Rustbelt RNA Meeting 2019

Poster abstracts

1. Developing an optimal AID-degron system to elucidate UPF protein function in human cells

Fuad Abbas (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University, Columbus), Zhongxia Yi (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University, Columbus), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology, The Ohio State University, Columbus)

Abstract:
The Nonsense Mediated mRNA Decay (NMD) pathway is of critical importance as a watchdog in the regulation of gene expression and selective degradation of erroneous mRNA across all eukaryotic species. NMD operates via several pathways in order to selectively degrade mRNA transcripts containing a premature termination codon (PTC), also known as a nonsense mutation. Implicated in the NMD pathway are several proteins, with the core being composed of the up-frameshift proteins UPF1, UPF2, UPF3A and UPF3B, which help mediate the degradation of mRNAs bearing a premature stop codon by recruiting and interacting with downstream exon junction complexes (EJCs). UPF2 has been observed to have a diverse array of functions across different cells, and at different stages of cell growth and differentiation. To help elucidate these functions, UPF2 proteins will be constitutively degraded in HCT116 human cancer cells and the resulting phenotype will be observed. Protein destabilization will be carried out using an auxin-inducible degron (AID) system. Upon addition of auxin, OsTIR1 expressed in the HCT116-OsTIR1 cell line will ubiquitinate the AID-domain on translated UPF2 proteins, thereby targeting them for destruction. This project aims to investigate parallel, alternative NMD pathways associated with the UPF2 gene and their potential mechanisms via knockout of UPF2 using an auxin-inducible degron (AID) system, and analysis of the knockout phenotype.

Keywords: NMD, UPF proteins, auxin-inducible degron

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

Sudeshi Abedeera (Department of Chemistry & Biochemistry, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry & Biochemistry, Kent State University)

Abstract not available online - please check the printed booklet.

3. Quantitative analysis of yeast Kozak variants for AUG and near-AUG start codons using a massively parallel reporter assay

Christina Akirtava (Department of Biological Sciences, Carnegie Mellon University), Hunter Kready (Department of Biological Sciences, Carnegie Mellon University), Lauren Nazzaro (Department of Biological Sciences, Carnegie Mellon University), Gemma E. May (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Translation typically initiates at AUG codons surrounded by favorable sequences first discovered by Marilyn Kozak and, hence, termed the “Kozak context” (Kozak, 1984). Interestingly, translation initiation can also occur at near cognate codons (NCCs) that differ from AUG at one nucleotide. Translation from NCCs regulates gene expression during stress and its misregulation has been implicated in neurodegeneration and cancer (Kearse & Wilusz, 2017). While the Kozak context for AUGs and NCCs has been experimentally tested in human cells (Diaz de Arce et al, 2017; Noderer et al., 2014), limited studies have been done in yeast (Cuperus et al., 2017; Dvir et al., 2013). As such, the extent to which Kozak sequence controls translation in yeast remains unclear. We used Fluorescence-Activated Cell Sorting and highthroughput sequencing (Noderer et al., 2014) to assay expression of YFP reporters harboring all Kozak variants surrounding AUG and NCCs in S. cerevisiae. Compared to human cells, we find less initiation at NCCs in yeast grown in rich media, with no NCC context supporting more than 10% of optimal AUG expression. Among AUGs, the Kozak context influences expression over a ~20-fold range, with a strong preference for -3 A. We further tested a leaky scanning model (LSM) using hundreds of reporters with tandem AUG codons in varied Kozak contexts. Surprisingly, tandem AUGs did not increase reporter expression as compared to a single start. However, incorporating the effects of tandem start codons, increased the predictive relationship between Kozak context and initiation from 5% to 35-75%. Finally, using our Kozak strengths in an LSM we predicted translation of endogenous yeast genes and found that Kozak context alone accounts for 9% of the variation in translation efficiency. These results comprehensively define the Kozak context for AUG and NCCs in yeast. While relative Kozak strengths explained much of the expression of our reporter library, our results suggest that other factors limit the rate of translation initiation in vivo.

Keywords: Translation start codon, Kozak

4. RNA drug discovery: developing computational models to predict ‘hit’ compounds

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

Abstract:
One solution to the increase in antibiotic resistance is targeting noncoding regulatory RNAs (ncRNAs). ncRNAs may be considered ideal drug targets as they would affect pathogens at the genomic level; most antibiotics currently target pathogens at the protein level. One ncRNA of interest is the T-box riboswitch, which regulates gene expression through transcription termination or translation inhibition and is found primarily in Gram-positive bacteria.¹ Previous studies have shown the ability to target structured elements in the T-box riboswitch, for example, using small synthetic compounds^{2, 3} or readily available commercial compound libraries⁴. While both these methods of targeting RNA have clear advantages; specifically target-based designs (synthetic compounds) or chemical space (compound libraries), they do pose challenges to experimental resources. We are developing computational prediction models to improve efficiencies in compound screening. We have begun generating 3D grids to predict compound binding characteristics. Using chemoinformatic approaches, we are correlating computational generated binding characteristics to experimental data to develop a pharmacophore model that may predict future ‘hit’ compounds and expand 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.
2. 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 Des 2016, 87 (2), 182.
3. Orac, C. M.; Zhou, S.; Means, J. A.; Boehm, 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 (19), 6786.
4. Frohlich, K. M.; Weintraub, S. F.; Bell, J. T.; Todd, G. C.; Vare, V. Y. P.; Schneider, R.; Kloos, Z. A.; Tabe, E. S.; Cantara, W. A.; Stark, C. J. et al. Discovery of Small-Molecule Antibiotics against a Unique tRNA-Mediated Regulation of Transcription in Gram-Positive Bacteria. ChemMedChem 2019, DOI:10.1002/cmdc.201800744 10.1002/cmdc.201800744.

Keywords: T-Box Riboswitch, ncRNA, Drug Discovery

5. Cytoplasmic recapping, a new epitranscriptomic modification targeting long non-coding RNA

Rolando Alvarado (Cardiovascular Regeneration, Houston Methodist), Daniel Kiss (Cardiovascular Regeneration, Houston Methodist)

Abstract:
Cytoplasmic recapping is an epitranscriptomic modification that adds a new 7-methylguanosine (m⁷G) cap onto RNA. Noncoding RNA share key characteristics with mRNAs and can possibly be targets for the cytoplasmic recapping machinery. Recapping of truncated ncRNA may significantly alter their stability and/or function(s). This proof of concept study utilized Clariom D Microarrays, qPCR and other molecular methods to assay RNAs harvested from control cells and those expressing a Dominant Negative Cytoplasmic Capping Enzyme (DN-cCE) which inhibits the cytoplasmic recapping machinery. Preliminary microarray data show over 1,000 ncRNAs had detectable changes when cytoplasmic capping was blocked by expressing DN-cCE. A more extensive study is required to build upon this proof of concept and to identify cytoplasmically recapped lncRNAs with greater certainty.

References:
Berger, M.R., Alvarado, R. and Daniel L. Kiss (2019) mRNA 5’ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced. In press: FEBS Letters.
Ulitsky, I., & Bartel, D. P. (2013). lincRNAs: genomics, evolution, and mechanisms. Cell, 154(1), 26-46.
Naemura, M., Kuroki, M., Tsunoda, T., Arikawa, N., Sawata, Y., Shirasawa, S., & Kotake, Y. (2018). The long noncoding RNA OIP5-AS1 is involved in the regulation of cell proliferation. Anticancer research, 38(1), 77-81.
Dagvadorj, A., Tan, S. H., Liao, Z., Xie, J., Nurmi, M., Alanen, K., ... & Nevalainen, M. T. (2010). N-terminal truncation of Stat5a/b circumvents PIAS3-mediated transcriptional inhibition of Stat5 in prostate cancer cells. The international journal of biochemistry & cell biology, 42(12), 2037-2046.

Keywords: Epitranscriptomic , RNA modification, cytoplasmic recapping

6. Exploring the global determinants of bacterial mRNA decay

James R. Aretakis (Wayne State University, Department of Biological Sciences, Detroit, MI 48202), Nadra Al-Husini (Wayne State University, Department of Biological Sciences, Detroit, MI 48202), Mohammed-Husain M. Bharmal (Wayne State University, Department of Biological Sciences, Detroit, MI 48202), Nisansala S. Muthunayake (Wayne State University, Department of Biological Sciences, Detroit, MI 48202), Jared M. Schrader (Wayne State University, Department of Biological Sciences, Detroit, MI 48202)

Abstract:
In eukaryotes global mRNA half-lives are strongly influenced by translation elongation¹ and by the rate of translation initiation². In bacteria it is unclear what mRNA features influence global mRNA half-lives. In addition, phase-separated BR-bodies, analogous to eukaryotic P-bodies, contribute to mRNA degradation by stimulating the rate of mRNA decay^3,4. Here we measure the genome-wide mRNA decay rates in Caulobacter crescentus. Then using this mRNA dataset, we investigate what features control mRNA degradation in bacteria. The strongest correlation (R=0.49) we found with mRNA half-life is codon usage, suggesting translation elongation is a dominant factor. Translation efficiency provides the next strongest correlation (R=0.30), and GC content (R=-0.18) and mRNA length (R=-0.06), which are associated with BR-body enrichment, are inversely correlated. As translation efficiency is a measure of both translation initiation and translation elongation, we are using synthetic mRNAs to separate and vary these two translation phases independently to determine which mRNA translation phase has the largest influence on mRNA degradation. Furthermore, we will follow up by probing the mechanisms by which translating ribosomes can either stabilize or destabilize mRNAs. Ultimately, this information will allow us to compare how mRNA decay is controlled between eukaryotic and bacterial cells.

References:
¹Presnyak, Vladimir, et al. "Codon optimality is a major determinant of mRNA stability." Cell 160.6 (2015): 1111-1124.

²Chan, Leon Y., et al. "Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability." eLIFE 7 (2018): e32536.

³Al-Husini, Nadra, et al. "α-Proteobacterial RNA Degradosomes Assemble Liquid-Liquid Phase-Separated RNP Bodies." Molecular cell 71.6 (2018): 1027-1039.

⁴Al-Husini, Nadra, et al. "BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates." bioRxiv (2019): 690628.

Keywords: mRNA Decay, translation elongation, translation initiation

7. Understanding the Novel Cleavage behavior of RNase Cusativin through Site-Specific Mutagenesis and Liquid Chromatography coupled with Mass Spectrometry

Jowad N. Atway (Department of Chemistry, University of Cincinnati ), Priti Thakur (Department of Chemistry, University of Cincinnati ), Patrick A. Limbach (Department of Chemistry, University of Cincinnati ), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati )

Abstract not available online - please check the printed booklet.

8. RAQ-NASBA: Rapid, Aptamer-detected, Quantitative Nucleic Acid Sequence Based Amplification

Lauren M. Aufdembrink (Department of Genetics, Cell Biology and Development, University of Minnesota), Nathaniel J. Gaut (Department of Genetics, Cell Biology and Development, University of Minnesota), Joseph M. Heili (Department of Genetics, Cell Biology and Development, University of Minnesota), Kate P. Adamala (Department of Genetics, Cell Biology and Development, University of Minnesota), Aaron E. Engelhart (Department of Genetics, Cell Biology and Development, University of Minnesota)

Abstract not available online - please check the printed booklet.

9. 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), Elizabeth V. Wasmuth (Structural Biology Program, Sloan Kettering Institute, New York, NY ), 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.

10. A transient RNA protonation state modulates oncogenic microRNA-21 maturation

Jared T Baisden (Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill), Joshua A. Boyer (Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill), Bo Zhao (Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill), Qi Zhang (Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill)

Abstract not available online - please check the printed booklet.

11. Temporal control of ESRP2 licenses fetal-to-adult switch in alternative splicing required for hepatocyte maturation

Sushant Bangru (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Jackie Chen (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Waqar Arif (Dept. of Biochemistry, University of Illinois, Urbana-Champaign), Frances Alencastro, Andrew Duncan (Department of Pathology, University of Pittsburgh), Russ P Carstens (Dept. of genetics, University of Pennsylvania), Auinash Kalsotra (Dept. of Biochemistry, and Institute for genomic biology, University of Illinois, Urbana-Champaign)

Abstract not available online - please check the printed booklet.

12. Cotranscriptional RNA strand invasion mediates ligand sensing in the E. coli thiB thiamine pyrophosphate riboswitch

Katherine Berman (1Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL 60208), Julius Lucks (2Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208)

Abstract:
RNA folds immediately as it is transcribed by RNA polymerase, thereby creating RNA structures faster than new nucleotides being added to an emerging RNA chain. Consequently, cotranscriptionally folded RNA structures can differ greatly from equilibrium folded structures. Riboswitches are structured RNA elements that respond to small molecules, ions, and other metabolites to regulate transcription, translation, splicing, and mRNA degradation. Riboswitches take advantage of cotranscriptional folding to regulate gene expression by altering the folding pathway depending on the presence or absence of a ligand. The thiamine pyrophosphate (TPP) riboswitch, a highly conserved riboswitch family found in all kingdoms of life, has been shown to regulate translation via changes in cotranscriptional RNA structure. In this work, we have investigated the hypothesis that the Escherichia coli thiB TPP riboswitch functions through an RNA strand invasion mechanism by which TPP binding blocks a key rearrangement event that allows ‘sequestering stem’ to form around the ribosome binding site, preventing translation. To examine this hypothesis, we have used cotranscriptional SHAPE-seq, a technique which combines RNA chemical probing, precise polymerase arrest and high-throughput sequencing, to study intermediate structures in the folding pathway of the E. coli thiB TPP riboswitch. A series of deletion mutants created to favor or disfavor an intermediate structure predicted to facilitate this mechanism demonstrated efficient strand invasion to be essential to thiB riboswitch function. This work along with recent work on the ZTP riboswitch and the signal recognition particle (SRP) RNA have demonstrated the prevalence of strand invasion in RNA cotranscriptional folding pathways.

Keywords: SHAPE-seq, Cotranscriptional RNA folding pathways, RNA structure

13. Comparative analysis of translational and transcriptional S-box riboswitches

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

Abstract not available online - please check the printed booklet.

