytic core of the spliceosome but its role in splicing is still undefined. Analysis of RNA cross-linking-immunoprecipitation-high-throughput-sequencing data demonstrated that DDX41 binds preferentially to exons and splice sites of pre-mRNAs. Furthermore, DDX41 binds to both major and minor class spliceosomal snRNAs. RNA-seq analyses of DDX41WT and DDX41R525H over-expression or DDX41 knockdown human cells have revealed subtle-yet global-effects on pre-mRNA splicing. For future studies, we are generating an auxin-inducible degron system to promote complete degradation of DDX41 protein to determine the function of DDX41 in splicing using in vitro splicing assays. These data support our hypothesis that DDX41 is a component of the spliceosome and plays an important role in pre-mRNA splicing. Mutations and/or deletions of DDX41 may result in the aberrant splicing of key leukemic drivers; in turn, this results in leukemogenesis.

Keywords: myelodysplastic syndrome, DEAD-box RNA helicases, pre-mRNA s

65. Investigating the role of the bulge and pentaloop for htrA RNA thermometer melting behavior

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 mRNA structures in the 5′-untranslated region of some bacterial heat-shock or pathogenic virulence genes. RNATs regulate the translation of these genes by changing their conformations in response to temperature. In our lab, we study an RNAT in the htrA gene of Salmonella enterica. The htrA RNAT is a 30-nucleotide hairpin structure with a single-nucleotide bulge and a five-nucleotide loop. Our research focuses on investigating the role of these two structural elements for htrA RNAT melting behavior. We synthesized several htrA RNAT sequences with mutations that are specific to these elements. We then performed SHAPE assays on the mutants at 30 and 37 ˚C, temperatures at which the wild type is closed and open, respectively; results suggest that some mutants remain folded at 37 ˚C. Next, we performed additional SHAPE assays over a wide range of temperatures and constructed melting curves for individual nucleotides. The melting curves confirm that these mutants also have slightly elevated melting points, thus implying that the loop and the bulge region are important in fine-tuning the melting temperature of the htrA RNAT to the physiological range.

Keywords: SHAPE, RNAT, htrA

66. Investigation of pseudouridine synthase 7 function in pathogenic yeast candida albicans

Ethan Pickerill (Biology, Ball State University), Douglas Bernstein (Biology, Ball State University)

Abstract not available online - please check the printed booklet.

67. L7Ae enhances the cleavage rate of archaeal RNase P

Stella M. Lai (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)

Abstract not available online - please check the printed booklet.

68. lncRNA Regulation by Protein Arginine Methyltransferase 5, Rmt5.

Murat C. Kalem (Department of Microbiology and Immunology, University at Buffalo School of Medicine), Hussain Odeh (Department of Microbiology and Immunology, University at Buffalo School of Medicine), John C. Panepinto (Department of Microbiology and Immunology, University at Buffalo School of Medicine)

Abstract not available online - please check the printed booklet.

69. Maize RMR12 is a CHD3 nucleosome remodeler required for maintaining paramutations and normal development

Natalie Deans (Department of Molecular Genetics, The Ohio State University, Columbus, OH), Brian Giacopelli, Daniel Hlavaty, Emily McCormic (Department of Molecular Genetics, The Ohio State University, Columbus, OH), Charles Addo-Quaye, Brian Dilkes (Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN), Jay Hollick (Department of Molecular Genetics, Center for Applied Plant Sciences and Center for RNA Biology, The Ohio State University, Columbus, OH)

Abstract not available online - please check the printed booklet.

70. Mass spectrometry-based mapping of RNA-binding molecules

Prabuddha Madubashitha (Department of Chemistry, Wayne State University), Rasoul Daliri (Department of Chemistry, Wayne State University), M. T. Rodgers (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
Recent advances in rational design of small molecules and biochemical screening methods have identified novel RNA targeting molecules including peptides and small molecules. Meanwhile, characterization of the binding sites of RNA targeting molecules remains a major challenge. In this study, we have employed a mass spectrometry (MS) based method to map the binding sites of a basic peptide and an aminoglycoside to the transactivation responsive (TAR) RNA as a model system.^1,2 Fourier Transform Ion Cyclotron Resonance (FT-ICR) MS was used to study complex formation and to map the binding sites of the peptide and aminoglycoside with TAR RNA. The MS data show concentration-dependent complex formation between the peptide and RNA. Moreover, 1:2 and 1:3 stoichiometric complexes between RNA and the peptide are formed with increasing peptide concentration. Similarly, our data show 1:1, 1:2, and 1:3 stoichiometric complexes of TAR RNA and the aminoglycoside. The complex between the aminoglycoside and TAR RNA was mapped by collision-induced dissociation, which allowed multiple binding sites to be identified. This MS-based mapping strategy is being extended to reveal important molecular details of the interactions between other small molecules and their RNA targets.

References:
1. Richter, S., Cao, H., Rana, T. M. Biochemistry 2002, 41, 6391-6397.
2. Sannes-Lowery, K. A., Mei, H-Y., Loo, J. A. ‎Int. J. Mass Spectrom. 1999, 193, 115–122.

Keywords: mass spectrometry, peptides, aminoglycosides, TAR RNA

71. Measures of ‘single’- versus multiple- round translation argue against a mechanism to ensure coupling of transcription and translation

Menglin Chen (Department of Microbiology, Ohio State Biochemistry Program, and Center for RNA Biology, The Ohio State University, 484 W. 12th Ave, Columbus, Ohio, 43210), Kurt Fredrick (Department of Microbiology, Ohio State Biochemistry Program, and Center for RNA Biology, The Ohio State University, 484 W. 12th Ave, Columbus, Ohio, 43210)

Abstract not available online - please check the printed booklet.

72. Metabolic labeling of RNA in synchronized S. cerevisiae daughter cells

Brittany N Stawicki (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Eckhard Jankowsky (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)

Abstract:
RNA metabolism markedly changes during the cell cycle. A large percentage of mRNAs undergo translational repression during cell division. Equivalent variations in mRNA stability are expected. However, it is currently unknown how exactly translational state and mRNA half lives change with the cell cycle in S. cerevisiae. To address this problem we are devising an approach to measure mRNA half lives and translational state of mRNAs during the cell cycle in S. cerevisiae. Given that mother and daughter cells in S. cerevisiae differ in cell size and cell cycle length, it is also important to separately analyze mother and daughter cells. To isolate synchronized S. cerevisiae daughter cells in a non-disruptive fashion, we attached unsynchronized yeast cells to a coated glass substrate and eluted budding daughter cells in short time intervals. This approach currently yields more than 80% synchronized daughter cells. We then combined this synchronization approach with optimized 4-thiouracil metabolic RNA labeling. We provide evidence that 4-thiouracil labeled RNA enters the translating RNA pool and is effectively translated. However, labeling times of more than 60 min compromise translation. Our combined cell synchronization and metabolic labeling approach is suitable for high-throughput analysis and likely for other S. cerevisiae strains, including those with genetic alterations. The approach sets the stage for quantitative measurements of translational states and mRNA stabilities over the course of the S. cerevisiae cell cycle.

Keywords: Metabolic Labeling, S Cerevisiae

73. Methylation of Ded1 affects its role in translation

McKenzie Murvin (Biology, University of Richmond), Alec DAlessandro (Biology, University of Richmond), Christian Freniere (Biology, University of Richmond), Angie Hilliker (Biology, University of Richmond)

Abstract not available online - please check the printed booklet.

74. MicroRNA stability is regulated by oncogenic protein kinase Akt1

Christina Z. Chung (Biochemistry, The University of Western Ontario), Emad Manni, Nileeka Balasuriya, Xuguang Liu (Biochemistry, The University of Western Ontario), Shawn S. C. Li (Biochemistry, Oncology and Child Health Research Institute, The University of Western Ontario), Patrick ODonoghue (Biochemistry, Chemistry, The University of Western Ontario), Ilka U. Heinemann (Biochemistry, The University of Western Ontario)

Abstract:
The deregulation of microRNAs (miRNAs) is associated with multiple human diseases, yet cellular mechanisms governing miRNA abundance remain largely elusive. We elucidated the first link between oncogenic kinase activity and the regulation of miRNA stability. Human miR-122 is required for Hepatitis C proliferation and low miR-122 abundance is associated with hepatic cancer. The adenylyltransferase Gld2 catalyzes the post-transcriptional addition of a single adenine residue (A+1) to the 3’-end of miR-122, enhancing its stability. We found that Gld2 activity is regulated by site-specific phosphorylation in its disordered N-terminal domain. We identified two phosphorylation sites (S62, S110) where phosphomimetic substitutions increased Gld2 activity and one site (S116) that markedly reduced activity. Using mass spectrometry, we confirmed that HEK 293 cells readily phosphorylate the N-terminus of Gld2. We identified protein kinase A (PKA) and protein kinase B (Akt1) as the corresponding kinases that site-specifically phosphorylate Gld2 at S116, abolishing Gld2-mediated nucleotide addition. Transfection of Gld2 and fully activated ppAkt1, but not inactive Akt1 into HEK 293 cells, destabilizes miR-122 in living cells. The data demonstrate a novel phosphorylation-dependent mechanism to regulate Gld2 activity and miRNA stability, revealing tumor suppressor miRNAs as a previously unknown target of Akt1-dependent oncogenic signaling.

Keywords: miRNA regulation, site-specific phosphorylation, Akt1

75. Modeling the effects of U4atac snRNA mutations using human iPS cell-derived brain organoids

Jagjit Singh (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic), Filomena Pirozzi (Department of Genetics, School of Medicine, Case Western Reserve University), Anthony Wynshaw-Boris (Department of Genetics, School of Medicine, Case Western Reserve University), Richard A. Padgett (Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic)

Abstract not available online - please check the printed booklet.

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

Safiya Khurshid (Centre for Childhood Cancer, Nationwide Childrens Hospital, Columbus OH), Matias Montes (Centre for Childhood Cancer, Nationwide Childrens Hospital, Columbus OH), Brianne Sanford (Centre for Childhood Cancer, Nationwide Childrens Hospital, Columbus OH), Dawn Chandler (Centre for Childhood Cancer, Nationwide Childrens Hospital, Columbus OH; Molecular, Cellular and Developmental Biology, Ohio State University, Columbus OH.)

Abstract not available online - please check the printed booklet.

77. Molecular function of ADAR3 in neuronal activity

Reshma Raghava Kurup (Genome, Cell and Developmental Biology Program, Indiana University), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN 47405)

Abstract:
ADARs (Adenosine deaminase that act on RNA) carry out adenosine (A) to inosine (I) editing, one of the most abundant RNA modifications [1]. In humans, there are three ADAR family members, ADAR1, ADAR2 and ADAR3. While ADAR1 and ADAR2 are ubiquitous, ADAR3 is specifically expressed in the nervous system. ADAR1 and ADAR2 are the major editing enzymes whereas ADAR3 lacks editing activity [2]. ADAR editing activity is required for normal neurological function, and loss of ADAR activity is associated with several pathologies including neurodegenerative diseases, cancer, auto-immune diseases and metabolic disorders. Recently our lab has demonstrated the role of ADAR3 as a negative regulator of RNA editing [3]. ADAR3 has the conserved domains of catalytically active deaminases and can directly bind to RNA, but ADAR3 inhibits editing of transcripts.
A recent study determined that ADAR3 deficient mice exhibit increased anxiety and deficits in memory formation [4]. In addition, KCl mediated depolarization of the neuroblastoma cell line, SH-SY5Y, reduced expression of a long non-coding RNA that regulates transcription of ADAR3 and resulted in differential expression of genes involved in proper neuronal function [5, 6]. Some of these genes were also differentially expressed in ADAR3 deficient mice that exhibited cognitive deficits. Therefore, we are investigating the role of ADAR3 in response to neuronal stimulation. We found that there is increased expression of ADAR3 upon KCl treatment. In addition, we observed changes in subcellular localization of ADAR3. The upregulated ADAR3 expression and translocation of ADAR3 in response to neuronal activity suggests ADAR3 plays an important role in regulation of signaling pathways linked to neuronal activation.

References:
1. Walkley, C. R. & Li, J. B. Rewriting the transcriptome : adenosine-to-inosine RNA editing by ADARs. Genome Biol. 18 (2017).
2. Chen, C. et al. A third member of the RNA-specific adenosine deaminase gene family ADAR3 contains both single and double stranded RNA binding domains. RNA 6 (2000).
3. Oakes, E. et al. Adenosine Deaminase That Acts on RNA 3 (ADAR3) Binding to Glutamate Receptor Subunit B PremRNA Inhibits RNA Editing in Glioblastoma. J Biol Chem 3 (2017).
4. Mladenova, D. et al. Adar3 Is Involved in Learning and Memory in Mice. 12 (2018).
5. Hirose, T. et al. NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies. 25 (2014).
6. Barry, G. et al. The long non-coding RNA NEAT1 is responsive to neuronal activity and is associated with hyperexcitability states. Sci. Rep. 7 (2017).

Keywords: ADAR3

78. Molecular Mechanisms to Regulate the Substrate Recognition of an A-to-I RNA Editing Enzyme

Suba Rajendren (Genome, Cell and Developmental Biology Program, Indiana University, Bloomington), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine, Bloomington)

Abstract:
RNA editing alters genomically encoded information to generate the transcriptomic diversity required for normal development and proper neuronal function in all animals. Aberrant RNA editing is associated with neurological disorders and cancers in humans. The adenosine deaminase acting on RNA (ADAR) family of enzymes catalyzes adenosine (A) to inosine (I) RNA editing. ADARs act on double stranded RNAs (dsRNAs), however not every adenosine is equally edited. The mechanisms that regulate ADAR specificity and editing efficiency are not well understood. Our lab utilizes Caenorhabditis elegans to study the cellular factors and molecular mechanisms which regulate RNA editing. In C. elegans, RNA editing is catalyzed by ADR-2. However, a dsRNA binding protein, ADR-1, promotes RNA editing at specific sites in vivo. Herein, our studies indicate ADR-2 has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein-protein interactions can guide substrate recognition for RNA editors. However, in the nervous system, ADR-1 is not required for ADR-2 to edit certain targets, which have one or two highly-edited sites. We hypothesize that protein-protein interactions with other neural RNA binding proteins promote ADR-2 binding to certain transcripts to catalyze editing at specific highly edited sites within the nervous system. Elucidating how ADR-2 binds to substrate mRNAs in specific tissues and determining the tissue specific co-factors of editing will result in identification of molecular mechanisms that regulate RNA editing. This will lead to the development of targeted therapeutics to restore editing in patients with heart and neurological diseases.

Keywords: RNA editing, Celegans, dsRNA binding domains (dsRBDs)

79. mRNA modifications alter translation elongation and fidelity

Daniel Eyler (Department of Chemistry, University of Michigan), Monika Franco (Program in Chemical Biology, University of Michigan), Taslima Khan (Program in Chemical Biology, University of Michigan), Kristin Koutmou (Department of Chemistry and Program in Chemical Biology, University of Michigan)

Abstract not available online - please check the printed booklet.

80. Native Mass Spectrometry of Ligand-Mediated Activation of RNA binding by TRAP

Melody Pepsi Holmquist (Department of Chemistry and Biochemistry, The Ohio State University), Benjamin J. Jones (Department of Chemistry and Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

81. Natural tRNA variation and visualizing mistranslation in human cells.

Jeremy T. Lant (Dept. of Biochemistry, Western University), Matthew D. Berg (Dept. of Biochemistry, Western University), Christopher J. Brandl (Dept. of Biochemistry, Western University), Patrick ODonoghue (Dept. of Biochemistry, Western University)

Abstract:
The human cytosolic tRNA pool is astoundingly complex. Over 600 tRNA or tRNA-like genes encode over 400 expressed tRNAs(1). Each tRNA contains an average of 13 post-transcriptional modifications, amounting to over 213 microspecies that must coordinate to faithfully decode only 61 sense codons(2). Despite this complexity, we demonstrated with a fluorescent reporter that a single tRNA variant expressed in HEK293 cells could elicit up to 2-5% mistranslation of a proline codon (Pro to Ala)(3). Interestingly, we could only observe significant levels of mistranslation after cells were deprived of serum and glucose for four days(3). Also, cells were surprisingly mistranslation-tolerant, as no induction of the heat shock response, or loss in cellular viability could be detected(3). Our findings point to a ‘hidden phenotypes’ model of cytosolic tRNA behavior, where mistranslating tRNAs can exist quietly until a combinatorial factor exacerbates their phenotype. Indeed, tRNA expression profiles vary between tissues, can respond to stimuli, and become altered in many diseases including cancer(4). More recently, we have completed high-coverage sequencing of all tRNA genes in a cohort of 48 healthy individuals to better understand the prevalence of natural tRNA variants in the human population. We have also been composing a review paper on the behaviors of cytosolic tRNAs in human disease. I discuss our early work on tRNA-dependent mistranslation in cell culture, and new perspectives on the roles of cytosolic tRNA variants in human disease.

References:
1.Chan, P. P. & Lowe, T. M. GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes. Nucleic Acids Res. 44, D184–D189 (2016).
2.Schimmel, P. The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis. Nat. Rev. Mol. Cell Biol. 19, 45–58 (2017).
3.Lant, J. T. et al. Visualizing tRNA-dependent mistranslation in human cells. RNA Biol. (2017).
4.Kirchner, S. & Ignatova, Z. Emerging roles of tRNA in adaptive translation, signalling dynamics and disease. Nat. Rev. Genet. 16, 98–112 (2015).

Keywords: Transfer RNA, mistranslation, human disease

82. NMR studies of conformational selection of hnRNP H on RNA recognition and its interaction with the HIV exonic splicing silencer ESS2P RNA

Liang-Yuan Chiu (Department of Chemistry, Case Western Reserve University), Srinivas Penumutchu (Department of Chemistry, Case Western Reserve University), Niyati Jain (Department of Chemistry, Case Western Reserve University), Andrew Sugarman (Department of Chemistry, Oberlin College), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
Alternative splicing in human immunodeficiency virus type 1 (HIV-1) is tightly regulated by the equilibrium between the spliced and unspliced primary transcripts. This equilibrium is maintained by the presence of inefficient splice sites that are further regulated by hnRNP and SR proteins. The A3 3’-splice site of HIV-1 is required for Tat mRNA production. The inefficient utilization of this splice site has been attributed to the presence of a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. HnRNP H binds to ESS2P element to inhibit the splicing process; however, there is no structural and mechanistic information about how hnRNP H recognizes ESS2P to inhibit the splicing. In our studies, we aim to characterize the three-dimensional structure of hnRNP H and ESS2P. The domain organization of hnRNP H/F proteins is modular consisting of N-terminal tandem quasi-RNA Recognition Motifs (HqRRM1,2) and a third C-terminal qRRM3 embedded within glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker with most of the amino acids strictly conserved between hnRNP H and F. We probed the structural dynamics of its HqRRM1,2 domain with x-ray crystallography, NMR spectroscopy, and Small Angle X-ray Scattering (SAXS). We observed that HqRRM12 contains multiple structures in solution by SAXS. These exchangeable conformations are located on the linker region and RNA recognition sites. Moreover, the three-dimensional structure of ESS2P has been determined using NMR spectroscopy and SAXS. Collectively, this work provides structural insight how hnRNP H recognizes the HIV ESS2P element.

References:
1. Srinivasa Penumutchu, Liang-Yuan Chiu, Jennifer L. Meagher, Alexandar L. Hansen, Jeanne A. Stuckey, and Blanton S. Tolbert. (2018) Differential Conformational Dynamics Encoded by the Inter-qRRM linker of hnRNP H, Journal of the American Chemical Society (Accepted Manuscript)

2. Sandrine Jacquenet, Agnès Méreau, Patricia S. Bilodeau, Laurence Damier, C. Martin Stoltzfus, and Christiane Branlant. (2001) A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H, J Biol Chem 276, 40464-404751

Keywords: hnRNP, ESS2P, HIV-1

83. Nucleolus-enriched repeat-containing noncoding RNA regulates rDNA transcription.

Qinyu Hao (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL), Rajneesh Srivastava, Sarath C. Janga (School of Informatics and Computing, Indiana University & Purdue University, Indianapolis, IN), Susan M. Freier (IONIS Pharmaceuticals, Carlsbad, CA), Brian McStay (Centre for Chromosome Biology, NUI Galway, Ireland), Sui Huang (Northwestern University, Chicago, IL), Supriya G. Prasanth, Kannanganattu V. Prasanth (Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL)

Abstract not available online - please check the printed booklet.

84. Optimization of a fluorescence-based assay for high throughput screening of small molecule inhibitors of the DEAD-box RNA helicase DDX3X

Kaba Tandjigora (The Center for RNA Science and Therapeutics), Eckhard Jankowsky (The Center for RNA Science and Therapeutics)

Abstract:
DEAD-box RNA helicases utilize ATP to bind and remodel RNA and RNA protein complexes. They are involved in virtually all aspects of RNA metabolism. Deregulation of many DEAD-box helicases have been associated with various diseases, particularly to cancer and infectious diseases. Although several DEAD-box helicases are attractive targets for anti-cancer and antiviral therapeutics, few efforts have been made to develop inhibitors for specific DEAD-box helicases. Here, we developed a fluorescence-based, endpoint assay suitable for High Throughput Screening (HTS) of small molecule inhibitors of the unwinding activity of DDX3X, a human DEAD-box RNA helicase. DDX3X has been linked to various cancers and is targeted by multiple viruses. We expressed and purified recombinant DDX3X and characterized enzymatic activities. We then developed a fluorescence-based assay for duplex unwinding that relies on fluorescence quenching of a cy3-labeled RNA strand by a label on the complementary RNA strand. The fluorescence signal increases upon unwinding. The assay was optimized for end-point determination and proved to be DMSO tolerant. Stability of the fluorescence signals and Z’ factor indicate high robustness, sensitivity, and reproducibility. The data set the stage for HTS screening of small molecule inhibitors of the helicase activity of DDX3X. The developed HTS assay is potentially applicable for other RNA helicases.

Keywords: DDX3X, HTS, Small molecules

85. Overcoming a “molecular ruler” mechanism: the unusual heterotrimeric tRNA splicing endonuclease of Trypanosoma brucei

Gabriel Silveira dAlmeida (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Christopher Trotta (PTC Therapeutics Inc), Arthur Gnzl (School of Medicine, University of Connecticut), Juan Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

86. Overexpression of a non-muscle RBFOX2 splice isoform induce cardiac arrhythmias in Myotonic Dystrophy

Chaitali Misra, Feikai Lin (Department of Biochemistry, University of Illinois, Urbana-Champaign, IL), Sushant Bangru, Darren J. Parker (Department of Biochemistry, University of Illinois, Urbana-Champaign, IL), Sara Koenig, Ellen Lubbers, Peter Mohler (Davis Heart and Lung Research Institute, Wexner Medical Center, Ohio State University, OH), Jamila Hedhli, Wawrzyniec L. Dobrucki (Department of Bioengineering), Thomas A. Cooper (Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX), Auinash Kalsotra (Department of Biochemistry, Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, IL)

Abstract not available online - please check the printed booklet.