14. Probing determinants of translation initiation in non-SD mRNAs

Mohammed Husain Bharmal (Biological Sciences, Wayne State university), Jared Schrader (Biological Sciences, Wayne State university)

Abstract:
A key step in gene expression is when ribosomes initiate mRNA at the start codon. In E. coli, translation initiation at the start codon is facilitated by base pairing of the 16S rRNA and a Shine-Dalgarno (SD) site in the mRNA. Surprisingly, recent genome surveys revealed that only half of bacterial genes contain SD sequences, with some bacterial species having as few as 8% of their genes encoded with upstream SD sequences¹. To understand the mechanism(s) of non-SD translation initiation, we utilize Caulobacter crescentus, an α-proteobacterium that is highly adapted to initiation in leadered non-SD mRNAs (lacking SD sequence in 5’ untranslated region (UTR)) and leaderless mRNAs (lacking 5’ UTRs)². We hypothesize that a lack in mRNA secondary structure increases the accessibility of the start AUG codon to the ribosome thereby facilitating initiation. To test this hypothesis, we used computational analysis to predict start codon regional accessibility by calculating ΔGunfold across the Caulobacter genome. The ΔGunfold predictions revealed that the start AUG codons are more accessible than elongating AUG codons within the body of mRNAs. Interestingly, leaderless mRNAs have a lower ΔGunfold than leadered RNAs, and generation of a set of synthetic leaderless RNAs with larger ΔGunfold strongly inhibits leaderless translation. Mutations in the 5′ UTR’s of SD and non-SD mRNAs revealed that ΔGunfold negatively correlates with translation levels although to a lower extent. To further establish the role of mRNA structure and start codon selection we are generating large ribosome binding site (RBS) libraries across diverse mRNAs with altered secondary structures whose translation will be assayed by flow-seq³.

References:
References:
1. Chang, B.; Halgamuge, S.; Tang, S. L., Analysis of SD sequences in completed microbial genomes: Non-SD-led genes are as common as SD-led genes. Gene 2006, 373, 90-99.
2. Schrader, J. M.; Zhou, B.; Li, G. W.; Lasker, K.; Childers, W. S.; Williams, B.; Long, T.; Crosson, S.; McAdams, H. H.; Weissman, J. S.; Shapiro, L., The coding and noncoding architecture of the Caulobacter crescentus genome. PLoS Genet 2014, 10 (7), e1004463.
3. Kosuri, S.; Goodman, D. B.; Cambray, G.; Mutalik, V. K.; Gao, Y.; Arkin, A. P.; Endy, D.; Church, G. M., Composability of regulatory sequences controlling transcription and translation in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (34), 14024-9.

Keywords: non-SD translation initiation, start codon accessibility

15. Hsa-miR-149 Mediated Posttransriptional Control of Steroid Biosynthesis Pathway in Prostate Cancer

Asmita Bhattarai (Cleveland State University), Girish C. Shukla (Cleveland State University)

Abstract not available online - please check the printed booklet.

16. Identification of Functional SMNs and Suppressors of the Nonfunctional SMNE134K

Anton J Blatnik III (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Vicki L. McGovern, Corey Ruhno, Thanh T. Le (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center), Veronica Pessino, Shawn Driscoll, Sam Pfaff (Salk Institute for Biological Studies, Howard Hughes Medical Institute, La Jolla CA), Shibi Likhite, Brian Kaspar (Department of Gene Therapy, The Research Institute at Nationwide Childrens Hospital), Arthur HM Burghes (Department of Biological Chemistry & Pharmacology, The Ohio State University Wexner Medical Center)

Abstract:
Spinal muscular atrophy (SMA) is caused by loss of SMN1 and retention of SMN2, leading to SMN deficiency. SMN functions in assembly of spliceosomal snRNPs and U7 snRNP, however other functions have been proposed. How deficiency of SMN results in SMA is unclear. To determine which functions are important to SMA, we have developed a cell line which can be conditionally deleted for SMN via Cre expression. In these lines, removal of SMN always results in lethality, thus they can be used to which SMN alleles are functional. Bernabo et al reported a cell line in which we determined that one mutation results in exclusion of exon 2B. Surprisingly, this results in a functional protein, as this allele rescues lethality of our cells upon SMN deletion. This cell line was used to characterize the role of SMN in translation, yet does produce functional SMN. We have used our cell lines to assay whether SMN missense mutations have any partial function in the absence of SMN2. We report the missense mutants SMNA2G, SMND44V, SMNA111G, SMNE134K, and SMNT274I are all nonfunctional in cells lacking SMN. To date no missense alleles have partial function in mammals, but instead work in concert with SMN2. Given SMN forms an oligomeric complex, we asked whether two different mutant alleles interact to produce a functional complex via allelic complementation. We have utilized our cell line to analyze this effect in more detail finding complementation between different SMN alleles to occur between several combinations. For example SMNE134K complements SMNA2G, indicating different functional domains between the N-terminus and Tudor domain. Complementation has been analyzed in mice, in which SMNA111G complements SMNT274I and SMNQ282A, rescuing survival, electrophysiology, and snRNP assembly. Furthermore, we have used the SMNE134K mutation in a screen for suppressors. While the SMNE134K mutation never rescues deleted SMN cell lines, ENU mutagenesis yielded five lines which do survive. 4 of these have alterations in SMN associated proteins and one has a mutation in an Sm protein. We reintroduced this Sm mutation into the SMNE134K cell line and confirmed its ability to suppress lethality when SMN is deleted. This implicates Sm assembly as the critical function affected by the SMNE134K mutation.

Keywords: SMN, allelic complementation, suppressor screen

17. Role of Upstream RNA Secondary Structures in an RNA Thermometer from Bradyrhizobium japonicum

Kathryn M. Bormes (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Elizabeth A. Jolley (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802)

Abstract:
Many heat shock genes in bacteria are regulated through a class of
noncoding temperature sensitive RNAs, called RNA thermometers (RNATs).
The most widely studied RNAT is the ROSE (Repression Of heat Shock
Expression) element. ROSE elements are short RNA sequences that
contain two to four hairpin loops and are associated with expression of
small heat shock proteins. This project is focused on determining the effects
of the additional hairpins within ROSE elements on the functionality of the
RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that is well
known for containing multiple different ROSE elements. The 5’ UTR of the
ROSE element that encodes for heat shock protein A (hspA) in B.
japonicum has an intricate secondary structure containing three hairpins
upstream of the RNAT. While the RNAT within the 5’UTR of B. japonicum
has been previously studied, it is not clear how the upstream stem-loops
contribute to the temperature-sensing function of the ROSE elements. This
relationship was studied using UV thermal denaturation in various buffer
conditions. The individual hairpins and the full-length sequence were
studied and compared. A mutant RNA sequence was also created that
disrupted the stability of the stem-loop immediately upstream of the RNAT.
To determine if the role of the upstream hairpins and RNAT in B. japonicum
is evolutionarily important, other ROSE elements sequences were studied
computationally by bioinformatics. In each Mg2+ concentration, the WT
sequence showed several transitions within the melting curve indicating that
the RNA sequence is not folding cooperatively. When studied in the mutant
sequence, increasing Mg2+ concentration caused some transitions to
collapse to the same temperature range. The melt curve experiments
suggested that the upstream hairpins are important for stabilizing the RNAT,
and disruption of the immediately upstream stem-loop leads to decrease
thermal stability. Based upon the initial results of the bioinformatics study, it
appears as if there is increased thermal stability of the upstream hairpins
compared to the RNAT as observed within B. japoncium’s ROSE element.

Keywords: ROSE Elements, Secondary Structure

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

Elaina Boyle (Center for RNA Biology and the Department of Chemistry and Biochemistry, The Ohio State University), David Gvarjaladze (Institute of Biophysics, Ilia State University), Nunu Metreveli (Institute of Biophysics, Ilia State University), Karin Musier-Forsyth (Center for RNA Biology and the Department of Chemistry and Biochemistry, The Ohio State University), Besik Kankia (Department of Chemistry and Biochemistry, The Ohio State University and Institute of Biophysics, Ilia State University)

Abstract not available online - please check the printed booklet.

19. Assessing the utility of a novel fluorescence turn-on assay for RNase P

Rachel M. Braun (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), Walter J. Zahurancik (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Sravya Kovvali (Department of Microbiology 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.

20. Elucidating the Kinetic Mechanism of Human METTL16

Kurtis Breger (Chemistry and Biochemistry; University of Notre Dame), Noah Springer (Chemistry and Biochemistry; University of Notre Dame), Agnieszka Ruszkowska (Chemistry and Biochemistry; University of Notre Dame), Jessica A. Brown (Chemistry and Biochemistry; University of Notre Dame)

Abstract not available online - please check the printed booklet.

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

Audrey Bui (Biology Department, St. Bonaventure University), Arpitha Pamula (Biology Department, St. Bonaventure University), Jordan Powers (Biochemistry Program, Biology Department, St. Bonaventure University), Joseph Hong (Biology Department, St. Bonaventure University), Claire Schaef (Biochemistry Program, Biology Department, St. Bonaventure University), Xiao-Ning Zhang (Biochemistry Program, Biology Department, St. Bonaventure University)

Abstract not available online - please check the printed booklet.

22. Role of Unique C-terminal Domain in a Plant Aminoacyl-tRNA Trans-editing Protein

Jun-Kyu Byun (Department of Chemistry and Biochemistry and The Ohio State University), William A. Cantara (Department of Chemistry and Biochemistry and The Ohio State University), John Vu (Department of Chemistry and Biochemistry and The Ohio State University), Jawad Abid (Department of Chemistry and Biochemistry and The Ohio State University), Jyan-Chyun Jang (Department of Horticulture and Crop Science and The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry and The Ohio State University)

Abstract:
Some aminoacyl-tRNA synthetases (aaRSs) are error-prone, mispairing noncognate amino acids with cognate tRNAs. To prevent mistranslation, many aaRSs have evolved quality control mechanisms. Bacterial prolyl-tRNA synthetases (ProRSs) mischarge noncognate Ala onto tRNA^Pro and possess an insertion (INS) domain that can deacylate this mischarged tRNA and avoid mistranslation. However, some bacteria and all eukaryotes lack an INS domain and instead, encode a free-standing trans-editing domain homolog, ProXp-ala. Sequence alignments revealed that all plant ProXp-ala contain a conserved C-terminal domain (CTD) that is missing from all other eukaryotic ProXp-ala. The CTD is predicted to fold into a long helical segment connected to the catalytic domain via a random coil. To determine the function of the CTD, we prepared a truncated Arabidopsis thaliana (At) ProXp-ala variant (ΔC-ProXp-ala). The in vitro Ala-tRNA^Pro deacylation rate of ΔC-ProXp-ala was decreased 16-fold relative to wild-type (WT) ProXp-ala. Electrophoretic mobility shift assays suggest that this decrease is primarily due to a tRNA binding defect. SEC-MALS showed that WT At ProXp-ala adopts several oligomeric states, while ΔC-ProXp-ala is exclusively monomeric. The CTD may therefore function to enhance tRNA binding and/or induce multimerization in vitro. Microscale thermophoresis and kinetic studies are currently underway to determine the effect of tRNA anticodon mutations on ProXp-ala binding. In vivo studies in At using split-GFP constructs do not support homo-oligomerization, but do not rule out the possibility that this domain promotes interactions with other binding partners. Therefore, yeast two-hybrid experiments are planned to identify potential interacting partners. At ProXp-ala disruption strains showed an early germination phenotype. Additional phenotypic experiments are being carried out under a variety of growth conditions to better understand the function of the WT enzyme and the role of the unique CTD in vivo. This work will reveal how plants have uniquely evolved to avoid mistranslation and may lead to the discovery of new ProXp-ala functions.

Keywords: aminoacyl-tRNA synthetase, trans-editing , plants

23. 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, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Kurt Fredrick (Department of Microbiology, Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
In prokaryotes, the synthesis of RNA and protein occurs simultaneously in the cytoplasm. A number of studies indicate that translation can strongly impact transcription, a phenomenon often attributed to physical coupling between RNA polymerase (RNAP) and the lead ribosome on the nascent mRNA. Whether there generally exists a mechanism to ensure or promote RNAP–ribosome coupling remains unclear. Here, we used an efficient hammerhead ribozyme and developed a reporter system to measure single- versus multiple-round translation in Escherichia coli. Six pairs of cotranscribed and differentially translated genes were analyzed. For five of them, the stoichiometry of the two protein products came no closer to unity (1:1) when the rounds of translation were severely reduced in wild-type cells. Introduction of mutation rpoB(I572N), which slows RNAP elongation, could promote coupling, as indicated by stoichiometric SspA and SspB products in the single-round assay. These data are consistent with models of stochastic coupling in which the probability of coupling depends on the relative rates of transcription and translation and suggest that RNAP often transcribes without a linked ribosome.

Keywords: ribosome, RNA polymerase, NusG

24. A mechanistic study of the pbuE adenine riboswitch from the perspective of cotranscriptional chemical probing

Luyi Cheng (Interdisciplinary Biological Sciences, Northwestern University), Robert T. Batey (Department of Biochemistry, University of Colorado, Boulder), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University)

Abstract not available online - please check the printed booklet.

25. Characterizing the Role of the DEAD-Box RNA helicase DDX5 in Small Cell Lung Cancer

Matthew Russon (Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA), Zheng (Cindy) Xing (Department of Biochemistry, Purdue University, West Lafayette, IN 47907, ), Elizabeth J. Tran (Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA)

Abstract not available online - please check the printed booklet.

26. Genome-wide analysis of R-loops and nascent transcript synthesis in S. cerevisiae

Sara C. Cloutier (Biochemistry Department, Purdue University), Jiale Deng (Biochemistry Department, Purdue University), Elizabeth J. Tran (BIochemistry Department, Purdue University; Purdue University Center for Cancer Research)

Abstract not available online - please check the printed booklet.