87. PEGylated Oligonucleotide Synthesis – From Research Scale to Production Scale

Dessalegn B Nemera (Process Development, Nitto Denko Avecia), Sibo Jiang (Process Development, Nitto Denko Avecia), Holli Palmer (Analytical Development, Nitto Denko Avecia), Eduardo Paredes (Process Development, Nitto Denko Avecia)

Abstract:
One of the powerful strategies of drug delivery system to targeted tissue or cells is “Ligand conjugated therapeutic oligonucleotide”. This technique has attracted significantly more interest in the past years due to its uptake-enhancing, pharmacokinetic-improving and receptor-specific targeting properties. PEGylated oligonucleotide, in which polyethylene glycol (PEG) is conjugated to therapeutic oligonucleotide, is one of the conjugation strategies used to prolongs half-life and increases retention of the therapeutic oligonucleotide in tissues.

Manufacturing of PEGylated oligonucleotide had been historically challenging in the process of synthesizing, scaling up and cost of good considerations. This poster discusses lab scale process development (PD) work performed for a 40-mer therapeutic PEGylated RNA oligonucleotides followed by a successful 300 g scale-up manufacture. Challenges and lesson learned from the PD work and scale-up manufacture are presented in great detail.

Keywords: PEGylation, Oligonucleotide

88. Phosphorylation is yeast eukaryotic translation initiation factor 4A drives stress-induced changes in growth

Ansuman Sahoo (Department of Biological Sciences, SUNY Buffalo), Marium Ashraf (Department of Biological Sciences, SUNY Buffalo), Sarah E. Walker (Department of Biological Sciences, SUNY Buffalo)

Abstract not available online - please check the printed booklet.

89. Pnrc2 regulates 3'UTR-mediated decay of cyclic transcripts during somitogenesis

Kiel T. Tietz (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University ), Thomas L. Gallagher (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University ), Monica C. Mannings (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University ), Zachary T. Morrow (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University ), Nicolas L. Derr (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University ), Sharon L. Amacher (Department of Molecular Genetics and Center for RNA Biology, The Ohio State University )

Abstract:
Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of embryonic segments (somites). This molecular oscillator generates cyclic gene expression in the tissue that generates somites and has the same periodicity as somite formation. Molecular components of the clock include the her/Hes family of transcriptional repressors, but additional transcripts also cycle. Maintenance of oscillatory gene expression requires that transcriptional activation and repression, RNA turnover, translation, and protein degradation are rapid (one cycle is 30 minutes in the zebrafish). Little is known about post-transcriptional control of cyclic transcripts during somitogenesis and our work employs genetic and biochemical approaches to better understand rapid cyclic transcript turnover. We have shown that loss of Proline-rich nuclear receptor coactivator 2 (Pnrc2) in zebrafish causes accumulation of cyclic transcripts like her1, deltaC, and deltaD, and that the her1 3’UTR confers instability to otherwise stable transcripts in a Pnrc2-dependent manner. To begin to identify her1 3’UTR cis-regulatory elements critical for Pnrc2-mediated decay, we show here that the last 179 nucleotides (nts) of the 725 nt her1 3’UTR is sufficient to confer rapid instability. Additionally, we show that the 3’UTR of the deltaC cyclic transcript also contains Pnrc2-mediated destabilizing elements. We have identified a Pumilio Response Element (PRE) within the last 179 nts of the her1 3’UTR that is also present in the deltaC 3’UTR and show here the PRE is necessary for the her1 3’UTR to convey rapid turnover. These results suggest Pnrc2 may function with Pumilio, a known decay factor, to regulate the rapid turnover of cyclic transcripts during somitogenesis. Our work explores mechanisms regulating oscillation dynamics during vertebrate segmentation and will further our understanding of pathways controlling post-transcriptional gene regulation.

Keywords: her1, Pumilio, Rapid mRNA turnover

90. Poly(A) Binding Proteins Tune Translation in the Heart

Joe Seimetz (Biochemistry, UIUC), Sandip Chorghade (Biochemistry, UIUC), Bo Zhang (Biochemistry, UIUC), Stefan Bresson (Microbiology, UTSW), Nicholas Conrad (Microbiology, UTSW), Auinash Kalsotra (Biochemistry, UIUC)

Abstract not available online - please check the printed booklet.

91. Polypyrimidine tract-binding protein 1 (PTBP1) is a key regulator of hepatocyte responses to toxin induced injury

Miranda Gurra (Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, ), Ullas Valiya Chembazhi (Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, ), Cody Lund (Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, ), Auinash Kalsotra (Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, )

Abstract not available online - please check the printed booklet.

92. Process Development for the Synthesis of Chemically Modified DNA Oligonucleotide

Mamta Kaura (Process Development Department, Nitto Denko Avecia), Arnie De Leon (Process Development Department, Nitto Denko Avecia), Parades Eduardo (Process Development Department, Nitto Denko Avecia)

Abstract:
Chemically modified oligonucleotides play a vital role in the field of medicine and biotechnology. Avecia has extensive experience in optimizing the manufacturing process of various modified oligonucleotides. Here, we present the synthesis optimization of a DNA oligonucleotide modified with hydrophobic lipid group at the 5'-end. Finding a suitable solvent system to ensure the solubility of the lipid phosphoramidite solution and the coupling mixture significantly increased the coupling efficiency thus increasing the final yield.

Keywords: Oligonucleotides, Lipid, Coupling efficiency

93. Process validation approach for oligonucleotides

Anna Nielander M.S. (Nitto Denko Avecia), Sibo Jiang Ph.D. (Nitto Denko Avecia), Richard Lombardy Ph.D. (Nitto Denko Avecia), James ODonnell Ph.D. (Nitto Denko Avecia), Eduardo Paredes Ph.D. (Nitto Denko Avecia)

Abstract not available online - please check the printed booklet.

94. Proximal 3'UTR introns elicit EJC-dependent NMD during zebrafish development.

Pooja Gangras (Department of Molecular Genetics, OSU), Thomas L. Gallagher (Department of Molecular Genetics, OSU), Robert D. Patton (Department of Molecular Genetics, OSU), Kiel T. Tietz, Natalie C. Deans (Department of Molecular Genetics, OSU), Ralf Bundschuh, Sharon L. Amacher (Center for RNA biology, OSU), Guramrit Singh (Department of Molecular Genetics, Center for RNA biology, OSU)

Abstract not available online - please check the printed booklet.

95. Pseudouridylation enzyme RsuA influence 30S ribosome assembly

Kumudie Jayalath (Chemistry & Biochemistry, Kent State University), Minhchau To (Chemistry & Biochemistry, Kent State University), Sanjaya Abey (Chemistry & Biochemistry, Kent State University)

Abstract:
Ribosome is the Ribonucleoprotein complex that 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. Furthermore, as observed with a reverse transcriptase based activity assay, RsuA is catalytically cooperative with ribosomal protein S17. Our data suggests that the RsuA enzyme binds preferably to non-pseudoknoted (extended) helix 18 that is also important for its function.

Keywords: Ribosome biogenesis, RNA modification enzyme , Pseudouridine

96. Pumilio (PUM) proteins mediated post-transcriptional regulation

Swetha Rajasekaran (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University Comprehensive Cancer Center, The Ohio State University), Raleigh Kladney (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University Comprehensive Cancer Center, The Ohio State University), Wayne O. Miles (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University Comprehensive Cancer Center, The Ohio State University)

Abstract not available online - please check the printed booklet.

97. Quadruplex-mediated affinity purification of DNA-induced slicing complex

Daniel M. Dayeh (The Ohio State University), Elaina P. Boyle (The Ohio State University), Besik Kankia (The Ohio State University), Karin Musier-Forsyth (The Ohio State University), Kotaro Nakanishi (The Ohio State University)

Abstract:
Budding yeast Argonaute, a central enzyme in the RNA interference pathway, was recently loaded with a DNA guide and shown to assemble a functional DNA-induced slicing complex (DISC) that can cleave highly-structured RNAs in vitro. A major limitation in the preparation of recombinant eukaryotic Argonaute is the difficulty of removing host-derived endogenous cellular RNAs that remain tightly bound to the majority of the population, leaving only small amounts of protein available for loading with synthetic RNA or DNA guides. This drawback has hampered the development of more advanced Argonaute-mediated applications. Here, we use the recently-described quadruplex-and-Mg2+ connection technology to develop a nucleic acid-based affinity tag for the isolation of homogeneous DISCs. This method allows active and desirable DISCs to be purified away from contaminating RNA-bound complexes that limit the current use of DISC. The simple, cost-effective method presented here increases the potential applications of Argonaute-based tools in biotechnology and therapeutics.

References:
Dayeh, D.M., Cantara, W.A., Kitzrow, J.P., Musier-Forsyth, K., Nakanishi, K. . (2018) Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA Nucleic Acids Res.

Keywords: RNAi, DISC , programmable RNase

98. R(A)PTOR - A tool for systematic identification of Poly(A) tails and 3’ unmapped regions from single molecule direct RNA-sequencing datasets

Aniruddhan Govindaraman (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University Indianapolis), Raja Shekar Varma Kadumuri (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University Indianapolis), Mir Quoseena (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University Indianapolis), Sarath Chandra Janga (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University Indianapolis)

Abstract:
The 3’ cleavage of pre-messenger RNA (mRNA) and successive polyadenylation is a fundamental cellular process in eukaryotes. Studies report poly-A tail as a long chain of adenine nucleotides added during RNA processing to 3’ terminal of a messenger RNA (mRNA) molecule, however, the terminal 3’ region is known to harbor additional unmappable regions (UMR) composed of poly uridylation and guanylation[1]. Although short read sequencing technologies are extensively used for study of 3’ terminal polyA regions, the major drawback of third generation sequencing technologies lies in their inability to detect full length homopolymeric sequences [1],[2]. Recent long read sequencing technologies like Nanopore sequencing enable sequencing of full length transcripts at a single molecule resolution, and currently there are no tools for systematically analyzing 3’ terminal unmapped regions from direct RNA-sequencing datasets. We present RAPTOR (https://github.com/aniram118/RAPTOR), a command line tool for 3’ terminal unmapped region analysis of nanopore direct RNA sequencing data. RAPTOR provides a comprehensive report of UMR length, sequences, conserved polyA hexamer regions, nucleotide base composition and transcript vs UMR length correlation analysis at a single molecule resolution. In our benchmarking studies, we sequenced mRNA samples obtained from HepG2 & K562 cell lines resulting in 243,802 & 598,428 reads respectively. RAPTOR identified UMRs exhibited a median length of 50-100 nt, in agreement with previous studies[1].Our results also support an enrichment of previously known conserved polyA hexamers [3]. Nucleotide composition analysis of the identified 3’ UMR regions showed an enrichment for A and U nucleotides in both HepG2 [A : 29%, U: 28%, G:20%, C:23% ] and K562 [A : 30%, U: 29%, G:19%, C:22%] and interestingly, guanylation was observed in upstream and downstream regions of UMR while uridylation was found to occur more in central regions, suggesting their characteristic role in mRNA stability. In addition, conserved motif analysis of UMR regions followed by RBP binding site analysis, identified several RBPs including HNRPK, PCB2, SART SRSF9, HNRPR and RBM4 to be enriched in the unmapped regions, suggestive of an unappreciated of role of these RBPs in binding to 3’ tails of mRNAs.