27. Role of Exonucleases in RNA Polymerase V Transcript Degradation

Adriana N. Coke (University of Michigan), Masayuki Tsuzuki (University of Tokyo), M. Hafiz Rothi (Department of Molecular, Cellular, and Developmental Biology at the University of Michigan), Andrzej Wierzbicki (Department of Molecular, Cellular, and Developmental Biology at the University of Michigan)

Abstract:
In Arabidopsis thaliana, RNA-directed DNA Methylation (RdDM) is the primary mechanism whereby de novo DNA methylation is added to silence transposable elements. RdDM relies on the transcriptional activity of a plant-specific RNA Polymerase, RNA Polymerase V (Pol V). Pol V transcribes long non-coding RNA which acts as a scaffold for other proteins in the RdDM pathway to bind, leading to de novo DNA methylation at the site of Pol V transcription. Despite the importance of Pol V transcripts for transcriptional gene silencing in plants, it is unknown how Pol V transcripts are degraded and whether this degradation plays a role in the regulation of RdDM. We conducted RT-qPCR experiments to detect changes in Pol V transcript levels across several RNA exonuclease mutants, and the results suggest that some exonucleases may degrade Pol V transcripts in a locus-specific manner. Our research aims to understand whether and how this locus-specific degradation is involved in the regulation of RdDM.

Keywords: RdDM, Exonucleases, long non-coding RNA

28. A quantitative model of temperature actuated DNA origami nanocaliper constructs

Kyle Crocker (Department of Physics, The Ohio State University), Joshua Johnson (Interdisciplinary Biophysics Graduate Program, The Ohio State University), Carlos Castro (Department of Mechanical and Aerospace Engineering, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University)

Abstract not available online - please check the printed booklet.

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

Noah J. Daniels (Cardiovascular and Metabolic Sciences, Cleveland Clinic), James M. Hiznay, Courtney E. Hershber, William DiPasquale, Devlin C. Moyer (Cardiovascular and Metabolic Sciences, Cleveland Clinic), Sukanya Srinivasan, Eckhard Jankowsky (RNA Science and Therapeutics, Case Western Reserve University), Jaroslaw P. Maciejewski (Translational Hematology and Oncology Research, Cleveland Clinic), Richard A. Padgett1 (Cardiovascular and Metabolic Sciences, Cleveland Clinic)

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 patients diagnosed with the bone marrow neoplasm, myelodysplastic syndrome (MDS). 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 DDX41 R525H displayed slightly tighter binding of double stranded RNA and a 4-fold decrease in RNA duplex unwinding compared with DDX41 WT 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 DDX41 WT and DDX41 R525H over-expression or DDX41 knockdown human cells have revealed subtle-yet wide-spread-effects on pre-mRNA splicing. These data support our hypothesis that DDX41 is a component of the spliceosome and plays an important role in pre-mRNA splicing. Alterations of DDX41 may result in the aberrant splicing of key leukemogenic drivers.

Keywords: DDX41, pre-mRNA Splicing, Myelodysplastic Syndrome

30. Insights into RNA recognition of small molecules using the Fragment Molecular Orbital (FMO) method

Indrajit Deb (Department of Biophysics, University of Michigan, Ann Arbor, Michigan - 48109, USA), Aaron T. Frank (Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan - 48109, USA)

Abstract:
Predicting the binding affinity between a ligand and a target is the key to optimizing target-ligand interactions and designing bioactive drug-like molecules. Unfortunately, current state-of-the-art empirical methods, when applied to RNA-ligand complexes, exhibit weak correlations between predicted and experimentally measured binding affinities between the RNAs and ligands. Fragment Molecular Orbital (FMO) method^1,2 is a divide-and-conquer technique that enables ligand binding energies to be computed using a high-level quantum mechanical (QM) description of the interactions between a ligand and a target receptor.³ Here we describe, to the best of our knowledge, the first reported use of FMO calculations to predict binding energies in RNA-ligand complexes⁴. We found that FMO calculations could be used to generate reliable estimates of the relative interaction strengths in RNA-ligand complexes. Also, using these FMO calculations, we were able to decompose the binding energies into its specific components and identify component(s) that are dominant. If our preliminary results hold, FMO calculations should find utility as a high-accuracy yet general approach for studying the molecular recognition of small molecule ligands by RNAs associated with human diseases, and in so doing aid is structure-guided design of RNA chemical probes.

References:
1. Kitaura K, Ikeo E, Asada T, Nakano T, Uebayasi M. Fragment molecular orbital method: an approximate computational method for large molecules. Chem. Phys. Lett. 1999, 313, 701-706.
2. Fedorov DG, Ishida T, Kitaura K. Multilayer formulation of the fragment molecular orbital method (FMO). J. Phys. Chem. A. 2005, 109, 2638-2646.
3. Otsuka T, Okimoto N, Taiji M. Assessment and acceleration of binding energy calculations for protein–ligand complexes by the fragment molecular orbital method. J. Comput. Chem. 2015, 36, 2209-2218.
4. Howe JA, Wang H, Fischmann TO, Balibar CJ, Xiao L, Galgoci AM, Malinverni JC, Mayhood T, Villafania A, Nahvi A, Murgolo N. Selective small-molecule inhibition of an RNA structural element. Nature 2015, 526, 672-677.

Keywords: Fragment Molecular Orbital (FMO) method, Binding energy, RNA-ligand complex

31. Kinetic Analysis of ATP Dissociation from wt-Rok1p and its truncated variants

Amanda DiLoreto (Biochemistry, Allegheny College), Kathryn Sutter (Biochemistry, Allegheny College), Lisa Yoder (Biochemistry, Allegheny College), Zachary Iezzi (Biochemistry, Allegheny College), Ivelitza Garcia (Biochemistry, Allegheny College)

Abstract:
Ribosomal RNA processing is a highly regulated pathway, requiring many trans-acting proteins; for example, 14 DEAD-box proteins play essential roles in rRNA folding. The various functions of DEAD-box proteins are strongly coupled to conformational changes that take place during ATP hydrolysis. Structurally, these proteins are modular, containing two Rec-A like domains that bind ATP and RNA, as well as N-terminal and C-terminal peripheral domains (NTD and CTD). The Saccharomyces cerevisiae DEAD-box protein Rok1p and Rok1p truncated variants were analyzed utilizing stopped-flow fluorescence spectroscopy in the absence of RNA to investigate peripheral domain effects on ATP dissociation. Dissociation rates were determined at various temperatures ranging from 16 °C to 40 °C. TNP-ATP fluorescence quenching as a function of time resulted in a double exponential decay curve, indicating a two-step dissociation mechanism (k_fast and k_slow). The slow dissociation step suggests that a conformational change is required prior to TNP-ATP release. A thermodynamic-comparative analysis of Rok1p truncation variants showed variable peripheral domain effects on nucleotide dissociation. In the presence of the CTD, the slow conformational change is entropically driven while the release of TNP-ATP is enthalpic. Protein constructs containing the NTD exhibited a gradual inversion in thermodynamic contributions. This study illustrates that both the NTD and CTD affect the ATP binding pocket.

Keywords: dead box protein

32. Investigation into the function and mechanism of an orphan 3’-5’ polymerase implicated in noncoding RNA processing.

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

Abstract not available online - please check the printed booklet.

33. In vivo RNA structure prediction by motif constraining: an integrative approach

Catherine Douds (BMMB Department, Pennsylvania State University, State College, PA), Phil Bevilacqua (Department of Chemistry, Pennsylvania State University, State College, PA)

Abstract not available online - please check the printed booklet.

34. Arrowtail RNA for Ligand Display on Ginger derived Exosome-like Nanovesicles to deliver siRNA for Cancer Suppression

Zhefeng Li (Center for RNA Nanobiotchnology and Nanomedicine; College of Pharmacy,The Ohio State University), Lili Du (Center for RNA Nanobiotchnology and Nanomedicine; College of Pharmacy,The Ohio State University), Hongzhi Wang (Center for RNA Nanobiotchnology and Nanomedicine; College of Pharmacy,The Ohio State University), Chad Benntt (Medicinal Chemistry Shared Resource, Comprehensive Cancer Center; The Ohio State University), Huang-ge Zhang (James Brown Cancer Center, Department of Microbiology & Immunology, University of Louisville), Peixuan Guo (Center for RNA Nanobiotchnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; College of Medicine,The Ohio State University)

Abstract:
Exosomes had shown increasing potential as delivery vesicle or therapeutic for gene therapy, but challenging like cost/yield, drug payload and targeting specificity still exist. Plant derived exosome-like nanoparticle were reported as a promising substitution and exhibit biocompatible through oral, intranasal administration. Recently, we reported the control of RNA orientation to decorate extracellular vesicles with cell targeting ligand for specific delivery of siRNA to tumors (Nature Nanotechnology, 13: 82-89, 2018). Here we introduce another application of arrowtail RNA nanoparticle to display ligand on ginger derived exosome-like nanovesicles (GDENs) for siRNA delivery and tumor inhibition through IV administration. Cushion ultracentrifugation coupled with equilibrium density gradient ultracentrifugation were used for purifying GDENs that displayed size, density and morphology similar to human derived exosome. As ligand to bind cancer, folic acid was displayed on the surface of GDENs for targeted delivery of survivin siRNA to KB cancer models. In vitro gene knockdown efficacy by folate-GDENs/siRNA complex was comparable to transfection. The observed inhibition of tumor xenograft after intravenously injection reveals the potential of GDENs as economic delivery system for siRNA.

References:
Pi F, Binzel D, Lee TJ, Li Z, Sun M, Rychahou P, Li H, Haque F, Wang S, Croce CM, Guo B, Evers BM, Guo P. Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression. Nature Nanotechnology. 2018 Jan; 13(1):82-89.
Li Z, Yin H, Wang H, Benntt C, Wang D, Zhang HG, Guo P. Arrowtail RNA for Ligand Display on Exosome-like Nanovesicles to deliver siRNA for Cancer Suppression. Under preparation.

Keywords: Exosome, RNA, FA

35. The RESC protein, MRB800 plays an important role in RNA editing progression by stabilizing protein-RNA and protein-protein interactions in Trypanosoma brucei

Ashutosh P. Dubey (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA ), Natalie M. McAdams (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA ), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA )

Abstract not available online - please check the printed booklet.

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

Glenn Duncan (Department of Biology, Pennsylvania State University), David Mitchell (Department of Chemisty, Pennsylvania State University), Andrew Veenis (Department of Chemistry, Pennsylvania State University ), Philip C. Bevilacqua (Department of Chemistry, Pennsylvania State University )

Abstract not available online - please check the printed booklet.

37. Maximizing quantitative structural information from high-throughput RNA structure probing

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

Abstract:
RNAs enact numerous cellular functions through the formation of intricately folded structures. High-throughput RNA structure probing experiments couple chemical probing of RNA structure with high-throughput sequencing. These experiments can be used to determine signatures of biologically relevant structures in order to construct models of functional RNA folds. While this experimental approach has so far yielded useful data, several major limitations have precluded our ability to obtain precise and quantitative RNA structural information. These limitations include a lack of standards for experimental and data processing steps that result in inconsistent generation and interpretation of the primary chemical probing ‘reactivity’ data that is collected from these experiments. This in turn has prevented rigorous comparison of experimental results within and between laboratories.

Here, we have designed and begun to characterize a standard benchmark panel of RNAs of known structure that can be used as experimental calibration standards to allow comparison of reactivities within and between experiments. By implementing calibration standards, measurements of reactivity in RNA structure probing experiments will become more quantitative, allowing the maximal amount of structural information to be extracted from these experiments. Our preliminary studies have shown the value of standards to correct for experimental variation, and we have additionally used these standard RNAs to compare and evaluate current experimental methods of high-throughput RNA structure probing. These RNAs serve as a strategy to develop and validate an accurate and quantitative definition of chemical probe reactivity that is directly linked to RNA structure.

Keywords: high-throughput structure probing, standardization, SHAPE-Seq

38. Pathogenic CLP1 p.R140H mutation alters mRNA processing in human motor neurons

Jordan S. Farr (Department of Genetics and Genome Sciences, Case Western Reserve University), Geneva R. LaForce (Department of Genetics and Genome Sciences, Case Western Reserve University), Cydni Akkesson (Department of Genetics and Genome Sciences, Case Western Reserve University), Eric J. Wagner (Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston), Ashleigh E. Schaffer (Department of Genetics and Genome Sciences, Case Western Reserve University)

Abstract:
Pontocerebellar Hypoplasia Type 10 (PCH10) is a rare pediatric neurodegenerative disorder caused by bi-allelic p.R140H mutations in the multifunctional RNA kinase CLP1^1,2. The disease has been characterized by cerebellar and brainstem atrophy, progressive microcephaly, intractable tonic-clonic seizures, and progressive motor neuron loss^1,2. However, new patients have revealed previously undocumented phenotypes while displaying little to no cerebellar involvement. A combination of work from our lab and others indicate that motor neuron degeneration is among the most penetrant phenotypes presented by mutations in CLP1, making the motor neuron a cell type of particular interest in the study of PCH10³. To study the effects of the PCH10 mutation in motor neurons, we reprogrammed primary fibroblasts from an affected patient (p.R140H/p.R140H) and unaffected parental control (p.R140H/+) into induced pluripotent stem cells (iPSCs), then directed them to a motor neuron fate using an established differentiation protocol⁴. Because CLP1 is involved in both the processing of intron-containing tRNAs as well as 3’-end processing of mRNAs, we collected bulk RNA from the mature motor neurons and assayed for changes in mRNA and tRNA processing. Unlike reports documenting the PCH10 mutation in other cell types^1-3, we find no tRNA processing defects in the affected motor neurons by Northern Blot. Instead, utilizing total RNA-seq and poly(A) Click-seq⁵, we have found broad, structural transcriptomic dysregulation. Affected motor neurons display a significant bias against the usage of intronic polyadenylation sites as well as a strong bias toward the expression of long genes, producing a gene expression signature that predicts neuronal hyperexcitability.

References:
1. Schaffer, A.E. et al. CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration. Cell 157, 651-63 (2014).
2. Karaca, E. et al. Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function. Cell 157, 636-50 (2014).
3. Hanada, T. et al. CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 495, 474-80 (2013).
4. Martinez, F.J. et al. Protein-RNA Networks Regulated by Normal and ALS-Associated Mutant HNRNPA2B1 in the Nervous System. Neuron 92, 780-795 (2016).
5. Routh, A. et al. Poly(A)-ClickSeq: click-chemistry for next-generation 3-end sequencing without RNA enrichment or fragmentation. Nucleic Acids Res 45, e112 (2017).