References:
1. Chang, Hyeshik; Lim, Jaechul; Ha, Minju; Kim, V Narry (2014) TAIL-seq: Genome-wide Determination of Poly(A) Tail Length and 3′ End Modifications. 10.1016/j.molcel.2014.02.007

2. Byrne, A., Beaudin, A. E., Olsen, H. E., Jain, M., Cole, C., Palmer, T., … Vollmers, C. (2017). Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. Nature Communications, 8, 16027. http://doi.org/10.1038/ncomms16027

3. Pesole, G., Liuni, S., Grillo, G., Licciulli, F., Mignone, F., Gissi, C., & Saccone, C. (2002). UTRdb and UTRsite: specialized databases of sequences and functional elements of 5′ and 3′ untranslated regions of eukaryotic mRNAs. Update 2002. Nucleic Acids Research, 30(1), 335–340.

Keywords: Polyadenylation, Nanopore Sequencing

99. Rbfox1: extraordinary RNA binding specificity by optimized discrimination against non-cognate sequences

Xuan Ye (Center for RNA Science and Therapeutics,Department of Biochemistry, Case Western Reserve University, Cleveland, OH), Fan Yang (Department of Chemistry, University of Washington, Seattle, WA), Ulf-Peter Guenther (European Molecular Biology Laboratory, Heidelberg, Germany), Gabriele Varani (Department of Chemistry, University of Washington, Seattle, WA), Eckhard Jankowsky (Center for RNA Science and Therapeutics,Department of Biochemistry, Case Western Reserve University, Cleveland, OH)

Abstract not available online - please check the printed booklet.

100. Reaction kinetics, nucleoside preference, and impact of amino-acid-linked platinum analogues on RNA and DNA

Bett Kimutai (Wayne State University Chemistry), Chenchen He (Wayne State University Chemisty), Andrew Roberts (Wayne State University Chemistry), Marcel Jones (University of Detroit Mercy), M. T. Rodgers (Wayne State University Chemistry), Christine S. Chow (Wayne State University Chemistry)

Abstract:
Cisplatin is known to preferentially coordinate to deoxyguanosine residues of DNA and forms adducts that inhibit DNA functions. RNA is also a competitive target of cisplatin and aquated cisplatin may have preferential target residues in RNA as observed with DNA. Since cisplatin has a number of drawbacks that reduce its effectiveness, including repair mechanisms that remove the platinum adducts from the DNA strands, there is still a need to develop compounds that are able to coordinate to different nucleic acid sites. RNA residues may serve as ideal targets for such platinum-based drugs. Platinum compounds that target residues other than deoxyguanosine may have alternative anticancer mechanisms that circumvent the undesired inactivation pathways of cisplatin. In this study, we demonstrate that amino-acid-linked platinum(II) analogues (AlaPt, OrnPt, and ArgPt) have altered reactivity compared to cisplatin. They preferentially coordinate and form adducts with adenosine and deoxyadenosine residues. ArgPt also forms adducts with poly(A) RNA and shows selective toxicity in cancer cells. Binding of AlaPt and OrnPt to adenosine leads to destabilization of the glycosidic bond for certain adducts. This phenomenon is dependent on the site of platination and the type of ligand present in the metal complex. The weakening of the nucleoside glycosidic bond, could have important biological consequences including depurination and degradation of nucleic acids.

Keywords: cisplatin, cell toxicity

101. Recognition of RNA substrates by Thg1 family proteins

Tracy M. Roach (The Ohio State University, Department of Chemistry and Biochemistry), Marie-Theres Pöhler (Institute for Biochemistry, Leipzig University), Krishna Patel (The Ohio State University, Department of Chemistry and Biochemistry), Mario Mörl (Institute for Biochemistry, Leipzig University), Jane Jackman (The Ohio State University, Department of Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

102. Regulation of alternative splicing by U5 small nuclear RNA variants

Justin Mabin (Biomolecular Chemistry, University of Wisconsin Madison), Katherine Senn (Biomolecular Chemistry, University of Wisconsin Madison), Heidi Dvinge (Biomolecular Chemistry, University of Wisconsin Madison)

Abstract:
Alternative splicing (AS) is a highly dynamic and multivariate process that shapes and expands the human transcriptome and proteome. To date, much of the research on AS regulation has focused on splicing co-factors. But, increasing evidence has shown that core-splicing proteins are also involved in regulating AS¹. In addition, the spliceosome contains five small nuclear RNAs (snRNAs) that are responsible for carrying out much of the splicing reaction. snRNAs are loaded with their own complement of proteins forming small nuclear ribonucleoprotein (snRNP) complexes that function in spliceosome assembly. Recently, it has been shown that snRNP proteins are differentially expressed across human tissues, hinting at a cell-type specific regulatory role of core snRNP proteins². However, there is little research aimed at how the core snRNAs may contribute. Here we focus on U5 snRNAs as there have been five U5 snRNA variants previously shown to incorporate into spliceosomes³. Human genome analysis has identified an additional large number of snRNA variant genes. However, their expression remains largely unknown. Our goal was to systematically characterize the expression of the U5 snRNA sequence variants. Preliminary data from our lab suggests that many of these snRNAs are actually expressed in human tissues. We have also identified several novel U5 variants capable of snRNP formation and are currently working to identify which variants are incorporated into spliceosomes. Other preliminary work has shown that perturbations in U5 snRNA variant levels can affect transcript-specific splicing events, suggesting that U5 snRNA variants may have a role in regulating AS. Overall, we hypothesize that U5 snRNA variants are capable of forming variant snRNPs that have a unique subset of splicing co-factors that may target snRNAs to specific splice-sites. Future work will aim to identify variant snRNP compositional differences and their transcriptome-wide AS effects.

References:
1. Papasaikas P, Tejedor JR, Vigevani L and Valcarcel J (2015) Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery. Mol Cell 57, 7–22.
2. Grosso AR, Gomes AQ, Barbosa-Morais NL, Caldeira S, Thorne NP, Grech G, von Lindern M and Carmo-Fonseca M (2008) Tissue-specific splicing factor gene expression signatures. Nucleic Acids Res 36, 4823–4832.
3. Sontheimer EJ and Steitz JA (1992) Three novel functional variants of human U5 small nuclear RNA. Mol Cell Biol 12, 734–746.

Keywords: Alternative splicing, small nuclear RNA, Core spliceosome

103. Regulation of translational fidelity in Salmonella Typhimurium

Rebecca E Steiner (Ohio State Biochemistry Program Ohio State university ), Michael Ibba (Microbiology Ohio State University )

Abstract not available online - please check the printed booklet.

104. RIPiT-Seq analysis suggests a compositional switch between structurally and functionally distinct exon junction complexes

Robert Patton (Department of Physics, The Ohio State University), Justin Mabin (Department of Molecular Genetics, The Ohio State University), Lauren Woodward (Department of Molecular Genetics, The Ohio State University), Ralf Bundschuh (Department of Physics, Center for RNA Biology, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University)

Abstract:
The exon junction complex (EJC) is a protein complex first deposited upstream of exon splice junctions during pre-mRNA splicing. The EJC plays a vital role in shaping the fate of an mRNA by nucleating the assembly of peripheral proteins which function in diverse post-transcriptional processes, especially nonsense-mediated decay (NMD). RIPiT-Seq footprint analysis of the EJC core bound to the mutually exclusive peripheral proteins RNPS1 and CASC3 reveals that alternate EJCs similarly bind to junctions on a gene by gene basis. Further analysis of genes which are found with significantly higher levels of RNPS1 or CASC3 shows heightened levels of RNPS1 containing EJCs on transcripts associated with the nucleus and CASC3 containing EJCs in transcripts associated with the cytoplasm. Treatment with the translation inhibitor cycloheximide also causes a large increase in the number of transcripts with CASC3 containing EJCs, indicating a build up of CASC3 EJCs in the cytoplasm. We conclude that when the EJC is first assembled in the nucleus it contains the protein RNPS1, but that some time before translation goes through a compositional change in which RNPS1 is replaced with CASC3. This switch leads to two distinct EJCs with separate NMD pathways.

Keywords: nonsense-mediated decay (NMD), pre-mRNA splicing, exon junction complex (EJC)

105. RISC assembly of Argonaute correlated with the formation of internal water clusters

Mi Seul Park (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Audrey C. Kehling, Raul Araya-Secchi, James A. Brackbill (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Hong-Duc Phan, Daniel M. Dayeh (Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.), Marcos Sotomayor (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA), Kotaro Nakanishi (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA)

Abstract not available online - please check the printed booklet.

106. RNA Aptamer Development for Biosensor by SPR-SELEX

Kritisha Bhandari (Department of Chemistry, Southern Illinois University Edwardsville), Sun Jeong Im (Department of Chemistry, Southern Illinois University Edwardsville), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville)

Abstract:
E. coli outbreak is not only a problem in the United States but is a global concern. A biosensor that can detect harmful microorganisms can be a very effective approach to prevent this issue. Aptamers, single-stranded nucleotides, have high affinity towards a specific target molecule. Biosensors, when developed with an aptamer, are capable of detecting microorganisms like E. coli. An aptamer can be selected from the process of SELEX (Systematic Evolution of Ligand by Exponential Enrichment). A SELEX with surface plasmon resonance (SPR-SELEX) was recently developed in our lab to accelerate the SELEX process. In this project, the SPR-SELEX will expand to RNA aptamer selection. RNA can form more diverse structures than DNA. This research emphasizes the identification of an RNA aptamer sequence for E. coli K-12 strain.

Keywords: SELEX, SPR

107. RNA drug discovery: optimizing chemoinformatic analysis workflow

Ali H. Aldhumani (Chemistry & Biochemistry, Ohio University), Jennifer V. Hines (Chemistry & Biochemistry, Ohio University)

Abstract:
Targeting noncoding regulatory RNA is an emerging field of drug discovery with successful examples in targeting specific small bulges and other structured regions with small molecules. A continued challenge, however, is the specific targeting of larger RNA motifs that are less structured and more flexible. The antiterminator element in the T-box riboswitch is one example. The T-box riboswitch regulates gene expression by transcription antitermination or inhibition of translation initiation and is a good target for antibacterial drug discovery.¹ The antiterminator element contains a highly conserved 14 nucleotide sequence and serves an essential role in the riboswitch function. Based on prior results,² we are optimizing chemoinformatic workflows combining experimental results from screening assays with molecular modeling to identify new ligand scaffolds with improved activity. Utilizing the Schrödinger suite of software, we are developing a pharmacophore binding model for the antiterminator by exploring ligand docking to multiple RNA conformers. Using the Glide function in Maestro, a grid is generated of the antiterminator (NMR derived structure), to which a model compound library (NIH Natural Products Repository IV) is docked. Chemoinformatic analysis of docking results and experimental data to determine quantitative structure-activity relationships will assist in predicting other compounds of interest and provide further insight of the druggability of the T-box riboswitch.

References:
1. Kreuzer, K. D.; Henkin, T. M., The T-box riboswitch: tRNA as an effector to modulate gene regulation. Microbiol Spectrum 2018, 6 (4), RWR-0028-2018.
2. (a) Liu, J.; Zeng, C.; Hogan, V.; Zhou, S.; Monwar, M. M.; Hines, J. V., Identification of spermidine binding site in T-box riboswitch antiterminator RNA. Chem. Biol. Drug Design 2016, 87, 182-189; (b) Orac, C. M.; Zhou, S.; Means, J. A.; Boehme, D.; Bergmeier, S. C.; Hines, J. V., Synthesis and stereospecificity of 4,5-disubstituted oxazolidinone ligands binding to T-box riboswitch RNA. J. Med. Chem. 2011, 54, 6786-6795.