Keywords: CLP1, motor neuron, intronic polyadenylation

39. Investigating the role of the DEAD-box helicase Drs1 in assembly of pre-60S subunits in Saccharomyces cerevisiae

Fiona Fitzgerald (Biological Sciences, Carnegie Mellon University), Stephanie Biedka (Biological Sciences, Carnegie Mellon University), Daniel M. Wilson (Biological Sciences, Carnegie Mellon University), Cate McCormick (Biological Sciences, Carnegie Mellon University), John L. Woolford (Biological Sciences, Carnegie Mellon University)

Abstract:
Ribosomes contain a large subunit (LSU) and a small subunit (SSU), made of both ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). rRNA is transcribed in the nucleolus and transits through the nucleus to the cytoplasm, while undergoing folding and binding of r-proteins, enabled by assembly factors (AFs). Properly assembled functional centers within the LSU- the peptidyl transferase center (PTC) and the polypeptide exit tunnel (PET)- are essential for ribosomes to carry out accurate and efficient protein synthesis. Thus, assembly of the PTC and the PET must be closely monitored.

Twelve RNA helicases are necessary for LSU assembly, presumably to chaperone or surveil proper folding of rRNA. RNA helicases utilize binding and hydrolysis of ATP to unwind or remodel RNA or RNP substrates. These helicases contain a core domain, as well as either or both N- and C-terminal extensions (tails). While the catalytic cores of these proteins are more conserved, their tails are less so- presumably providing substrate specificity.

Drs1, a DEAD-box helicase necessary for LSU biogenesis, enters pre-60S ribosomes early, when nascent rRNA is starting to fold, and exits as pre-ribosomes transit from the nucleolus to the nucleoplasm. By learning more about the presence, activity, and specificity of Drs1, we can learn more about early stages of LSU biogenesis, rRNA folding, as well as formation of the PTC and PET.

Using chemical cross-linking, together with truncation mutants, we have found that the C-terminal tail of Drs1 is necessary for its entry into pre-ribosomes, through interactions made with other AFs. We are also able to determine the role that Drs1 plays in pre-ribosome remodeling events through both random and site-directed mutations predicted to alter ATP binding or hydrolysis. In these mutants, Drs1 binds but is not released from pre-ribosomes, preventing pre-mature particles from exiting the nucleolus while also negatively affecting the recycling of various early AFs.

Keywords: helicase, rRNA

40. Pseudouridinylation of mRNA coding sequences alters translation

Monika K. Franco ( University of Michigan, Program in Chemical Biology), Daniel E. Eyler, Kristin S. Koutmou (University of Michigan, Department of Chemistry), Zahra Batool, Malgorzata Dobosz-Bartoszek, Yury S. Polikanov (University of Illinois, Chicago, Department of Biological Sciences), Monica Z. Wu, Bijoyita Roy (New England Biolabs Inc., Ipswich, MA), Michelle L. Dubuke (University of Massachusetts, Proteomics and Mass Spectrometry Facility. University of Massachusetts, Department of Biochemistry and Molecular Pharmacology), Joshua D. Jones (University of Michigan, Department of Chemistry)

Abstract not available online - please check the printed booklet.

41. Maternal THC exposure leads to large-scale downregulation of genes in pre-frontal cortex of developing mouse brain

Gayathri Panangipalli (Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana, 46202), Izaque de Sousa Maciel (Department of Psychological and Brain Sciences, Indiana University, 702 North Walnut Grove Ave, Multidisciplinary Science Building, Bloomington, Indiana, 47405-2204), Quoseena Mir (Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana, 46202), Rajneesh Srivastava (Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana, 46202), Hui-Chen Lu (Department of Psychological and Brain Sciences, Indiana University, 702 North Walnut Grove Ave, Multidisciplinary Science Building, Bloomington, Indiana, 47405-2204), Sarath Chandra Janga (Department of Biohealth Informatics, Department of Medical and Molecular Genetics, Centre for Computational Biology and Bioinformatics, Indiana University School of Medicine)

Abstract:
Tetrahydrocannabinol (THC) is the major psychoactive component of marijuana. With the significant increase of marijuana consumption in young adults, especially expected mothers, the understanding of its lasting impacts on their children’s brains have never become more critical. This study focuses on elucidating the effect of maternal THC exposure on prefrontal cortex of the developing mouse brain by conducting unbiased transcriptome analysis. Six pregnant CD1 mice were included in this study of which three dams were injected subcutaneously daily with 3 mg/kg THC (Tetrahydrocannabinol) from the 5th gestational (GD5) day till 10 days after birth (postnatal day 10-P10). Three pregnant dams were injected with control in the same manner. Total RNAs were isolated from prefrontal cortex at P14 and subjected for RNA-sequencing. 15-25 million 75bp paired-end reads generated from sequencing were mapped and quantified against the mouse reference genome (mm10). It was observed that of the 871 most significantly differentially expressed genes(p <0.01, FDR<0.01), majority of them are down-regulated. Functional analysis revealed an enrichment of the gene sets associated with “cell motility”, “cell-cell signaling” and “nervous system development”, suggestive of the role of neuromaturation, cilia formation, cell migration, myelination, axon morphology, and neurotransmission processes to be dysregulated upon THC exposure in developing prefrontal cortex. Transcription factors belonging to POU domain and SOX superfamily linked to regulation of progenitor and stem cells were also found to be significantly dysregulated indicative of large-scale changes in the expression of their target genes. L1cam, a gene essential for axon growth and brain development [1], was also observed to be expressed differently. Genes including Adcy1 and MapK linked with signal transduction between neurons were found to be significantly perturbed upon THC exposure. Adcy1 expression is restricted to the central nervous system implying its significance in perception and cognition [2]. Such improper transmission of signals among cells can lead to differences in regulation of essential developmental pathways. Hence, our results suggest that maternal THC exposure disrupts the prefrontal cortex development of their progenies and may result in lasting changes in their brain functions and behaviors.

References:
1. Nakamura, Y et al. Role of the cytoplasmic domain of the L1 cell adhesion molecule in brain development. J Comp Neurol. 2010 April 1; 518(7):1113-32.
2. Xia, Z et al. Type I calmodulin-sensitive adenylyl cyclase is neural specific. J Neurochem. 1993 Jan; 60(1):305-11.

Keywords: Tetrahydrocannabinol, neuromaturation, pre-frontal cortex development

42. Characterizing FMN-analogs binding the FMN riboswitch by native ion mobility-mass spectrometry

Varun V. Gadkari (University of Michigan Department of Chemistry), Elizabeth Tidwell (University of Michigan Department of Biophysics), Markos Koutmos (University of Michigan Department of Biophysics), Brandon T. Ruotolo (University of Michigan Department of Chemistry)

Abstract not available online - please check the printed booklet.

43. Lethal Mutation in 16S rRNA cause defects in RNA Chaperone Activity of RsmC

Tyler Galbraith (The University of Texas at Dallas), Keshav GC (Kent State University), Sanjaya Abeysirigunawardena (Kent State University)

Abstract not available online - please check the printed booklet.

44. The role of Myotonic Dystrophy Type 1 in the Liver

Zac Dewald (Department of Biochemistry, University of Illinois at Urbana-Champaign), Andrew Gupta (Department of Biochemistry, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, University of Illinois at Urbana-Champaign)

Abstract:
Myotonic Dystrophy type 1 (DM1) is multi-systemic muscular dystrophy, affecting 1 in 3000 people. DM1 is caused by a (CTG)n repeat expansion in the 3’ UTR of the ubiquitously expressed gene DMPK. The (CUG)n containing RNAs resulting from the transcription of this diseased DMPK gene aggregate in the nucleus, forming foci which sequester various RNA binding proteins (RBPs). The most pivotal of the RBPs sequestered are the muscleblind-like (MBNL) family proteins, a group of splicing factors that play significant roles in the juvenile-to-adult development of many tissues. Their sequestration in DM1 severely inhibits this maturation process in many tissues. Recent studies show that DM1 patients have increased susceptibility to non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome. Furthermore, DM1 patients are abnormally sensitive to a wide range of analgesics and anesthetics, with complications ranging from prolonged anesthesia recovery to heightened pulmonary dysfunction. These findings suggest a predisposition for liver damage and dysfunction in DM1 patients; however, this possibility has not been investigated.
To understand the effects of DM1 in the liver, we generated a hepatocyte-specific DM1 mouse model, in which we can induce the expression of CUG containing RNA specifically in the liver. Through these mice, we have already shown how the expression of the toxic RNA sequester MBNL proteins in hepatocytes and causes a significant shift in the transcriptome of the liver as well as liver lipid accumulation and defects in glucose metabolism. We have begun to characterize the transcriptomic changes driven by DM1 in the liver, and have shown these lead to changes in liver morphology, inflammation, and necrosis. Ultimately, our findings will how ablating MBNL activity causes several unique metabolic, digestive, and drug clearance pathologies that jeopardize the health of DM1 patients and complicate the treatment of DM1.

Keywords: Myotonic Dystrophy Type 1, Alternative Splicing, Liver

45. Moonlighting of RsmC as RNA chaperone and annealer protein

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:
Ribosome is the ribonucleoprotein machine that carries out protein biosynthesis in all kingdoms of life. Excellent coordination of rRNA tanscription, folding, modification, processing and assembly of r-proteins during ribosome biogenesis is essential to form functional ribosome. Involvement of assembly factors is critical to synchronize various processes that co-occur with ribosome assembly. Here we report moonlighting of RsmC (ribosomal RNA small subunit methyltransferase C) as an RNA chaperone and RNA strand annealer during ribosome synthesis in cells. RsmC methylates an exocyclic amine of guanine (G) located at 1207 of helix 34 (h34) of 16S rRNA (E. coli standard nucleotide numbering) from SAM to m2G. A 25-fold increase in rate of annealing of h34 was observed. Furthermore, RsmC denatures the predicted hairpin structure of its substrate strand. Chaperone activity of RmsC may be critical for the formation of 30S head domain.

Keywords: Ribosome biogenesis, RNA methyltransfease, chaperone and annealing protein

46. 3′UTR-mediated localization of mRNAs to the rear of migrating aortic endothelial cells

Prabar K. Ghosh (Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Debjit Khan (Equal contribution, Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Jennifer Taylor, Vasu Kommireddy (Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Dalia Halawani (Department of Neuroscience, Icahn School of Medicine, Mount Sinai), Linda Graham (Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic), Paul L. Fox (Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic)

Abstract:
Endothelial cell (EC) migration is the rate determining step for wound healing in injured blood vessels as well as angiogenesis. Defects in repair contribute to vascular pathogenesis including atherosclerosis. Although the importance of asymmetric protein localization in cell migration is well known, e.g., caveolin-1 (Cav-1) accumulation in the cell rear, essentially nothing is known about polarization of mRNA to cell rear, or mechanisms directing localization. To investigate the global significance of rear-localized transcripts, we isolated forward and rear segments of planar-migrating ECs by laser-capture microdissection, and analyzed localized mRNA abundance by microarray. Remarkably, transcripts with highest rear-to-front ratios encoded proteins that, like Cav-1, are associated with cholesterol metabolism, e.g., HMG-CoA reductase (HMGCR, rate-determining enzyme of cholesterol biosynthesis). Deletion of the 3'UTR from CAV1 mRNA prevents localization of Cav-1 protein to EC-rear, indicating localization of the protein is RNA-dependent. Fusion of either HMGCR or CAV1 3′UTR is sufficient to drive reporter expression to the rear of a migrating EC. We identified the first 112 nt of HMGCR 3′UTR as a minimal element for its rear localization, while the cis-element of CAV1 is present within the first 483 nt of its 3′UTR. In a candidate approach to discover protein interactors of rear-localized 3′UTRs, we observed reporter-3′UTR mRNA association with NSAP1. NSAP1 was localized in the EC-rear and endogenous rear-localized mRNAs were shown to bind NSAP1. Its depletion markedly reduced wound closure of an EC monolayer, confirming a critical role of NSAP1 in cell migration. Colchicine, an inhibitor of microtubule (MT) polymerization, disrupted rear-localization of HMGCR and Cav-1 proteins, implicating the MT system in cell-rear transcript localization. Association of HMGCR and CAV1 mRNAs as well as NSAP1 with MT-directed kinesin-1 motor complexes was observed suggesting a NSAP1-kinesin 1 transport complex for MT-dependent rear-localization of mRNAs. These results elucidate a novel regulatory system in motile cells that establishes mRNA polarization.

Keywords: mRNA polarization, cell migration, 3UTR

47. Functional analysis of BipA in E. coli reveals the plasticity of 50S ribosome assembly

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), Benjamin R. Warner (Department of Microbiology and Center for RNA Biology, The Ohio State University), Ralf Bundschuh (Department of Physics and Center for RNA Biology, The Ohio State University), Leonard J. 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.

48. A Single Molecule Study of the Impact of Small Molecules on Intermolecular G-Quadruplex Formation

Prabesh Gyawali (Department of Physics, Kent State University, Kent, OH, USA ), Keshav GC (Department of Chemistry and Biochemistry, Kent State University, OH, USA ), Yue Ma (Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University, OH, USA ), Kazuo Nagasawa (Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo), Hamza Balci (Department of Physics, Kent State University, Kent, OH, USA )

Abstract:
We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3 on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.

Keywords: G-quadruplex, Small Molecule , FRET

49. Interactions of structured regulatory RNAs

Ian Hall (Department of Chemistry, University of Michigan), Huaqun Zhang (Biophysics Program, University of Michigan), Amos Nissley (Department of Chemistry, University of Michigan), Sarah Keane (Department of Chemistry & Biophysics Program, University of Michigan)

Abstract:
Listeria monocytogenes is a bacterium causing ~1,600 cases of Listeriosis annually in the USA. The mortality rate of this illness is 20-30% with pregnant women and immune compromised at elevated risk. This pathogen’s virulence is controlled by the PrfA transcription factor which regulates the expression of many virulence genes¹. PrfA expression is regulated at the mRNA level by an RNA thermosensor (RNAT) in its 5’ untranslated region. The prfA RNAT is a structured element of the mRNA which sequesters the ribosome binding site (RBS) and start codon at low temperatures suppressing translation of downstream genes. At higher temperatures the RNAT undergoes structural changes, exposing the RBS and promoting translation. Through this mechanism L. monocytogenes uses temperature to trigger virulence². Additionally, L. monocytogenes harbors a SAM-I riboswitch, sreA, which regulates the expression of a methionine transport system at the transcriptional level. Both sreA and prfA function in cis to control the expression of their respective downstream genes. Interestingly these RNAs also interact in trans to regulate virulence gene expression³. This interaction links nutrient availability and temperature sensing to the virulence of L. monocytogenes⁴. Additional structure-function work is necessary to understand L. monocytogenes’ virulence regulation. I have initiated a thermodynamic characterization of the interaction by isothermal titration calorimetry. This work will provide quantitative information about the interaction and will guide structural studies by X-ray crystallography, NMR spectroscopy, and small angle X-ray scattering.

References:

1 De Las Heras, A., Cain, R. J., Bielecka, M. K. & Vázquez-Boland, J. A. Regulation of Listeria virulence: PrfA master and commander. Current Opinion in Microbiology 14, 118-127, doi:10.1016/j.mib.2011.01.005 (2011).
2 Loh, E. et al. Temperature triggers immune evasion by Neisseria meningitidis. Nature 502, 237-240, doi:10.1038/nature12616 (2013).
3 Loh, E. et al. A trans-Acting Riboswitch Controls Expression of the Virulence Regulator PrfA in Listeria monocytogenes. Cell 139, 770-779, doi:https://doi.org/10.1016/j.cell.2009.08.046 (2009).
4 Loh, E., Righetti, F., Eichner, H., Twittenhoff, C. & Narberhaus, F. RNA Thermometers in Bacterial Pathogens. 6, doi:doi:10.1128/microbiolspec.RWR-0012-2017 (2018).

Keywords: Thermosensor, Riboswitch, RNA-RNA Interaction

50. Improving cell permeability of a ribosome targeting peptide as a potential antibiotic

Sherrell K. Haney (Chemistry, Wayne State University), Mackenzie J. Olbrys (Chemistry, Wayne State University), Prabuddha Waduge (Chemistry, Wayne State University), Dr. Christine S. Chow (Chemistry, Wayne State University)

Abstract:
The development of bacterial resistance against currently available antibiotics is a major problem. Therefore, it is important to develop novel strategies to overcome the bacterial resistance. Towards this end, a previous in vitro selection study in our lab identified several peptides targeting a functionally important region of the bacterial ribosome, helix 31 (h31). Even though several in vitro studies have been conducted previously, it is important to examine the in vivo activity of the selected peptides. The poor cell permeability of these peptides is a major challenge. In this study, we are specifically focusing on the peptide TLWDLIP, and fusing a cell-penetrating peptide (CPP), which is expected to improve its ability to cross the cell membrane, particularly for bacterial species. The objective of our study is to chemically synthesize TLWDLIP-CPP fused peptides and investigate their in vivo inhibitory activity against E. coli. The overall goal of this study is to assess the effect of fusing a CPP to h31-targeting peptide, which is important in the development of peptide-based potential antibiotics. First, TLWDLIP and TLWDLIP+CPP were chemically synthesized using solid-phase peptide synthesis. Then, the synthesized peptides were purified by employing high-performance liquid chromatography (HPLC) and characterized using matrix-assisted laser desorption/ionization (MALDI). Next, the minimum inhibitory concentration (MIC) studies were carried out against E. coli using the broth dilution method. The short-term goal is to complete the MIC studies and to assess the effect of the CPP on TLWDLIP’s activity. The long-term goal is to find new drug target sites that may effectively combat antibiotic resistance.

Keywords: peptide, drug delivery

51. Toehold Mediated Shape Transition of Nucleic Acid Nanoparticles

Jordan Hartung, Daniel Miller (Department of Chemistry, Ball State University, Muncie, IN 47306 ), Nathan McCann, Kheiria Benkato (Department of Chemistry, Ball State University, Muncie, IN 47306 ), Emil F. Khisamutdinov (Department of Chemistry, Ball State University, Muncie, IN 47306 )

Abstract:
Development of novel materials possessing structural transformations in response to an external stimuli such as environmental changes (pH, temperature etc.), presence of a particular proteins or short oligonucleotides are of great interest for a variety of applications ranging from medicine to electronics. The dynamic operations of most Nucleic Acid (NA) devices rely on networks of NA strand displacement processes in which an external or stimulus strand displaces a target strand from a DNA or RNA duplex. The rate of strand displacement can be greatly increased by the use of “toeholds,” single-stranded regions of the target complex to which the invading strand can bind to initiate the reaction forming additional base pairs that provide a thermodynamic driving force for transformation. Herein we demonstrate development of highly robust nanoparticle shape transition approach that enables sequential transformatino of DNA polygons from one shape to another. Using gel-shift assay, we demonstrated that transformation is highly effective and occurs at isothermal conditions (37 °C) that can be potentially implemented within living cells as a reporter molecules. The shape transformation was also by atomic force microscopy. This work intended to provide robust approach for the development of stimuli responsive regulatory nano-devices for nucleic acid nanotechnology.

Keywords: Nucleic Acid Nanoparticles, Shape Transition

52. Activation of caged functional RNAs by oxidative desulfurization of 2-thiouridine

Joseph Heili (BMBB & MCDBG, University of Minnesota), Wakana Sato (BMBB & MCDBG, University of Minnesota), Nathan Gaut (BMBB & MCDBG, University of Minnesota), Tanner Hoog (BMBB & MCDBG, University of Minnesota), Kate Adamala (BMBB & MCDBG, University of Minnesota), Aaron Engelhart (BMBB & MCDBG, University of Minnesota)

Abstract:
The discovery, about 35 years ago, that RNA can act as both a heritable information source and as a functional molecule has led many to consider the possibility there was an RNA world during the origins of life. Researchers in this field have demonstrated high-fidelity, rapid templated nonenzymatic polymerization of RNA rich in GC sequences using activated nucleotide derivatives. In contrast, AU-rich sequences exhibit diminished fidelity and speed. This is due in part to G-U wobble pairing (which is ca. isoenergetic with A-U base pairing) as well as the lower stability of the A-U base pair relative to the G-C base pair.
Recently, it was shown that the diminished wobble pairing, and stronger base pairing with A, exhibited by 2-thiouridine (2sU) allows it to substitute for uridine in nonenzymatic RNA polymerization, enabling faster, higher-fidelity base pairing. While this solves the problem of wobble pairing in nonezymatic copying, it brings about a second. In functional RNA, G-U wobble pairs are common secondary structure elements and metal ion binding motifs. Furthermore, the stronger base pairing of 2sU-containing duplexes resulting from such copying reactions inhibit strand separation, which is required to produce a folded, single-stranded functional RNA.
We sought to take advantage of a recent finding showing that 2sU can be oxidatively desulfurized to U, reasoning that we could exploit this phenomenon to create switchable functional RNAs by employing RNAs with critical G-U wobble pairs (or O2 interactions). In preliminary work with a fluorescent aptamer and ribozyme nuclease containing critical interactions with the O2 of U, we have observed that RNAs transcribed with 2sU exhibit diminished function, which can be rescued by the oxidative desulfurization reaction. We also have investigated the kinetics of the oxidative desulfurization reaction, finding the optimal reaction conditions to protect RNA while activating the caged function. Capitalizing on this phenomenon, we are developing switchable functional RNAs for both prebiotic chemistry and synthetic biology applications using engineered functional RNAs.

Keywords: Functional RNA Aptamer, Ribozyme, 2-thiouridine

53. Complex landscape of splicing signatures in myeloid neoplasms

Courtney Hershberger (Cardiovascular and Metabolic Sciences, Cleveland Clinic), Devlin Moyer (Cardiovascular and Metabolic Sciences, Cleveland Clinic), Wencke Walter, Stephan Hutter, Claudio Haferlach, Torsten Haferlach (MLL Munich Leukemia Laboratory), Vera Adema, Jaroslaw Maciejewski (Translational Hematology and Oncology Research, Cleveland Clinic), Richard Padgett (Cardiovascular and Metabolic Sciences, Cleveland Clinic)

Abstract not available online - please check the printed booklet.

54. Nucleolin Potentially Regulates G4-Duplex Equilibration During Ribosome Maturation Through Chaotropic Residues

Tanner G. Hoog (Department of Genetics, Cell Biology, and Development, University of Minnesota), Matthew R. Pawlak (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota), Katarzyna P. Adamala (Department of Genetics, Cell Biology, and Development, University of Minnesota), Aaron E. Engelhart (Department of Genetics, Cell Biology, and Development, University of Minnesota)

Abstract:
Ribosomes are assembled in the nucleolus of cells where nucleolin, a nucleolus localizing protein, aids in ribosome maturation. Recently, it has been empirically shown that G-quadruplexes, a secondary structure of nucleic acid formed by the non-canonical base pairing of four guanines, fold on the ES27 segment of rRNA¹. Additionally, nucleolin is known to bind and stabilize G-quadruplex conformation². Given these overlapping findings, we seek to confirm the presence of G-quadruplex structures as the determinant of nucleolin mediated ribosomal biogenesis. Nucleolin’s binding domain is rich in arginine which has guanidinium groups, a chaotrope. In related work, we have observed that perchlorate, a Hofmeister chaotrope, selectively denatures duplexes relative to G-Quadruplexes. We are investigating whether nucleolin can modulate the G4-duplex equilibrium of rDNA (as has been shown to be the case in the anticancer drug quarfloxin³) or rRNA.

References:
1. Mestre-Fos, S. et al. G-quadruplexes in Human Ribosomal RNA. J. Mol. Biol. 1–16 (2019).
2. Lago, S., Tosoni, E., Nadai, M., Palumbo, M. & Richter, S. N. The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids. Biochim. Biophys. acta. Gen. Subj. 1861, 1371–1381 (2017).
3. Drygin, D. et al. Anticancer Activity of CX-3543: A Direct Inhibitor of rRNA Biogenesis. Cancer Res. 69, 7653–7661 (2009).

Keywords: Nucleolin, G-quadruplex, Ribosome Maturation

55. piRNA mediated dual gene knockdown via the ping-pong mechanism

Mohammed Enamul Hoque (Chemistry and Biochemistry, Kent State University), Nicole West (Chemistry and Biochemistry, Kent State University), Aiswarya Mukundan Nair (Biological Sciences, Kent State University), Sumirtha Balaratnam (Chemistry and Biochemistry, Kent State University), Helen Piontkivska (Biological Sciences, Kent State University), Soumitra Basu (Chemistry and Biochemistry, Kent State University)

Abstract:
PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that play an essential role in the defense system against the deleterious action of mobile genetic elements (transposons) by cooperating with PIWI proteins. A subset of piRNAs are generated and amplified through the secondary biogenesis of piRNA, or the ping-pong cycle, where one piRNA is utilized for the successive knockdown of at least two target genes. To identify the target genes, we used bioinformatics analysis to search sequences of human protein-coding genes for potential hits that contain 10-nucleotide-long matches at the 5' end. The vast majority of piRNAs contained multiple
hits to the first and second genes in the sequence, up to 142 matches to the first target and up to 283 matches to the second target. Thus, we designed an endogenous-reporter gene system (piR-A → MDM2 → piR-B → luciferase). In this system, piR-A binds at 3’-UTR of MDM2 mRNA and cleaves it by the slicer activity of the PIWI protein which produces the second piRNA (piR-B). piR-B then binds to the complementary target region inserted in the 3’ UTR of the Renilla luciferase gene and represses the expression of the Renilla gene. Quantitative PCR and western blot data indicate the knock-down of both MDM2 and Renilla genes in triple-negative breast
cancer cells with the treatment of piR-A, indirectly suggesting the generation of piR-B. Currently, we are examining longer matches to identify more attainable oncogene targetsto investigate the ping-pong cycle using endogenous genes.

Keywords: piRNA, Ping-Pong Cycle, Bioinformatics

56. Modulation of T-box riboswitch function by analogs of 4,5-disubstituted oxazolidinones

Md Ismail Hossain (Chemistry & Biochemistry, Ohio University), Ali Aldhumani (Chemistry & Biochemistry, Ohio University), Rumita Laha (Chemistry & Biochemistry, Ohio University), Jennifer V. Hines (Chemistry & Biochemistry, Ohio University)

Abstract:
The number of antibiotics that were once used efficiently to treat bacterial infections are becoming resistant at an alarming rate. Multi-drug resistant bacteria are now common and may cause the death of individuals with simple infections. Fighting against the super-bugs globally has become a challenge. Fortunately, new drug targets like the T-box riboswitch have been identified in bacteria, which are not found in the human genome. These non-coding regulatory RNAs are found in the 5´ untranslated regions of the bacterial mRNA which codes for proteins crucial for their survival. The T-box riboswitch controls gene expression through interaction with its natural ligand tRNA, and the antiterminator RNA element of the T-box riboswitch is highly conserved.¹ We are investigating new classes of compounds targeting this regulatory RNA element. Previous studies of 4,5-disubstituted oxazolidinones showed promising binding specificity with different antiterminator model RNAs.² Analogs of these compounds were tested to determine their ability to a) modulate the stability of the antiterminator RNA, b) disrupt the antiterminator binding to the tRNA, and c) inhibit the T-box riboswitch function using different fluorescence monitored assays developed in our lab.^3,4 Four of these compounds disrupted the tRNA-antiterminator complex formation, three of which also inhibited riboswitch function, indicating possible antiterminator specific inhibition. The results indicate the potential to discover novel molecules that would modulate the riboswitch function with significant binding affinity and specificity with the antiterminator RNA.

References:
1. Kreuzer, Kiel D., and Tina M. Henkin. "The T box riboswitch: tRNA as an effector to modulate gene regulation." Microbiology spectrum 6, no. 4 (2018).
2. Anupam, Rajaneesh, Abhijit Nayek, Nicholas J. Green, Frank J. Grundy, Tina M. Henkin, John A. Means, Stephen C. Bergmeier, and Jennifer V. Hines. "4, 5-Disubstituted oxazolidinones: high affinity molecular effectors of RNA function." Bioorganic & medicinal chemistry letters 18, no. 12 (2008): 3541-3544.
3. Zhou, Shu, George Acquaah‐Harrison, Karen D. Jack, Stephen C. Bergmeier, and Jennifer V. Hines. "Ligand‐Induced Changes in T Box Antiterminator RNA Stability." Chemical biology & drug design 79, no. 2 (2012): 202-208.
4. Zeng, C., S. Zhou, S. C. Bergmeier, and J. V. Hines. "Factors that influence T box riboswitch efficacy and tRNA affinity." Bioorganic & medicinal chemistry 23, no. 17 (2015): 5702-5708.