Keywords: T-box riboswitch , drug discovery, ncRNA

108. Role of hepatic RNA silencing in the pathogenesis of obesity

Takahisa Nakamura (Pediatric Endocrinology, Cincinnati Childrens Hospital )

Abstract:
The worldwide prevalence of obesity has reached pandemic proportions, and with it have come other associated metabolic diseases. Despite the urgent need for treatment, the pathogenic mechanisms underlying this cluster of metabolic disease are still unclear, and effective preventive and/or therapeutic strategies are limited. Recent studies have revealed significant roles for microRNA (miRNA)-mediated events in the development and progression of metabolic diseases. For instance, the global dysregulation of miRNA biogenesis is triggered in an obese condition, of which specific miRNAs could drive pathophysiological changes and disrupt energy metabolism. However, a fundamental question if obesity evokes functional changes in miRNA-regulatory machinery, resulting in the abnormal biogenesis of miRNAs participating in the pathogenesis of obesity and metabolic disturbance, has not yet been addressed, despite drastic changes in global miRNA expression profile in the obese condition. We have recently discovered novel roles of hepatic miRNA-regulatory machinery in the pathogenesis of obesity and regulation of systemic energy homeostasis. These components in the liver regulate glucose- and fatty acids-driven energy production via RNA silencing of genes critical for mitochondrial functions and affect systemic energy homeostasis. These results have significant implications for understanding the role of hepatic miRNA and its regulatory machinery in the regulation of metabolic homeostasis and for defining an entirely novel class of therapeutics for metabolic diseases.

References:
Cai Zhang et al., "Hepatic Ago2-mediated RNA silencing controls energy metabolism linked to
AMPK activation and obesity-associated pathophysiology" Nature Communications (In press)

Keywords: Obesity , RNA silencing, Argonaute 2

109. Role of hsa- miR-149-5p in Steroid Biosynthesis pathway

Asmita Bhattarai (Center for Gene regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University ), Girish C. Shukla (Center for Gene regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University )

Abstract not available online - please check the printed booklet.

110. Role of the conserved GTPase BipA in assembly of the 50S subunit of the ribosome

Michelle R. Gibbs (Department of Microbiology and Center for RNA Biology, The Ohio State University), Kyung-Mee Moon (Department of Biochemistry and Molecular Biology, University of British Columbia), Leonard Foster (Department of Biochemistry and Molecular Biology, University of British Columbia), Kurt Fredrick (Department of Microbiology and Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

111. Roles of multiple polymerases in trypanosome mitochondrial non-encoded mRNA tail addition

Clara M. Smoniewski (Department of Biomedical Sciences, University of Minnesota Medical School, Duluth Campus), Miranda Galey (Department of Biology, University of Minnesota Duluth), Marshall Hampton (Department of Mathematics and Statistics, University of Minnesota Duluth), Sara L. Zimmer (Department of Biomedical Sciences, University of Minnesota Medical School, Duluth Campus)

Abstract:
Trypanosoma brucei is a parasitic protozoa that causes devastating vector-borne human and livestock diseases. The parasite modifies its metabolism to utilize different nutrients available in its insect vector and mammalian host, requiring extensive gene expression regulation, particularly in the mitochondria. In the trypanosome mitochondrion, gene expression is regulated post-transcriptionally because genes are polycistronically transcribed. Non-encoded 3' tails on trypanosome mitochondrial mRNAs have transcript and life-stage specific differences and are known to play a role in gene expression regulation. There are two types of heterogeneous A/U tails: initially added (in-tails), which are short and likely stability elements; and extended (ex-tails), which are longer and seem to have a role in translation. Our mathematical modeling of the distribution of As within the tails suggest that there may be multiple different ways that As are being added. This is consistent with two different poly(A) polymerases being active in the mitochondria: the primary polymerase kPAP1, and a putative polymerase kPAP2 that has been minimally characterized to date. By genetically manipulating these polymerases, we will investigate their effect on tail composition and length. A combination of hidden Markov modeling and genetic manipulations should clarify how the tails are differentially added to transcripts and across life-stages, and may lead to a greater understanding of the tails' roles in gene expression regulation.

Keywords: Post-transcriptional regulation, mitochondrial 3 non-encoded tails, trypanosomes

112. RsmC shows RNA annealing activity during ribosome biogenesis

Keshav GC (Department of Chemistry and Biochemistry, Kent State University), Prabesh Gyawali (Department Of Physics, Kent State University), Hamza Balci (Department Of Physics, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)

Abstract not available online - please check the printed booklet.

113. Selectivity and editing mechanisms of an 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 domain 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 and maintain high fidelity in protein synthesis. ProXp-ala, a structural homolog of the editing domain found in most bacterial prolyl-tRNA synthetases can deacylate mischarged Ala-tRNA^Pro. We have provided evidence that Caulobacter crescentus ProXp-ala recognizes features on the acceptor stem of tRNA^Pro, while three overlapping mechanisms contribute to discrimination between Ala-tRNA^Pro and Pro-tRNA^Pro: conformational selection, size exclusion and chemical selection. However, the structural basis for this discrimination is incompletely understood. We are using X-ray crystallography, NMR spectroscopy and enzymatic assays to probe the structural and thermodynamic details of conformational selection by studying the structures of ProXp-ala in complex with uncharged tRNA^Pro and bound to a non-hydrolyzable Ala-tRNA^Pro analog. We are also assessing the catalytic role played by the 2’OH of the terminal 76A in tRNA^Pro. These studies will facilitate our understanding of both substrate recognition and hydrolysis by the proofreading enzyme ProXp-ala.

References:
1. Das M., Vargas-Rodriguez O., Goto Y., Novoa, E., Pouplana L., Suga H., Musier-Forsyth K., (2014). Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation. Nucl. Acids Res. 42 (6):3943-3953.

2. Ling J, Reynolds N, Ibba M (2009) Aminoacyl-tRNA synthesis and translational quality control. Annu Rev Microbiol 63:61–78.

3. 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, RNA-protein interaction

114. SHAPE analysis of wild type and mutant htrA RNA thermometer in Salmonella enterica

Madeline Su, Anna Chee (Denison University, Department of Chemistry and Biochemistry), Rachel Mitton-Fry (Denison University, Department of Chemistry and Biochemistry)

Abstract not available online - please check the printed booklet.

115. SliceIt: RNA-Binding Protein (RBP) centric genome-wide in silico library of sgRNAs for CRISPR/Cas9 screens

Sasank Vemuri (BioHealth Informatics, IUPUI), Rajneesh Srivastava (BioHealth Informatics, IUPUI), Seyedsasan Hashemikhabir (BioHealth Informatics, IUPUI), Sarath Chandra Janga (BioHealth Informatics, IUPUI)

Abstract:
Several UV cross linking protocols such as eCLIP have been established to delineate the molecular interaction of RNA Binding Protein (RBP) and their target RNAs. CRISPR/ Cas9 system has been employed to verify such localized interactions in cells. With the advancement of pooled CRISPR/Cas9 screens, it is possible to study the global impact of these proteins in human cells. Here, we present SliceIt, a database of in silico sgRNA (or guideRNA) library that helps the researchers to conduct such high throughput screens. We used CRISPR-DO to design ~4.8 million unique sgRNAs targeting all possible RBP binding sites from the eCLIP experiment of 123 RBP in HepG2 and K562 cell lines from ENCODE. SliceIt provides a user friendly environment, developed in highly advanced search engine framework called Elasticsearch. It is available in both table and genome browser view that facilitates the easy navigation of RBP binding sites, sgRNAs, SNPs and GWAS. It also provides the exon expression profile across 53 tissues from GTEx to examine the locus specific changes proximal to the binding sites. User can also upload custom tracks of various file formats (in browser) to navigate additional genomic features in hg38 genome and cross compare with our profiling. All the binding site centric information is dynamically accessible via “search by gene”, “search by coordinate” and “search by RBP” and readily available to download. Finally, SliceIt is a one-stop repertoire of guideRNA library, RBP binding sites along with several layer of functional information to design the high throughput CRISPR cas9 screens for studying the phenotypes and diseases associated to RBP centric post transcriptional regulation.

URL: https://sliceit.soic.iupui.edu/

References:
1 Van Nostrand, E. L. et al. Nature methods, doi:10.1038/nmeth.3810 (2016).
2 Van Nostrand, E. L. et al. Methods (San Diego, Calif.) 118-119, 50-59, doi:10.1016/j.ymeth.2016.12.007 (2017).
3 Dixit, A. et al. Cell 167, 1853-1866 e1817, doi:10.1016/j.cell.2016.11.038 (2016).
4 Datlinger, P. et al. Nature methods 14, 297-301, doi:10.1038/nmeth.4177 (2017).
5 Adamson, B. et al. Cell 167, 1867-1882 e1821, doi:10.1016/j.cell.2016.11.048 (2016).
6 Rauscher, B. et al. Nucleic Acids Res 45, D679-D686, doi:10.1093/nar/gkw997 (2017).
7 Ma, J. et al. Bioinformatics 32, 3336-3338, doi:10.1093/bioinformatics/btw476 (2016).
8 Davis, C. A. et al. Nucleic Acids Res 46, D794-D801, doi:10.1093/nar/gkx1081 (2018).
9 Segura B et al. JMIR Med Inform 5, e48, doi:10.2196/medinform.7059 (2017).

Keywords: RNA binding proteins, in silico library, CRISPRCas9 pooled screens

116. SPR-SELEX: New Method to Determine DNA Aptamers that Targets E.coli for Biosensor

Sun Jeong Im (Department of Chemistry, Southern Illinois University Edwardsville), Hailey Bowen (Department of Chemistry, Southern Illinois University Edwardsville), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville)

Abstract:
Escherichia coli (E. coli) outbreak is a serious health issue in the United States today. These outbreaks are caused by pathogenic E. coli strains. One possible way to prevent outbreaks is by detecting the harmful E. coli via biosensor with a DNA aptamer. Using the systematic evolution of ligands by exponential enrichment (SELEX) technique, the DNA aptamer sequence with a high affinity and specificity to the target E. coli strain K12 can be determined. However, the current SELEX procedure is time-consuming. Also, the selection progress cannot be monitored in real time with the conventional SELEX method without radiolabeling. Surface plasmon resonance (SPR) can resolve these disadvantages of the current SELEX technique by measuring the molecular binding kinetics in real-time without any labeling. In this project, we present the development of a faster and safer SELEX method combined with surface plasmon resonance (SPR-SELEX) to identify DNA aptamers.

Keywords: SELEX, SPR

117. sRNA-target Prediction Organizing Tool (SPOT) integrates computational and experimental data to facilitate functional characterization of bacterial small RNAs

Alisa M. King (Department of Microbiology, University of Illinois at Urbana-Champaign), Carin K. Vanderpool (Department of Microbiology, University of Illinois at Urbana-Champaign)

Abstract not available online - please check the printed booklet.

118. SRSF2: A Splicing Factor that Positively Regulates the Alternative Splicing of MDM2

Matias Montes (Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University), Daniel Comiskey ( Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital), Rehan Hussain (Center for Molecular and Human Genetics, Nationwide Childrens Hospital), Dawn Chandler ( Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital)

Abstract not available online - please check the printed booklet.