Keywords: Riboswitch, Drug discovery, 4,5-disubstituted oxazolidinones

57. Effects of cellular concentrations of nucleotide diphosphate-chelated Mg2+ on RNA and DNA enzymes

Ruochuan Huang (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Ryota Yamagami (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802)

Abstract:
Our recent studies demonstrate that cellular concentrations of weakly amino acid-chelated magnesium (aaCM) stimulate RNA folding and catalysis while protecting RNAs from magnesium ion-induced degradation.¹ However, the effects of other metabolites that have an affinity for Mg²⁺ are not yet clarified. This study focuses on the effect of nucleotide-chelated Mg²⁺ since nucleotides also comprise a great part of metabolome in vivo (37 mM total).² Nucleotides are some of the major metabolites in cells and have one to three phosphates, which have increasingly tight binding of magnesium. On the basis of binding calculations, ∼85% ATP, ∼40% ADP, and only 5% AMP are estimated to possess a magnesium ion under cellular conditions of 0.5 mM Mg_free²⁺. We tested the self-cleaving activity in hammerhead ribozyme (HH16) in the presence of these chelated magnesium species. Our results indicate that NTP-chelated magnesium and NMP-chelated magnesium do not appreciably stimulate RNA catalysis, whereas NDP-chelated magnesium (NDPCM) promotes RNA catalysis up to 6.5-fold. Inspired by NDP, we observed similar stimulatory effects for several other Mg²⁺ diphosphate-containing metabolites, including UDP-GlcNAC and UDP-Glc. Additionally, we found similar stimulatory effects for a DNAzyme. Thus, rate stimulatory effects appear to be general with respect to the diphosphate and nucleic acid enzyme. These results implicate magnesium-chelated diphosphate metabolites as general facilitators of RNA function inside cells.

References:
1. Yamagami R, Bingaman JL, Frankel EA, Bevilacqua PC. 2018. Cellular conditions of weakly chelated magnesium ions strongly promote RNA stability and catalysis. Nat Commun 9: 2149
2. Bennett, B. D., Kimball, E. H., Gao, M., Osterhout, R., Van Dien, S. J., and Rabinowitz, J. D. 2009 Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5: 593−599.

Keywords: Ribozyme, Chelated Mg2+, In vivo-like condition

58. Pseudouridylation enzyme RsuA binding and its activity is modulated by ribosomal proteins and local rRNA structures

Kumudie Jayalath (Department of Chemistry and Biochemistry, Kent State University), Sean Frisbie (Department of Chemistry and Biochemistry, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)

Abstract not available online - please check the printed booklet.

59. Reverse (3'-5') RNA polymerases: Applications beyond tRNAHis maturation and non-coding RNA processing

Malithi I. Jayasinghe (Chemistry and Biochemistry, Ohio State University), Jane E. Jackman (Chemistry and Biochemistry, Ohio State University)

Abstract:
The recent discovery of the reverse (3'-5') RNA polymerase family, comprised of tRNA^His guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), opened a whole new world of possibilities, with potential applications in developing novel nucleic acid 5'-end labeling tools that are not supported by traditional forward (5'-3') polymerases.^{1, 2} Previous studies on many TLPs from a variety of organisms have shown their flexibility in accommodating various RNA substrates, consistent with their varied physiological roles.^{3, 4} However, early studies on templated nucleotide addition catalyzed by this enzyme family focused mainly on nucleotide addition to highly structured RNAs such as tRNAs and non-coding RNAs. Therefore, a clear understanding of the nucleotide addition by the Thg1/TLP enzymes on other RNAs, especially on model unstructured RNA substrates was lacking. Previous work in our lab demonstrated the feasibility of using various members of the TLP family to incorporate specific 5'-nucleotides into a short (26 nucleotide) model RNA substrate in the presence of an RNA oligonucleotide that serves as a template for the nucleotide addition. The RNA oligonucleotide template in this initial system is complementary to the 5'-end of the model RNA substrate and creates a 3'-overhang with a nucleotide combination of interest that serves as the template for incorporation of specific nucleotides. As evidenced from early experiments using this system, some TLPs are capable of incorporating a given combination of nucleotides across from the tested templates. Thus, we hypothesize that Thg1/TLP enzymes are amenable to be engineered as a versatile nucleic acid (DNA or RNA) 5'-end labeling tool. However, general principles governing how well specific substrate/template sequences can be accommodated by different TLPs have not been defined. The goal of this project is to define these principles by determining optimal nucleotide preference that is required for efficient nucleotide incorporation by TLPs. Methods to overcome challenges associated with separation of resulting long, highly stable RNA duplexes for accurate analysis purposes will also be developed as a goal of this work.

References:
[1] Gu, W., Jackman, J. E., Lohan, A. J., Gray, M. W., and Phizicky, E. M. (2003) tRNA [His] maturation: An essential yeast protein catalyzes addition of a guanine nucleotide to the 5' end of tRNA [His], Genes & Development 17, 2889-2901
[2] Jackman, J. E., and Phizicky, E. M. (2006) tRNA (His) guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition, Proceedings of the National Academy of Sciences of the United States of America 103, 8640-8645
[3] Rao, B. S., Maris, E. L., and Jackman, J. E. (2011) tRNA 5'-end repair activities of tRNA (His) guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea, Nucleic Acids Research 39, 1833-1842
[4] Long, Y. C., Abad, M. G., Olson, E. D., Carrillo, E. Y., and Jackman, J. E. (2016) Identification of distinct biological functions for four 3'-5' RNA polymerases, Nucleic Acids Research 44, 8395-8406

Keywords: 3-5 polymerase, nucleic acid 5-end labeling, tRNAHis guanylyltransferase

60. Understanding the biological significance of multiple zebrafish Trm10 homologs

Benjamin Jepson (MCDB at OSU), Jane Jackman (OSBP at OSU)

Abstract not available online - please check the printed booklet.

61. Theoretical pKa determinations of modified nucleobases using an implicit-explicit solvation model

Evan Jones, Evan Jones (Department of Chemistry, Wayne State University), Sebastien Hebert, H. Bernhard Schlegel (Department of Chemistry, Wayne State University), John SantaLucia (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
Nucleobase modifications play an important role in modulating the structural characteristics of RNA. Common examples of modifications include methylation of nucleobases, which changes the electronic structure and introduces non-polar character. Due to our interest in higher-order RNA structure and function, the specific characteristics of modifications need to be determined. One characteristic, the pKa value, plays an important role in determining the strength of hydrogen-bonding interactions by a modified nucleotide. Nucleobases contain one or more sites that can undergo protonation/deprotonation, and modifications to the nucleobase may result in changes to the pKa values. For RNA base pairs to form, they must displace waters in the first solvation sphere. A qualitative approximation may be achieved by combining implicit and explicit solvation to mimic the properties of the first solvation sphere. In this study, we used ab initio quantum mechanical calculations using a B3LYP/6-31+g(d,p) level of theory and SMD implicit solvation model with explicit water molecules to simulate the first solvation sphere (1). Calculations were performed using the Gaussian 09 program to predict pKa values for modified nucleobases and compared the results to experimentally determined pKa values. This approach gives improved theoretical pKa values for unmodified nucleosides and demonstrates that modifications have a wide range of effects on the pKa values of functionalized nucleobases. Understanding the pKa values of modified nucleobases will give insight into the specific structural and energetic impacts 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: pKa, modified nucleobases

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

Joshua D. Jones (University of Michigan Department of Chemistry), Robert T. Kennedy (University of Michigan Department of Chemistry), Kristin S. Koutmou (University of Michigan Department of Chemistry)

Abstract not available online - please check the printed booklet.

63. Pushing the boundaries of ribonucleoside detection and discovery by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

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), Ruoxia Zhao (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati), Patrick A Limbach (Department of Chemistry, University of Cincinnati)

Abstract not available online - please check the printed booklet.

64. Post-transcriptional regulation of caspofungin resistance in Cryptococcus neoformans.

Murat C. Kalem (Department of Microbiology and Immunology - SUNY University at Buffalo), Harini Subbiah (Department of Microbiology and Immunology - SUNY University at Buffalo), Jay Leipheimer (Department of Microbiology and Immunology - SUNY University at Buffalo), John C. Panepinto (Department of Microbiology and Immunology - SUNY University at Buffalo)

Abstract not available online - please check the printed booklet.

65. Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors

Michael G. Kearse (Department of Biological Chemistry & Pharmacology, Center for RNA Biology, The Ohio State University), Daniel H. Goldman (Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine), Aaron C. Goldstrohm (Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota), Peter K. Todd (Department of Neurology, University of Michigan), Rachel Green (Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine), Jeremy E. Wilusz (Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine)

Abstract:
Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

Keywords: translation, ribosome, inhibitor

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

Krishnendu Khan (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Camelia B. Gogonea (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Belinda Willard (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Valentin Gogonea (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic), Paul L. Fox (Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic)

Abstract not available online - please check the printed booklet.

67. A novel single molecule microscopy technique for extremely specific sensing of microRNAs with greater sensitivity

Kunal Khanna (University of Michigan - Chemistry), Alex Johnson-Buck (University of Michigan - Chemistry), Muneesh Tewari (University of Michigan - Department of Internal Medicine, Division of Hematology/Oncology), Nils Walter (University of Michigan - Chemistry)

Abstract not available online - please check the printed booklet.

68. Modulation of insulin receptor alternative splicing to develop cancer therapeutics

SAFIYA KHURSHID (Nationwide Childrens Hospital, Ohio State University, Columbus OH), Matias Montes (Nationwide Childrens Hospital, Ohio State University, Columbus OH), Brianne Sanford (Nationwide Childrens Hospital, Ohio State University, Columbus OH), Cenny Taslim (Nationwide Childrens Hospital, Ohio State University, Columbus OH), Frank Rigo (Ionis Pharmaceuticals, Carlsbad CA), Dawn Chandler (Nationwide Childrens Hospital, Ohio State University, Columbus OH)

Abstract not available online - please check the printed booklet.

69. CASowary: a tool for systematic design and optimization of cas13 guide RNAs to facilitate disrupting mRNA function

Alexander M. Krohannon (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana 46202), Simone Rauch (Department of Chemistry, The University of Chicago, Chicago, Illinois, USA), Bryan C. Dickinson (Department of Chemistry, The University of Chicago, Chicago, Illinois, USA), Sarath C. Janga (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University, 719 Indiana Ave Ste 319, Walker Plaza Building, Indianapolis, Indiana 46202)

Abstract:
Recent discovery of the gene editing system - CRISPR associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems^1,2,3. Cas13, a lesser studied cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The cas13 system utilizes base complementarity between a crRNA/sgRNA and a target RNA transcript, to preferentially bind to the target transcript. Unlike targeting the regulatory regions of genes on the genome, the transcriptome is significantly more redundant. Additionally, transcripts exhibit complex 3-dimensional structures and interact with an array of RBPs, both of which further limit the scope of effective sgRNAs. As a result, there currently exists no method to predict the efficacy of a specific sgRNA. Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA; using RNA knockdown data from cas13 characterization experiments⁴ for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information on RNA⁵. CASowary utilizes a Decision Tree architecture with a set of 112 features, to classify sgRNAs into 1 of 4 classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 90%; additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting cas system – CIRTS⁶, confirming the robustness and reproducibility of CASowary’s sgRNA predictions. In particular, several highly efficient sgRNA’s designed using CASowary against SMARCA4 gene exhibited strong agreement with experimental data. Applications of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of treatments for diseases linked with aberrations in RNA regulatory processes.

References:
¹ Hart, T. et al. Evaluation and design of genome-wide CRISPR/SpCas9 knockout screens. G3 (Bethesda) 7, 2719–2727 (2017)

² Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).

³ Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

⁴ Abudayyeh O. O., et al. (2017). RNA targeting with CRISPR-Cas13. Nature. 550 280–284. 10.1038/nature24049.

⁵ Schueler M, et al. Differential protein occupancy profiling of the mRNA transcriptome. Genome Biol. 2014;15:R15. doi: 10.1186/gb-2014-15-1-r15.

⁶ Rauch, S. et al. Programmable RNA-guided RNA effector proteins built from human parts. Cell 178, 122–134 (2019).

Keywords: CRISPRCas13, mRNA regulation, machine learning

70. RNase H1 is required for Arabidopsis thaliana embryonic development

Jan Kucinski (University of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor, MI, USA), Sebastian Chamera, Aleksandra Kmera (Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, Warsaw, Poland), M. Jordan Rowley (Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE), Pragya Khurana (University of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor, MI, USA), Marcin Nowotny (Laboratory of Protein Structure, International Institute of Molecular and Cell Biology, Warsaw, Poland), Andrzej T. Wierzbicki (University of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor, MI, USA)

Abstract:
RNase H1 is an endonuclease specific towards the RNA strand of RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. We show that Arabidopsis thaliana contains at least three RNase H1-like proteins originating from two gene duplication events and alternative splicing. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for enzymatic activity. One of those proteins is nuclear, one is localized to plastids, one is localized to mitochondria. While the nuclear RNase H1 is dispensable for development under normal growth conditions, the presence of at least one organellar RNase H1 is required for embryonic development. The plastid protein RNH1C affects plastid DNA copy number and sensitivity to replicative stress. This suggests that three genomes present in each plant cell are served by at least one specialized RNase H1 protein.

Keywords: RNase H1, Arabidopsis, RNADNA hybrid

71. Stability of RNA•DNA-DNA triple helices depends on base triple composition and length of the RNA strand

Charlotte N. Kunkler (Department of Chemistry and Biochemistry, University of Notre Dame), Jacob P. Hulewicz (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:
Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA-DNA triple helix that regulates gene expression. However, base triple composition of pyrimidine motif RNA•DNA-DNA triple helices is not well understood beyond the canonical U•A-T and C•G-C base triples. Using native gel-shift assays, the relative stability of 16 different base triples (Z•X-Y, where Z = C, U, A, G and X-Y = A-T, G-C, T-A, C-G) at a single position in an RNA•DNA-DNA triple helix was determined. The canonical U•A-T and C•G-C base triples were the most stable, while three non-canonical base triples completely disrupted triple-helix formation. We further show that our RNA•DNA-DNA triple helix can tolerate up to two consecutive non-canonical A•G-C base triples. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA-DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triples in DNA•DNA-DNA and RNA•RNA-RNA triple helices was distinctly different from those in RNA•DNA-DNA triple helices, showing that base triple stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.