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

Charlotte N. Kunkler (Department of Chemistry and Biochemistry, University of Notre Dame), Sarah C. Hickman (Department of Chemistry and Biochemistry, University of Notre Dame), Matthew C. Wang (Department of Chemistry and Biochemistry, University of Notre Dame), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)

Abstract not available online - please check the printed booklet.

120. STARzyme: a ribozyme with tRNA synthetase activity

Rui Gan (Biochemistry, Purdue University), Hillary Aguilar (Biochemistry, Purdue University), Blair Chen (Biochemistry, Purdue University), Barbara Golden (Biochemistry, Purdue University)

Abstract:
Unnatural amino acids (UAAs) are widely used in the field of post-translational modification, biospecific antibody, replication-incompetent virus, therapeutics enzymes, etc. Currently, the incorporation of UAAs is mainly relied on the engineering of the protein tRNA synthetases. However, the chemical diversity of the introduced UAAs is restricted by the specificity of the protein’s active site, thus, only a limited number of UAAs have been successfully incorporated. In this work, a ribozyme with tRNA synthetase activity was created and named STARzyme (specific tRNA aminoacylation ribozyme). There are two modules in the STARzyme, one module is the T-box riboswitch RNA that binds to a specific tRNA through codon-anticodon interaction; the other module is the flexizyme (flexible ribozyme) that catalyzes the aminoacylation through binding the CCA tail of tRNAs. By fusing the two modules together, the STARzyme is able to put a variety of UAAs into a specific tRNA. And thus, the chemical diversity of UAA substrates has the potential to be greatly expanded.

Keywords: unnatural amino acids, aminoacylation, ribozyme

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

Phillip J. McCown (Department of Chemistry and Biochemistry, University of Notre Dame), Matthew C. Wang (Department of Chemistry and Biochemistry, University of Notre Dame), Luc Jaeger (Department of Chemistry and Biochemistry, Biomolecular Science and Engineering Program, University of California at Santa Barbara), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)

Abstract not available online - please check the printed booklet.

122. Structural analysis of RNA in the gas phase using ion mobility coupled to native MS

Mengxuan Jia (Department of Chemistry and Biochemistry, The Ohio State University), Samantha Sarni (Department of Chemistry and Biochemistry, The Ohio State University), Andrew Norris (Department of Chemistry and Biochemistry, The Ohio State University), Edrick Choi (Department of Chemistry and Biochemistry, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University), Vicki Wysocki (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
Native mass spectrometry (MS) has emerged as a powerful bioanalytical tool to characterize proteins and protein complexes in the past few decades1,2. However, the application of native MS on RNA is limited for the following reasons. First of all, whether the RNA ions can maintain a native-like structure in the gas phase as protein ions do is still unclear. Research shows that the RNA ions might collapse in the gas phase3. Secondly, RNA refolding often requires a certain concentration of divalent metal ions, e.g. Mg2+. This non-volatile additive, Mg2+, would cause signal suppression and peak broadening which make the data hard to interpret. Thirdly, there is no RNA calibrant database for collision cross section (CCS) measurement from the commercialized traveling wave ion mobility MS. In this work, we optimized the instrument settings for the direct CCS measurement of two model RNAs(PDB 2PCV and 2DRB)3, tRNA Phe and its mutant4, in the gas phase by using drift tube ion mobility MS and compared the experimental CCS with the theoretical values. We also investigated the influence of charge state and the amount of Mg2+ titrated in using collision and surface induced unfolding profiles via on a traveling wave ion mobility MS. In conclusion, with proper conditions, the tested RNA retains its native-like structure in the gas phase.

References:
1. Boeri Erba, E. & Petosa, C. The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes. Protein Sci. 24, 1176–1192 (2015).
2. Heck, A. J. R. Native mass spectrometry: a bridge between interactomics and structural biology. Nat. Methods 5, 927–933 (2008).
3. Devine, P. W. A. et al. Investigating the Structural Compaction of Biomolecules Upon Transition to the Gas-Phase Using ESI-TWIMS-MS. J. Am. Soc. Mass Spectrom. 28, 1855–1862 (2017).
4. Strulson, C. A., Boyer, J. A., Whitman, E. E. & Bevilacqua, P. C. Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions. RNA 20, 331–347 (2014).

Keywords: Native MS, Ion mobility, RNA structure

123. Structural Determination of the Interaction Between Two Critical RNA-Binding Proteins

Taylor N. Ayers (Department of Biological Sciences, University of Pittsburgh), Hiba A. Al-Ashtal (Department of Biological Sciences, University of Pittsburgh), Andrea J. Berman Ph.D. (Department of Biological Sciences, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

124. Structural Imprints of in vivo RNA Folding in human Parasites

Abhishek Dey (Department of Biological Sciences, University of North Carolina-Charlotte), Kausik Chakrabarti (Department of Biological Sciences, University of North Carolina-Charlotte)

Abstract not available online - please check the printed booklet.

125. Structural insights into the RNA methyltransferase domain of METTL16

Agnieszka Ruszkowska (Department of Chemistry and Biochemistry, University of Notre Dame), Milosz Ruszkowski (Synchrotron Radiation Research Section of MCL, National Cancer Institute), Zbigniew Dauter (Synchrotron Radiation Research Section of MCL, National Cancer Institute), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)

Abstract:
N⁶-methyladenosine (m⁶A) is an RNA modification that plays a central role in biology. Most m⁶A marks are catalyzed by a heterodimeric complex comprised of methyltransferase-like protein 3 and methyltransferase-like protein 14 (METTL3/METTL14), which specifically methylates adenine within a RRACH (R = A or G; H = A, C, or U) sequence motif. Recently, methyltransferase-like protein 16 (METTL16) was confirmed to be an m⁶A RNA methyltransferase that modifies U6 spliceosomal RNA and the MAT2A mRNA encoding S-adenosylmethionine (SAM) synthase. Unlike METTL3/METTL14, METTL16-dependent m⁶A marks do not occur within the RRACH sequence motif, and they are found in introns and at intron-exon boundaries. Multiple studies report that METTL16 uses a combination of sequence and structure to recognize its RNA substrates and binding partners, some of which are implicated in cancer. For example, METTL16 interacts with an RNA triple helix located at the 3' end of the long non-coding RNA, MALAT1 (metastasis-associated lung adenocarcinoma transcript 1). METTL16 binding depends on both the nucleotide composition and structure of the MALAT1 triple helix.

Here, we present two X-ray crystal structures of the N-terminal methyltransferase domain (residues 1-291) of human METTL16 (METTL16_291): an apo structure at 1.9 Å resolution and S-adenosylhomocysteine-bound complex at 2.1 Å resolution. The structures revealed a highly conserved Rossmann fold that is characteristic of Class I S-adenosylmethionine-dependent methyltransferases and a large, positively charged groove which represents the RNA-binding site. In-depth analysis of the active site led to a model of the methyl transfer reaction catalyzed by METTL16. A native gel-shift assay shows that in contrast to full-length METTL16, METTL16_291 does not bind to the MALAT1 triple helix. Our results provide insights into the molecular structure of METTL16, which is distinct from METTL3/METTL14.

Keywords: METTL16, RNA methyltransferase , N6-methyladenosine

126. Structural Integrity Of rRNA Central Junction Is Essential For Ribosome Function

Arnab Ghosh (Department of Cell biology and Neuroscience, Rowan University School of Osteopathic Medicine), Jessica A. Zinskie (Department of Cell biology and Neuroscience, Rowan University School of Osteopathic Medicine), Daniel Shedlovskiy (Department of Cell biology and Neuroscience, Rowan University School of Osteopathic Medicine), Dimitri G. Pestov (Department of Cell biology and Neuroscience, Rowan University School of Osteopathic Medicine), Natalia Shcherbik (Department of Cell biology and Neuroscience, Rowan University School of Osteopathic Medicine)

Abstract not available online - please check the printed booklet.

127. Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles.

Emil F. Khisamutdinov (Chemistry, Ball State University), Jake K. Durbin (Biology, Ball State University), Nathan McCann (Chemistry, Ball State University), E. Hong (Chemistry, Ball State University), JR Halman (Chemistry, Ball State University)

Abstract:

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics that could potentially be used in immunomodulation therapy to treat diseases by enhancing, inducing, or suppressing an immune response that would be beneficial to the host. Despite tremendous advancements in NANP development, their immunotoxicity has never been fully characterized. Here, we describe a systematically studied immunological recognition of representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on the NANP's shape, connectivity, and composition. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Keywords: RNA and DNA nanoparticles, immunorecognition, interferons

128. Synthesis of higher order oligonucleotides for therapeutic investigations

Michael Brennan (Oligrow, Nitto Denko Avecia, Cincinnati, OH), Tim Efthymiou (Oligrow, Nitto Denko Avecia, Cincinnati, OH), Carol Kirby (Oligrow, Nitto Denko Avecia, Cincinnati, OH), Amanda Magrino (Oligrow, Nitto Denko Avecia, Cincinnati, OH), Daniel Tener (Oligrow, Nitto Denko Avecia, Cincinnati, OH), Huihe Zhu (Oligrow, Nitto Denko Avecia, Cincinnati, OH)

Abstract:
Aside from promising developments of siRNA and antisense technologies, higher order oligonucleotide structures are becoming a new field of interest for therapeutic purpose. Among them, tRNAs have been found to potentially treat nonsense mutation-associated diseases; tRNAs can override premature stop mutations and reproduce functional proteins. Another area is circular DNA/RNAs. With finding more circular RNAs in humans, cyclic DNA/RNA oligonucleotides are explored as both biological tools and potential therapeutics due to their good serum and thermal stabilities. To keep up with these new potentials in the field, we explored different routes, such as chemical ligation, enzymatic ligation, and/or linear synthesis, to manufacture such compounds from a process perspective. This poster has two parts. We will show the successful process development for a complete circular loop oligonucleotide (100 mg) using chemical ligation. The second part compares production of a modified tRNA using the standard linear construction through solid phase approach to an enzymatic approach where two smaller sequences are joined to make the longer 76-mer. From the analysis of the initial process development activities, a process for a 5 gram manufacture was chosen based on yield, impurity profile, and cost of goods.

Keywords:

129. Synthesis of single guide RNAs for CRISPR/Cas

Arun Kalliat Thazhathveetil (Process Development, Avecia), Daniel L. Tener (Small Scale Oligo Unit, Avecia)

Abstract:
Recent advent in genome-editing technologies has resulted in renewed interest in long oligonucleotides, especially RNA longmers that are used as single guide RNAs (sgRNA) for CRISPR-Cas9 system. These RNA longmers can be synthetically synthesized or produced in vitro or in vivo. Chemically synthesizing sgRNA provides versatility in incorporating modifications that can potentially improve the CRISPR gene editing efficiency. However, even with the recent advances in automated solid-phase synthesis, the production of long RNA oligomers is still difficult and time consuming. Poor coupling efficiency as the chain length increases resulting in poor yields, instability under cleavage and deprotection (C & D) conditions, difficulty in separating the full length product from truncated impurities are some of the main factors that affects the efficiency of longmer RNA manufacture. In this poster, we present the syntheses, C & D and purification studies of a series of RNA hundred-mers. These experiments were carried out as proof of concept experiments, to develop a process for the efficient and economic syntheses of RNA longmers. The information gleaned from these experiments was successfully used in a scale-up manufacturing process at Avecia, Cincinnati.

Keywords: RNA longmer, CRISPR, sgRNA

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

Lauren Peltier (Department of Molecular Genetics, The Ohio State University), Alicia Bao (Department of Molecular Genetics, The Ohio State University), Anita K. Hopper (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University)

Abstract not available online - please check the printed booklet.

131. The conserved debranching enzyme, Dbr1, also decaps RNA in vitro

Daoming Qin (Molecular Genetics and Cell Biology, University of Chicago), Aiswarya Krishnamohan (Molecular Genetics and Cell Biology, University of Chicago), Jonathan Staley (Molecular Genetics and Cell Biology, University of Chicago)

Abstract not available online - please check the printed booklet.

132. The DEAD-box helicase Drs1 remodels nucleolar pre-60S subunits prior to C2 cleavage

Stephanie Biedka (Department of Biological Sciences, Carnegie Mellon University), John Woolford (Department of Biological Sciences, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

133. The DEAD-box protein Dbp2p is linked to non-coding RNAs, the helicase Sen1p, and R-loops

Frank A. Tedeschi (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH), Sara C. Cloutier (Department of Biochemistry, Purdue University, West Lafayette, IN), Elizabeth J. Tran (Department of Biochemistry, Purdue University, West Lafayette, IN), Eckhard Jankowsky (Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH)

Abstract not available online - please check the printed booklet.

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

Elan A. Shatoff (Department of Physics, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University)

Abstract not available online - please check the printed booklet.

135. The effects of Ultraviolet radiation on Ribosomal RNA and its post-transcriptional modifications

Mariana Bonafim Piveta (Chemistry - University of Cincinnati), Patrick A. Limbach (Chemistry - University of Cincinnati), Balasubrahmanyam Addepalli (Chemistry - University of Cincinnati)

Abstract:
The genotoxic effects of Ultraviolet Radiation (UVR) on DNA have been widely described. We are beginning to understand the impact of UVR on tRNA, where modified nucleosides are susceptible to oxidative degradation upon UVA exposure1. The post-transcriptional modifications on ribosomal RNA (rRNA) are important to maintain the structure of ribosome and to ensure efficient translation of mRNAs, so that, cell homeostasis is maintained. These nucleoside modifications are also associated with antibiotic resistance2 and their dysregulation could cause human diseases3. In the current study, we are interested in understanding the impact of UVR on Ribosomal RNA under various conditions and their potential impact to cellular translation. We will present preliminary data documenting the variations in levels of nucleoside modifications through liquid chromatography tandem mass spectrometry (LC-MS/MS) following UVR exposure. Elucidation of how rRNA and its post-transcriptional modifications are affected upon stress (UVR exposure) will help understand the behavior of cellular translation, when subjected to environmental insults.

References:
1. Congliang Sun, Manasses Jora, Beulah Solivio, Patrick A. Limbach and Balasubrahmanyam Addepalli. The Effects of Ultraviolet Radiation on Nucleoside Modifications in RNA. ACS Chemical Biology 13 (3), 567-572 (2018). DOI: 10.1021/acschembio.7b0089
2. Arenz, S. & Wilson, D.N. Blast from the past: reassessing forgotten translation inhibitors, antibiotic selectivity, and resistance mechanisms to aid drug development. Mol. Cell 61, 3–14 (2016).
3. Sergiev, Petr V, Aleksashin Nikolav A, Chuguniva Anastasia A, Polikanov Yurv S, Dontsova Olga A; Structural and evolutionary insights into ribosomal RNA methylation, Nature Chemical Biology 14, 226–235 (2018).

Keywords: Ribosomal RNA, Ultraviolet Radiation, LC-MSMS

136. The GTPase activity of the ribosome assembly factor Nog1 is required for its release from the large ribosomal subunit

Amber J. LaPeruta (Carnegie Mellon University), Dan M. Wilson (Carnegie Mellon University), John L. Woolford (Carnegie Mellon University), Vikram Panse (ETH Zurich)

Abstract:
Efficient and accurate protein synthesis requires proper assembly of ribosomal functional centers. Defective ribosome assembly disrupts protein homeostasis and causes ribosomopathies and cancer. One essential functional center, the peptidyl transferase center (PTC), regulates protein synthesis. Mechanisms involved in PTC assembly have remained elusive due to lack of structural data. However, recent advances in cryo-EM technology enabled us to visualize the Nog2-TAP, Arx1-TAP and Nmd3-TAP pre-60S structures, an unprecedented feat. These structures informed hypotheses regarding PTC construction. The Nog2-TAP structure shows that the essential assembly factor Nog1 probes the rRNA of the PTC with its N-terminal helix bundle domain. Adjacent to the helix bundle, Nog1 has an Obg-like GTPase domain whose function in ribosome assembly remains unknown. To give mechanistic insight into PTC assembly, we are assessing ribosome biogenesis defects that result from the nog1G223A mutation in the GTPase domain of Nog1. In murine cell lines, the analogous mutation is a dominant lethal mutation that causes early, 27SB pre-rRNA processing defects. Using northern blot analysis, affinity purification of assembly intermediates and western blot analysis, we show that the GTPase activity of Nog1 is essential for its release from the large ribosomal subunit during assembly. We hypothesize GTP hydrolysis creates structural rearrangements in Nog1 that remove the helix bundle of Nog1 from the PTC to allow the release of Nog1.

Keywords: Ribosome, GTPase, Large Subunit

137. The Intron Annotation and Orthology Database (IAOD)

Devlin C. Moyer (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University), Graham Larue (Department of Biology, San Francisco State University), Courtney E. Hershberger (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University), Scott W. Roy (Department of Biology, San Francisco State University), Richard A. Padgett (Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner College of Medicine, School of Medicine, Case Western Reserve University)

Abstract:
During maturation of nearly all eukaryotic messenger RNAs and long non-coding RNAs, sequences called introns are removed in the process of RNA splicing. Two complexes of proteins and small nuclear ribonucleoprotein particles—the U2- and U12-dependent spliceosomes—are responsible for excising introns. Regulation of splicing has played an important role in eukaryotic evolution and is crucial to the etiologies of many human diseases. Many databases of intron annotation information have been created, but few remain accessible. Very few databases have ever annotated U12-dependent introns, and those that do have major limitations that restrict their relevance to many pursuits. The Intron Annotation and Orthology Database (IAOD) is a comprehensive database of intron annotation information in 24 evolutionarily diverse organisms. The website and database are both entirely open-source, and it has been specifically designed to be simple to update, so that it can be a useful genomics resource for many years. We believe that IAOD will be a valuable tool for many inquiries into the evolutionary history of introns.

Keywords: introns, alternative splicing, comparative genomics

138. The Musashi 1/2 proteins are required for photoreceptor maintenance and development

Fatimah Matalkah (Department of Biochemistry, West Virginia University), Bohye Jeong (Department of Biochemistry, West Virginia University), Jesse Sundar (Department of Biochemistry, West Virginia University), Visvanathan Ramamurthy (Departments of Biochemistry and Ophtalmology and Center for Neuroscience, West Virginia University), Peter Stoilov (Department of Biochemistry, Cancer Institute Research Program, West Virginia University)

Abstract not available online - please check the printed booklet.

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

Alan C. Kessler (Department of Neuroscience, The Ohio State University ), Hao Le (Department of Neuroscience, The Ohio State University ), Min An (Department of Neuroscience, The Ohio State University ), Sharon L. Amacher (Department of Molecular Genetics, The Ohio State University ), Christine E. Beattie (Department of Neuroscience, The Ohio State University ), Stephen J. Kolb (Department of Neurology, The Ohio State University )

Abstract not available online - please check the printed booklet.

140. The translational landscape of glioblastoma stem cells

Christine H. Lee (Center for RNA Science & Therapeutics, Case Western Reserve University- School of Medicine ), Deepak Sharma (Center for RNA Science & Therapeutics, Case Western Reserve University- School of Medicine ), Bo-Jhih Guan (Department of Genetics & Genome Sciences, Case Western Reserve University- School of Medicine ), Maria Hatzaglou (Department of Genetics & Genome Sciences, Case Western Reserve University- School of Medicine ), Jeremy N. Rich (Division of Regenerative Medicine, UC San Diego ), Eckhard Jankowsky (Center for RNA Science & Therapeutics, Case Western Reserve University, School of Medicine )

Abstract not available online - please check the printed booklet.

141. Theoretical pKa Calculations of Modified Nucleobases using Explicit Waters and a Polarizable Continuum Model

Evan L Jones (Wayne State University, Chemistry), Alan Mlotkowski (Wayne State University, Chemistry), Sebastain Hebert (Wayne State University, Chemistry), H. Bernhard Schlegel (Wayne State University, Chemistry), John SantaLucia, Jr. (Wayne State University, Chemistry), Christine S. Chow (Wayne State University, Chemistry)

Abstract:
Modified nucleobases are often responsible for key structural roles and are found in functionally important regions of RNA. Several nucleobase modifications such as methylations are dynamically regulated in nature with each playing a different regulatory role. The detailed characteristics of modifications are still poorly understood. One important feature of a nucleobase that could impact structure and function is the pKa value. Nucleobases contain one or more protonation/deprotonation sites, all with unique pKa values. Modifications to nucleobases lead to changes in the protonation/deprotonation sites. To fully understand the impacts of nucleobase modifications, the pKa values of modified nucleobases first have to be determined. Of more than 100 known nucleobase modifications, only a small percentage has reported pKa values. In this study, we determined the pKa values of various modified nucleobases from RNA and compared them to their unmodified counterparts by performing Gaussian calculations with a B3LYP 6-31 +G(d,p) basis set.(1) This method involved the use of explicit water molecules surrounded by an implicit solvation field along with a methyl group that is substituted in the place of the ribose sugar. This approach indicates that modifications have a wide range of effects on the pKa values of nucleobase functional groups. Understanding the pKa values of modified nucleobases will give insight into the specific structural impact of these modifications.

References:
Thapa, B; Schlegel, H. B.; Calculations of pKa’s and Redox Potentials of Nucleobases with Explicit Waters and Polarizable Continuum Solvation. J. Phys. Chem. A, 2015, 119 (21), pp 5134-5144

Keywords: RNA Modifications, Computational Chemistry

142. Thermal stability analyses of RNA duplexes with 2'-branches

Frances Franky Moore (Chemistry, Carnegie Mellon University), Stephanie Wang (Chemistry, Carnegie Mellon University), Stephanie Mack (Chemistry, Carnegie Mellon University), Timothy Chad Ratterman (Chemistry, Carnegie Mellon University), Subha R. Das (Chemistry, Carnegie Mellon University)

Abstract:
In RNA splicing, exons are ligated together and introns are removed as lariats by the spliceosomal machinery. The removal of introns is catalyzed by RNA portions of the spliceosome – particularly the U2 and U6 snRNAs that hybridize to the sequences of the branched RNA intron.

Here we describe the use of melting point (T_m ) to investigate the stability of distinctive duplexes which resemble those found within the spliceosome. Although the T_m is a well-established method for determining duplex stability, no data exists on duplex RNAs which include a backbone-branched RNA strand as would be found in splicing intermediates. This investigation of duplex stabilities will increase knowledge of the structure and function of the catalytic core of the spliceosome, and facilitate the development of in vitro splicing assays.

Keywords: Tm , RNA duplex, Spliceosome

143. Thermodynamic Analysis of the Rate of ATP Dissociation in a Truncated Variant of the DEAD-Box Protein Rok1p

Lisa Yoder (Department of Biochemistry, Allegheny College), Zach Iezzi (Department of Biochemistry, Allegheny College), Amanda Diloreto (Department of Biochemistry, Allegheny College), Kate Sutter (Department of Biochemistry, Allegheny College), Ivelitza Garcia (Department of Biochemistry, Allegheny College)

Abstract not available online - please check the printed booklet.