Keywords: triple helix, triplex, base triple

72. A Tale of Two Types of Archaeal RNase P: Of Rate-Limiting Steps and Subunit Stoichiometries

Stella M. Lai (Department of Chemistry and Biochemistry, Center for RNA Biology, and Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University), Walter J. Zahurancik (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, Center for RNA Biology, and Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University)

Abstract:
RNase P is an essential housekeeping enzyme that catalyzes the Mg²⁺-dependent 5′-maturation of precursor (pre)-tRNAs in all three domains of life. Defective tRNA processing in animals has been associated with a variety of diseases, including neurodegeneration, which underscores the need for a mechanistic understanding of RNase P assembly and catalysis. The canonical form of RNase P is a ribonucleoprotein (RNP) complex that is exemplified by archaeal RNase P, which consists of one catalytic RNA (RNase P RNA; RPR) and up to five protein subunits (RNase P proteins; RPPs): POP5, RPP21, RPP29, RPP30, and L7Ae. POP5•RPP30 and RPP21•RPP29 function as binary complexes to enhance the cleavage rate and improve substrate binding, respectively. However, the role of monomeric L7Ae, which binds RNA structural modules called kink-turns (K-turns) to induce axial bending, is unclear. We recently reported that L7Ae binds three K-turns in type A [e.g., Pyrococcus furiosus (Pfu)] and two K-turns in type M [e.g., Methanocaldococcus jannaschii (Mja), Methanococcus maripaludis (Mma)] euryarchaeal RNase P, which are classified based on the secondary structure of their RPRs. To determine whether the number and location of the K-turns affect the ability of L7Ae to contribute directly to RNase P catalysis, we are using rapid quench-flow-based single-turnover and manual quench-based multiple-turnover assays to measure the kinetics of pre-tRNA cleavage by Pfu, Mja, and Mma RNase P assembled in the absence and presence of L7Ae. In parallel, we are using native mass spectrometry to study the composition and stoichiometry of these RNase P assemblies to establish a structural context for the biochemical results. Together, these complementary analyses provide valuable insights into protein-aided catalysis in this ancient, multi-subunit RNP.

Keywords: RNase P, native mass spectrometry, tRNA processing

73. Stem-loop Antisense RNA and DNA Exclude Mismatched Target Sequences

Samuel D. Stimple (Department of Chemical and Biomolecular Engineering, The Ohio State University), Elizabeth Traverse (Department of Chemistry and Biochemistry, The Ohio State University), Si Yu Tan (Department of Chemistry and Biochemistry, The Ohio State University), Valerie Zaino (Department of Chemistry and Biochemistry, The Ohio State University), Richard A. Lease (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

74. CstF complex regulates an unique polyadenylation machinery in Arabidopsis thaliana

Kuan-Chieh Leu (Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan, ROC., Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546, USA.), Chun-Min Wang (Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan, ROC.), Huei-Jing Wang (Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan, ROC.), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546, USA.), Guang-Yuh Jauh (Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan, ROC.)

Abstract not available online - please check the printed booklet.

75. Strategies for utilizing NMR to gain insights into an intrinsically disordered domain of hnRNP A1

Jeffrey D. Levengood (Department of Chemistry, Case Western Reserve University), Julien Roche (Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University), Blanton S. Tolbert (Department of Chemistry, Case Western Reserve University)

Abstract:
The hnRNP A1 protein is involved in numerous processes of nascent RNA transcripts. These roles include, but are not limited to, translational control, splicing regulation, and mRNA stabilization. The protein performs its functions by first binding the RNA at a high affinity site before recruiting other proteins to aid in its RNA processing activities.

hnRNP A1 is composed of three domains, two structurally identical RRMs that form the nucleic acid binding protein UP1, and a disordered, glycine rich C-terminal domain (G-CTD). As the more structured domain, UP1 has been studied extensively. Its RNA binding properties have been examined through numerous biochemical and biophysical methods. Its structure, both free and bound, has been determined through NMR, X-ray crystallography, and small-angle X-ray scattering (SAXS).

In contrast to UP1, the G-CTD has not been very well characterized. However, its roles in hnRNP A1 activity requires that it be studied with just as much detail as UP1. The G-CTD is responsible for assembling the multi-protein complexes the protein utilizes to carry out its various functions. It has been shown to be a low complexity sequence domain (LCD) capable of mediating liquid-liquid phase separation (LLPS). This partitioning leads to the formation of stress granules, cytosolic bodies which are believed to be formed by mRNAs stalled in translation.

Experiments in high-pressure NMR on full length hnRNP A1 have revealed an interesting property of the G-CTD; its HSQC peaks disappear at high pressure. At 2,500 bar, the hnRNP A1 peaks that are part of the UP1 domain overlap with peaks from the isolated UP1 domain at identical pressure. The two spectra are completely identical as all peaks from the G-CTD are not present. The reason for this is currently unknown. Further experiments with the isolated G-CTD domain will be performed in order to gain further insight into this phenomenon.

Keywords: RRM, Stress Granules

76. Conformationally-sensitive cleavage of human telomere DNA via molecular recognition of the loop region

Yufeng Liang (Department of Chemistry and Biochemistry, The Ohio State University), Shiqin Miao (Department of Chemistry and Biochemistry, The Ohio State University), Dennis Bong (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

77. Determining the mechanism of cooperative binding of trp to TRAP using Bacillus halodurans

Katie Lichtenthal (Biological Sciences, University at Buffalo), Weicheng Li (Biochemistry, The Ohio State University), Mark Foster (Biochemistry, The Ohio State University), Paul Gollnick (Biological Sciences, University at Buffalo)

Abstract:
TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of tryptophan (trp) biosynthetic genes in several Baccilli. In response to excess trp levels, the amino acid binds to the 11 or 12-subunit TRAP ring. When activated by trp, TRAP binds to the mRNA of trp genes and down-regulates expression. Prior studies have focused mainly on B. subtilis (11 subunit TRAP), but recently we have been investigating the biophysical properties of TRAP (12 subunits) from the thermostable bacterium, B. halodurans. Trp binding is positively cooperative in both species, but the mechanism remains unclear. To further examine the mechanism of cooperative binding, we constructed B. halodurans TRAP 12mers from covalently linked dimers. The dimers were composed of all WT subunits or substitutions that abolish trp binding to the first or second subunit of each dimer. In addition, we created a TRAP protein composed of 1 WT subunit and 11 subunits containing substitutions that also abolish trp binding. K_ds and Hill coefficients (n) for the WT dimer were determined to be 2uM and 0.6, respectively, which agree with the K_ds value and n of 2uM and 1.7 for B. halodurans WT monomeric TRAP, respectively. In contrast, heteromeric TRAP (1WT:11Mut) gave an undetectable K_ds value. Further approaches to study trp binding to the substituted versions of the TRAP dimer and heteromer are being developed.

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

Keywords: Tryptophan, TRAP, Cooperativity

78. Androgen Receptor messenger RNA Metabolism

Eviania M. Likos (Biological, Geological, and Environmental Sciences - Cleveland State University)

Abstract not available online - please check the printed booklet.

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

Yichen Liu (Department of Chemistry, University of Michigan), Indrajit Deb (Department of Biophysics, University of Michigan), Aaron T. Frank (Department of Biophysics and Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

80. Stability factors of monomolecular DNA and RNA quadruplexes

Levan Lomidze (Center for RNA Biology, The Ohio State University, Columbus, OH 43210; Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Elaina Boyle (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Besik Kankia (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210 Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia)

Abstract:
One of the most stable quadruplexes is formed by the G3T sequence (GGGTGGGTGGGTGGG) that folds into a parallel quadruplex with three G-tetrads and chain-reversal T-loops containing anti glycosidic conformations of all guanines. In 1 mM K+, G3T unfolds at 75 °C whereas under physiological conditions, it unfolds above 100 °C. The RNA analog, ggguggguggguggg (g3u), which has the same folding topology, demonstrates even higher thermal stability. To understand the higher stability of RNA quadruplexes, we previously performed a comparative thermodynamic study of G3T and g3u quadruplexes, as well as chimeric constructs containing both DNA and RNA nucleotides. The all-RNA sequence was ~13 °C more stable than its DNA analog and the majority of G-g (DNA-for-RNA) substitutions destabilized the quadruplex. Only three G-g substitutions in the top (3'-end) tetrad and loop T-u substitutions increased the stability of the structure. The g3u quadruplex was also characterized by additional stabilizing interactions that are absent in the DNA analog. Taken together, this previous work revealed that stacking interactions are more favorable in DNA quadruplexes, while the chain-reversal loops play an important role in the higher stability of RNA quadruplexes. Here, we characterize the stabilizing factors of another monomolecular quadruplex, the thrombin-binding aptamer (TBA) GGTTGGTGTGGTTGG. In contrast to G3T, the TBA quadruplex adopts an antiparallel topology that requires half of the guanines to be in the syn conformation (two per G-tetrad). Our study reveals that DNA to RNA G-g substitutions in positions with an anti conformation stabilize the TBA quadruplex, while the same substitutions for Gs in the syn conformation inhibit quadruplex formation. The results of this study have implications for the rational design of RNA and DNA quadruplexes for biotechnological applications and for therapeutics targeting quadruplex structures.

Keywords: Quadruplexes, RNA, DNA

81. Diversity and plasticity of human spliceosomal small nuclear RNA variants

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

Abstract:
Splicing is a highly dynamic and regulated process that shapes the human transcriptome. To date, much of the research on splicing regulation has focused on auxiliary splicing factors. But, increasing evidence has shown that core-splicing proteins are also involved in regulating transcript-specific splicing events (1,2). 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 splice site recognition, spliceosome assembly and splicing catalysis. 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 proteins (3). Recent work has also shown that snRNAs are expressed in a cell-type specific manner in humans, and the relative abundance of snRNAs has been implicated in establishing cellular splicing programs (4). In addition, the human genome has over 1,600 annotated snRNA sequence variant genes. However, due to their numerous insertions, deletions and base changes, many of these variants have been given the label of pseudogenes as they are supposedly not expressed. But, it has been found that some snRNA sequence variants are expressed in human cells (5,6), and may play a role in regulating cellular differentiation (7). Yet, it is not known how many snRNA sequence variants are expressed in humans and if they can form functional snRNPs. In order to determine which snRNA sequence variants are expressed and potentially functional, we have analyzed human tissue and pooled cell lines for the expression of over eighty snRNA sequence variants. This data shows that many more snRNA gene variants are actually expressed in human cells than had originally been appreciated. Intriguingly, we have found that a subset of these expressed sequence variants are not able to form snRNPs. This indicates that expression does not necessarily correlate with snRNP function. However, we have also identified a number of novel variants that are able to incorporating into spliceosomes. It remains unknown how these variants impact cellular splicing, as snRNA variants themselves have remain largely uncharacterized.

References:
1. Papasaikas et. al. (2015) Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery. Molecular Cell
2. Saltzman et. al. (2011) Regulation of alternative splicing by the core spliceosomal machinery. Genes Dev.
3. Grosso et. al. (2008) Tissue-specific splicing factor gene expression signatures. Nucleic Acid Res.
4. Dvinge et. al. (2018) RNA components of the spliceosome regulate tissue- and cancers-specific alternative splicing. bioRxiv
5. O’Reilly et. al. (2013) Differentially expressed, variant U1 snRNAs regulate gene expression in human cells. Genome Res.
6. Sontheimer EJ and Steitz JA (1992) Three novel functional variants of human U5 small nuclear RNA. Mol Cell Biol.
7. Vazquez-Arango P and O’Reilly D (2017) Variant snRNPs: new players within the spliceosome system. RNA Biol.

Keywords: snRNAs, variant snRNPs, pre-mRNA splicing

82. Pnrc2 promotes 3'UTR-dependent oscillatory transcript decay during vertebrate segmentation

Monica Mannings (The Ohio State University Molecular Genetics Department), Kiel Tietz (The Ohio State University Molecular Genetics Department), Thomas Gallagher (The Ohio State University Molecular Genetics Department), Sharon Amacher (The Ohio State University Molecular Genetics Department)

Abstract:
Vertebrate segmentation is regulated by the segmentation clock, a biological oscillator that controls periodic formation of embryonic segments, or somites, within the presomitic mesoderm (PSM). Molecular components of the clock include the her/Hes family of transcriptional repressors, but additional transcripts also cycle. While transcriptional regulation of clock-associated genes has been studied comprehensively, post-transcriptional mechanisms governing oscillatory gene expression are not well-understood. Our previous work demonstrated that Proline rich nuclear receptor coactivator 2 (Pnrc2) promotes decay of clock-associated transcripts like her1 and dlc during segmentation, and loss of pnrc2 results in stabilization of oscillatory mRNAs. Microinjection of pnrc2 mRNA and pnrc1 mRNA, which codes for a protein that shares sequence similarity with Pnrc2, both restore normal her1 expression. Pnrc2 and Pnrc1 contain highly conserved C-terminal domains known to be important for mediating interactions among mRNA processing and decay factors, such as DCP1A and UPF1. Additionally, we utilized a reporter-based assay that showed that Pnrc2 promotes decay in a her1 and dlc 3'UTR-dependent manner. Regulatory motifs present within both 3’UTRs, a Pumilio Response Element and AU-rich Element, are known to bind to proteins that promote decay and translational repression. Mutation of the PRE and ARE within the full-length her1 3’UTR resulted in increased stability of reporter transcripts, suggesting both motifs likely bind to proteins that degrade cyclic transcripts. Interestingly, while cyclic transcripts accumulate in pnrc2 mutants, cyclic protein does not accumulate. Discordant expression of clock-associated mRNA and protein in pnrc2 mutants suggests that multiple levels of post-transcriptional gene regulation are implemented to sustain molecular oscillations during somitogenesis, and we are currently investigating trans factors that may be involved in decay and translation of oscillatory transcripts during vertebrate segmentation.