144. Trypanosoma brucei TTAGGG repeat binding factor interacts with TERRA independent to its DNA binding activity.

Amit Kumar Gaurav (GRHD, BGES, Cleveland State University), Vishal Nanavaty (Genomic Medicine Institute, Cleveland Clinic), Ranjodh Sandhu (Department of Microbiology and Molecular Genetics, University of California, Davis), Unnati Pandya (Vincent Center for Reproductive Biology, Massachusetts General Hospital), Bibo Li (GRHD, BGES, Cleveland State University)

Abstract:
Protozoan parasite Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. T. brucei evades the host immune response by regularly switching its major surface antigen, Variant Surface Glycoprotein (VSG). VSGs are exclusively expressed from sub-telomeric expression sites in a monoallelic manner. We have previously shown that telomere proteins play essential roles in the regulation of VSG switching and silencing. We identified telomeric proteins TbTRF, a TTAGGG repeat binding factor and TbRAP1. We have shown that TbRAP1 is essential for VSG silencing and it suppresses VSG switching. We have also shown that TbTRF is essential for protection of telomeric G-overhang structure and suppression of VSG switching. Recently we showed that the T. brucei telomere is transcribed into a long non-coding RNA called TElomeric Repeats-containing RNA (TERRA). It appears that higher levels of TERRA is linked to more telomeric RNA-DNA hybrids, which causes increased sub-telomeric DNA double strand breaks and more frequent VSG switching. Now we show that TbTRF associates with G-rich TERRA in vivo. Additionally, TbTRF depletion leads to higher TERRA levels. With electrophoretic mobility shift assays, we demonstrate that TbTRF binds directly to G-strand TERRA in vitro and its RNA binding activity is independent of its double-stranded telomere DNA binding activity. These results suggest a possible mechanism of TbTRF regulated VSG switching and telomere stability maintenance through TERRA interaction.

Keywords: TERRA, Telomere, Trypanosoma brucei

145. Trypanosoma brucei ZFP3 is a RNA binding protein that regulates the transcriptome and is a substrate for numerous protein arginine methyltransferases

Kyle Smith (University at Buffalo), Lucie Kafkova (University at Buffalo), Laurie K. Read (University at Buffalo)

Abstract:
Trypanosoma brucei is a single celled parasite and the causative agent of African sleeping sickness. The genome of T.brucei is transcribed in a polycistronic and unregulated manner. Due to this lack of transcriptional control, T.brucei relies heavily on RNA binding proteins for gene regulation. ZFP3 is an RNA binding protein that has been shown to stabilize specific transcripts as well as promote the translation of other transcripts.

Keywords: Trypanosome, Zinc Finger, Arginine methylation

146. Uncovering the Role of Cotranscriptional Folding in Riboswitches

Luyi Cheng (Interdisclipinary Biological Sciences Graduate Program, Northwestern University), Julius Lucks (Department of Chemical and Biological Engineering, Northwestern University)

Abstract not available online - please check the printed booklet.

147. Understanding the dsRBD mediated ADR-1 and ADR-2 interaction

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.

148. Understanding the role of telomeric long non-coding RNA, TERRA, in Trypanosoma brucei

Arpita Saha (GRHD, BGES), Bibo Li (GRHD, BGES)

Abstract:
The flagellated protozoan parasite Trypanosoma brucei causes human African trypanosomiasis. The parasite utilizes sophisticated antigenic variation – regular switching of its major surface antigen coat (VSGs) – to survive the immune response of the host. There are > 2,500 VSG genes in the T. brucei genome although only one is expressed at any time from one of the ~ 20 VSG expression sites (ESs) located at the subtelomeric regions. Telomeres are nucleoprotein complexes at chromosome ends. They are essential for genomic integrity and chromosome stability. Telomeric RNA, TERRA, has heterogenous sizes that range from 100 bp to 9 kb. It was first reported in T. brucei and later in yeast and vertebrates. We recently showed that T. brucei TERRA is transcribed as a readthrough transcript from the active ES-adjacent telomere. In addition, excessive amount of TERRA leads to more frequent VSG switching events. As a lncRNA, TERRA may also play an important role in VSG expression regulation. To better understand the functions of TERRA in antigenic variation, we intend to determine the TERRA protein interactome by performing iDRIP (direct RNA-protein interaction in vivo), which is a comprehensive approach aiming to capture the whole RNA-protein interactome. We are currently testing various pull-down conditions. Additionally, our preliminary IF and RNA FISH results showed that TERRA co-localizes with TbTRF, the duplex telomere DNA binding protein in T. brucei, which is consistent with the observation that TbTRF binds TERRA directly. Our study will help better understand the role of TERRA in antigenic variation and in maintaining telomere/subtelomere integrity.

Keywords: TERRA, Trypanosoma brucei, telomere integrity

149. Understanding the substrate specificity and biological functions of cytoplasmic tRNA methyltransferase (Trm10) enzymes

Ben Jepson (The Department of Chemistry and Biochemistry at The Ohio State University), Jane Jackman (The Department of Chemistry and Biochemistry at The Ohio State University)

Abstract not available online - please check the printed booklet.

150. Unique repression domains in Pumilio accelerate destruction of target mRNAs

Rene Arvola (Department of Biological Chemistry, University of Michigan), Joseph Buytendorp (Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota), Aaron Goldstrohm (Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota)

Abstract not available online - please check the printed booklet.

151. Unlocking the mechanism of HIV-1 viral assembly nucleation with native mass spectrometry

Samantha Sarni (Chemistry and Biochemistry, The Ohio State University, 100 West 18th Ave., Columbus OH, 43210-1340 OSU), Erik Olson (Chemistry and Biochemistry, The Ohio State University, 100 West 18th Ave., Columbus OH, 43210-1340 OSU), Shuohui Liu (Chemistry and Biochemistry, The Ohio State University, 100 West 18th Ave., Columbus OH, 43210-1340 OSU), Karin Musier Forsyth (Chemistry and Biochemistry, The Ohio State University, 100 West 18th Ave., Columbus OH, 43210-1340 OSU), Vicki Wysocki (Chemistry and Biochemistry, The Ohio State University, 100 West 18th Ave., Columbus OH, 43210-1340 OSU)

Abstract not available online - please check the printed booklet.

152. Use of chemical modification and tandem mass spectrometry to identify RNA-protein contact sites in archaeal RNase P

Hong Duc Phan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210), Andrew Norris (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Tien-Hao Chen (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210), Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210)

Abstract:
RNase P is a ribonucleoprotein (RNP) that catalyzes cleavage of the 5ʹ-leader in precursor tRNAs. While RNase P is present in all domains of life, its composition varies between Bacteria, Archaea, and Eukarya. In Archaea, RNase P is composed of a catalytic RNA subunit (RNase P RNA; RPR) and up to five proteins (RNase P proteins; RPPs), including RPP21, RPP29, RPP30, POP5, and L7Ae. In vitro reconstitution studies have demonstrated that the RPP21-RPP29 binary complex enhances substrate affinity and the POP5-RPP30 binary complex increases the RPR’s cleavage rate. L7Ae has been found in other RNPs, and its functional role in archaeal RNase P remains to be defined. Although structures of the RPPs are available, the RNA-contacting residues and the dynamic changes that accompany assembly of the RPPs to form the RNase P complex have not been characterized. Little is known about the binding modes of the RPPs, except for L7Ae that binds an RNA structural motif called a kink-turn. In this study, we sought to covalently modify, either in the absence or presence of the RPR, the abundant lysine residues in RPPs and detect the modification sites by bottom-up tandem mass spectrometry (MS). To separate the RNase P RNP sub-assemblies from free RPPs, we engineered a sequence at the 5’-end of the RPR and used a biotinylated DNA oligonucleotide that is complementary to this sequence to pull-down the RNase P assembly using streptavidin-coated magnetic beads. The advantage of this MS-based approach over other higher resolution structural techniques is that its high-throughput allows examination of all the RNase P sub-assemblies en route to the final holoenzyme. Using archaeal RNase P as a model, we demonstrate the utility of chemical modification, affinity purification, and tandem MS to identify RNA-contacting residues on proteins in multi-subunit RNP complexes.

Keywords: RNase P, mass spectrometry, chemical modification

153. Using LC-MS/MS to detect tRNA modification profile differences in Bacillus subtilis

Christina Psihountas (UC Chemistry), Manasses Jora (UC Chemistry), Patrick A. Limbach (UC Chemistry)

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 a comparison of the tRNA profiles of vegetative cells and spores. A comparison of profiles between domesticated and undomesticated strains are also discussed. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we can detect and characterize modifications from total tRNA samples obtained at different phases of the cell life cycle. Abundance of nucleosides from Bacillus subtilis is being analyzed to learn the effects of culturing conditions on the tRNA profile.

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.
Wetzel, Collin. (2015). Dissertation. 50-60.
http://bioinformatics.psb.ugent.be
Salazar, J. C., A. Ambrogelly, P. F. Crain, J. A. McCloskey and D. Söll (2004). Proceedings of the National Academy of Sciences of the United States of America 101(20).
Dumelin, C. E., Y. Chen, A. M. Leconte, Y. G. Chen and D. R. Liu (2012). Nature chemical biology 8(11).

Keywords: tRNA, Nucleosides, Mass Spectrometry

154. Using Limited Proteolysis to Understand Structural Changes of DEAD-box protein Rok1p

Megan Arnold (Allegheny College Biochemistry Department), Katelyn Perroz (Allegheny College Biochemistry Department), Yueting Xu (Allegheny College Biochemistry Department), Ivelitza Garcia (Allegheny College Biochemistry Department)

Abstract not available online - please check the printed booklet.

155. Using RNA interference to identify the acp3U tRNA modification enzyme in higher eukaryotes

Jamison Burchett (Chemistry and Biochemistry, Northern Kentucky University), Justen Mamaril (Chemistry and Biochemistry, Northern Kentucky University), Maggie Thomas (Chemistry and Biochemistry, Northern Kentucky University), Regan Bales (Chemistry and Biochemistry, Northern Kentucky University), Thomas Vornheder (Chemistry and Biochemistry, Northern Kentucky University), Michael Guy (Chemistry and Biochemistry, Northern Kentucky University)

Abstract:
Cellular enzymes form dozens of post-transcriptional tRNA modifications to increase tRNA stability and function. tRNA modification defects are linked to various diseases, including intellectual disability and cancer. The enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) tRNA modification, which is found in animals and plants, but not in yeast, has not been identified. Because of its high conservation among plants and animals, acp3U is likely to be important for tRNA function. To identify the acp3U enzyme, we are silencing candidate genes in cultured Drosophila melanogaster cells by RNA interference (RNAi), analyzing acp3U levels on tRNA from treated cells using primer extension, and then verifying results using quantitative real time PCR (qRT-PCR). Candidate genes are identified through BLAST searches of predicted human methyltransferase genes of unknown function that are also found in D. melanogaster and plants, but not in yeast. To date, we have identified 30 candidate genes, treated cells with double stranded RNA (dsRNA) to induce RNAi for over half of these genes, and then analyzed tRNA from treated cells for the presence of acp3U by primer extension. To verify silencing of gene expression, qRT-PCR is being performed. Identification of the acp3U gene in animals, including humans, will increase our understanding of the link between tRNA modifications and disease.

Keywords: tRNA , modification, translation

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