Keywords: decay, 3UTR, segmentation

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

Ila A. Marathe (Department of Microbiology, Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Paul D. Carlson, Molly E. Evans (Department of Chemical and Biological Engineering, Northwestern University), Stella M. Lai, Vicki H. Wysocki (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry-Guided Structural Biology, Center for RNA Biology, The Ohio State University), Michael G. Poirier (Department of Physics, The Ohio State University), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University), Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)

Abstract:
RNase P catalyzes the Mg²⁺-dependent 5'-maturation of tRNAs in all life forms. The ribonucleoprotein (RNP) form of RNase P consists of a catalytic RNase P RNA (RPR) and a variable number of RNase P Protein (RPP) cofactors: one in bacteria, and up to five in archaea and ten in eukaryotes. We use archaeal RNase P as a model to understand how multiple proteins independently and collectively modulate the functional scope of ribozymes, especially under physiological conditions. This objective gains significance due to an increased appreciation for the roles of RNPs in tissue complexity and human disease. Because the archaeal RPR alone requires ~500 mM Mg²⁺ even to support weak activity and needs RPPs for robust activity at physiological [Mg²⁺], we postulated that structural remodeling of the RPR by RPPs likely mirrors some of the changes promoted by high [Mg²⁺]. To test this hypothesis, we leveraged the powerful suite of RNase P assays, SHAPE-Seq, and FRET studies to identify functional and structural changes in the Pyrococcus furiosus (Pfu) RPR. First, we measured Pfu RPR’s activity in the presence of 10 to 500 mM Mg²⁺ to determine the [Mg²⁺] required for function. Second, we used SHAPE-Seq to interrogate the RPR at varying [Mg²⁺] and with single-nucleotide resolution to obtain insights into RPR structure (e.g., new inter-domain tertiary contacts). Finally, using dual-fluor-labeled Pfu RPR (currently in preparation), we plan to perform ensemble FRET experiments to determine the [Mg²⁺] required to establish long-range contacts and to use RPR mutants to validate any inter-domain contacts. This multi-pronged strategy helps parse the dual roles of Mg²⁺ in RNase P, wherein diffusely-bound Mg²⁺ promotes RPR structure and site-specific Mg²⁺ enables RPR catalysis. Collectively, our results suggest a model to link RPR structural changes to functional outcomes and establish a baseline to evaluate how archaeal RPPs forge a functional holoenzyme through concerted RPR remodeling.

Keywords: RNase P, RNA-protein interactions, Ribonucleoprotein assembly

84. A glance at the role of KHDRBS3 in the tumorigenesis of TNBC

Fatimah K Matalkah (Biochemistry/WVU), Bohye Jeong (Biochemistry/WVU), Peter Stoilov (Biochemistry/WVU)

Abstract:

TNBC has the worst prognosis among other breast cancer subtypes, and women diagnosed with the disease lack targeted treatments therapy. To identify possible molecular targets for treatment of TNBC, we analyzed the gene expression and alternative exon inclusion in more than 900 breast carcinoma and 120 normal breast tissue samples provided by the cancer genome atlas project (TCGA). We found that both the molecular basal like and the histological triple negative breast cancer (TNBC) subtypes display a distinct and very similar splicing profile. We also noticed high expression in the transcript level of the splicing factor KHDRBS3 in both subtypes. We examined the expression of KHDRBS3 in patient tumor samples and in a panel of breast cancer cell lines. We observed a higher protein level expression in the patient tumors and cell lines of TNBC relative to the ER/PR positive or normal mammary epithelial cells. To investigate, if the higher expression of KHRBS3 protein level would translate to an enhanced oncogenic property in the TNBC. We used two independent shRNA to stably knockdown the expression of KHDRBS3 in the cellular model of MDA-231-LN. Knocking down KHDRBS3 had no impact on cell proliferation or migration in vitro. To test if depletion of KHDRBS3 would have a different impact on TNBC progression and metastasis in vivo. MDA-231-LN expressing shKHDRBS3 were orthotopically transplanted into the mammary fat pad of female NSG mice. KHDRBS3 exhaustion had no significant outcome in the xenograft tumor growth rate. Neither it had a significant impact on the experimental or spontaneous metastasis to the lungs. While depletion of KHDRBS3 had no effect on the oncogenic properties of the selected cellular model, nevertheless we pursued to investigate the complete ablation of KHDRBS3 on spontaneous tumor formation of TNBC animal model. Indeed, we utilized the C3.1-Tag mouse model of triple negative breast cancer and back-crossed it to animals with either KHDRBS3 -/+, or KHDRBS3 -/- background. Surprisingly, complete ablation of KHDRBS3 from the mammary gland significantly hinder the formation of spontaneous mammary tumor. In conclusion, our data show that KHDRBS3 expression can probably be used as a molecular signature in TNBC.

Keywords: TNBC, KHDRBS3

85. Opioid-induced long noncoding transcriptome of human CD8+ T cells reveals opioid receptor subclass specific responses

Claire Mazahery (Pathology, Case Western Reserve University), Braulio Llorens (Molecular Biology and Microbiology, Case Western Reserve University), Saba Valadkhan (Molecular Biology and Microbiology, Case Western Reserve University), Alan D. Levine (Pathology, Molecular Biology and Microbiology, Case Western Reserve University)

Abstract:
Endogenous opioid peptides are released at sites of injury, and their cognate G-protein coupled opioid receptors (OR) are expressed on immune cells. Conflicting reports attribute immunostimulatory (opioids activate innate immune cells) and immunosuppressive (opioid users have elevated infection risk) activity to opioids. Intravenous drug use transmits viral infections, making the study of CD8+ T cells timely. From a cohort of methadone patients, we found that chronic opioid use disrupts CD8+ T cell subset balance, with decreases in T Effector Memory RA+ cells. Our findings from ex vivo T cell culture suggest that transcriptional mechanisms regulate the outcome from opioid receptor ligation, by modifying the coding and non-coding transcriptome. When T cells are activated through the T cell receptor (TCR) after 18 hours of opioid exposure, the noncoding RNA profile differs substantially from that seen with TCR stimulation without opioid pre-exposure. Globally, μ-OR signaling robustly inhibits or upregulates expression of lncRNAs, while δ-OR signaling has modest effects on expression. Compared to the response of protein coding genes, noncoding RNA expression showed more OR subclass specific regulation. Additionally, we identified >30 unannotated lncRNAs regulated by opioids ex vivo. We are silencing and overexpressing the noncoding RNAs we identified to define their ability to regulate OR specific responses. We are also investigating the contribution of these non-coding RNAs to the immunostimulatory and immunosuppressive perturbations observed in opioid users by contrasting lncRNA expression and activity in CD4 vs. CD8 T cells.

Keywords: noncoding transcriptome , human CD8+ T cells , opioid

86. 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, 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.

87. Investigating the role of the Anti-Shine Dalgarno in the Bacteroidetes

Zakkary McNutt (Ohio State Biochemistry Program and Department of Microbiology), Mai Dang (Ohio State University Department of Microbiology), Bappaditya Roy (Ohio State University Department of Microbiology), Aishwarya Devarag (Ohio State University Department of Microbiology), Kurt Fredrick (Ohio State University Department of Microbiology)

Abstract:
Translation initiation in bacteria often entails pairing between the Shine Dalgarno (SD) sequence of mRNA and the anti-SD (ASD) at the 3’ end of the 16S rRNA. Recent genomic studies have revealed that certain bacterial lineages, including those of the Bacteroidetes phylum, completely lack SD sequences [1]. Despite this, the conserved ASD is retained, suggesting an alternative function. Here, we present our initial work to probe the function of the ASD in Flavobacterium johnsoniae, a genetically tractable member of the Bacteroidetes. In E. coli, systematic mutagenesis of nucleotides 1534– 1542 of 16S rRNA suggests that five nucleotides (1535 CCUCC 1539) represent the functional core of the ASD. These mutant alleles fail to support cell growth and cause dominant lethality. We have begun to generate and characterize analogous mutations in the 16S rRNA of F. johnsoniae. This organism contains six virtually identical rrn operons. Three rrn operons have been deleted so far. This Δ3 strain exhibits a reduced growth rate, which can be partially complemented by a plasmid containing an rrn operon. In cells expressing heterogeneous 16S populations, mutant ribosomes can be tracked in sucrose gradients to assess changes in ribosome assembly and function. These experiments show that 30S subunits with mutations to the ASD largely retain function in F. johnsoniae, although these subunits are underrepresented in polysomes and accumulate in the 30S region of the gradient, indicating some defect in assembly or initiation.

References:
[1] Nakagawa, S., Niimura, Y., Miura, K. & Gojobori, T. Dynamic evolution of translation initiation mechanisms in prokaryotes. Proc Natl Acad Sci USA 107, 6382-6387, doi:10.1073/pnas.1002036107 (2010).

Keywords: Ribosome, RNA, Shine Dalgarno

88. Nucleotide level resolution of RNA folding interactions within peptide-based complex coacervates

McCauley O. Meyer (Biochemistry, Microbiology, and Molecular Biology, Penn State University), Saehyun Choi (Chemistry, Penn State University), Fatma P. Cakmak (Chemistry, Penn State University), Christine D. Keating (Chemistry, Penn State University), Philip C. Bevilacqua (Biochemistry, Microbiology and Molecular Biology, Chemistry, Penn State University)

Abstract:
Understanding how life arose is one of the great questions facing humanity. The RNA World Hypothesis partially addresses this by providing a genetic component as well as an enzymatic one, but does not provide for how RNA-compatible cells arose. A potential RNA-compatible protocell model that has recently garnered attention is complex coacervates. Coacervates are molecule-rich droplets formed via liquid-liquid phase separations (LLPS) primarily by electrostatic interaction of poly-cationic and poly-anionic polymers leading to a reduced water content inside of the droplets. Coacervates have been demonstrated to form with both biotic and abiotic polymers and have also been shown to strongly partition nucleic acids, amino acids and metal ions. As protocells, they have been mostly studied from a physical/chemical point of view; however, there has been little characterization of them with regard to whether RNA catalysis and replication could occur within them.
Herein I describe our early efforts toward characterizing RNA folding interactions at the nucleotide level within complex coacervates made from lysine homo-polymers and aspartic acid homo-polymers, as well as lysine homo-polymers and ATP. Nucleotide-level resolution of the folding status of the model RNA, tRNAphe from S. cerevisiae, was assessed by in-line probing. Through this method, a variety of additional coacervates made from both lysine and aspartic acid homo-polymers of different lengths were also investigated. Some coacervates promoted native folding of the tRNA, while others led to strand-specific denaturation. The effects on folding also differed based on polymer length and identity. These studies serve as a proof-of-concept that nucleotide-level resolution of RNA folding states within complex coacervates can be obtained and that this technique should be applicable to other functional RNAs including ribozymes.

Keywords: Liquid-Liquid Phase Separation, RNA folding, Early Earth

89. Duplex Stem Replacement with bPNA+ Triplex Hybrid Stems Enables Reporting on Tertiary Interactions of Internal RNA Domains

Shiqin Miao (Department of Chemistry and Biochemistry, The Ohio State University), Yufeng Liang (Department of Chemistry and Biochemistry, The Ohio State University), Ila Marathe (Department of Chemistry and Biochemistry, The Ohio State University), Jie Mao (Department of Chemistry and Biochemistry, The Ohio State University), Chris DeSantis (Department of Chemistry and Biochemistry, The Ohio State University), Dennis Bong (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

90. Development of novel tools to characterize mRNA chemical modifications

Luke Miller (University of Michigan, Program in Chemical Biology), Kristin S. Koutmou (University of Michigan, Department of Chemistry)

Abstract:
The combined application of high-throughput tools to rapidly quantify nucleoside modification levels with RNA-seq based technologies for mapping the location of modifications transcriptome-wide form a powerful approach for the discovery and characterization of modified nucleosides in mRNAs. We are using such an approach to broadly screen and characterize mRNA modifications. Ultimately, our goal is to create and adapt methods for high resolution, quantitative characterization of the mRNA epitranscriptome. We have performed a high-throughput UHPLC MS/MS screen for mRNA modifications, and identified three potential previously unreported mRNA modifications. Currently, we are undertaking UHPLC-MS/MS studies to investigate the levels of these modifications in mRNAs purified from yeast cells containing or lacking enzymes that we hypothesize are responsible for incorporating the additional modifications that we have identified. Furthermore, we are developing a method for mapping the location of an mRNA modification that we previously reported (5-formylcytidine) to the transcriptome. Ultimately, this work has the potential to further our understanding of mRNA modifications by discovering and quantitatively characterizing new nucleoside modifications in protein coding RNAs.

Keywords: mRNA, Epitranscriptome, mRNA modifications

91. Contribution of mRNA 3’ UTRs in substrate recognition by the nonsense-mediated mRNA decay pathway

Savannah F Mills (Department of Biochemistry, Case Western Reserve University), Kristian E Baker (Department of Genetics and Genome Sciences, Case Western Reserve University)

Abstract:
Nonsense-Mediated mRNA Decay (NMD) is a conserved RNA quality control process that identifies and rapidly degrades aberrant mRNAs harboring premature termination codons (PTCs), thereby preventing the expression of c-terminally truncated proteins. In addition to targeting PTC-containing mRNA, NMD also regulates the expression of hundreds of additional transcripts that encode full-length polypeptides. The mechanism by which the NMD machinery identifies its targets remains poorly understood; notwithstanding, key observations indicate that the length of RNA sequence downstream of a termination codon is an important determinant. Consistent with this, endogenous mRNAs lacking a nonsense codon but harboring lengthy 3’ untranslated regions (UTRs) are commonly regulated by the pathway. Despite this observation, a strict correlation between 3’ UTR length and NMD sensitivity at a transcriptome-wide level is lacking, indicating that additional RNA features contribute to discrimination between NMD sensitive and insensitive mRNAs.

As a means to generate a more comprehensive understanding of the mechanism underlying NMD substrate identification, we are characterizing long 3’ UTRs from endogenous yeast transcripts to identify RNA features that elicit, or confer resistance to, recognition by the NMD machinery. Preliminary data employing RNA immunoprecipitation of the core NMD factor, Upf1, confirms observations of a correlation between NMD sensitivity and the amount of Upf1 associated with mRNAs targeted to the NMD pathway. Notably, NMD insensitive mRNAs showed a drastic reduction in Upf1 association, despite harboring equivalently long 3’ UTRs. The observation that Upf1 fails to associate with these NMD insensitive mRNAs suggests that Upf1 is precluded from binding or stably associating with these transcripts. Ongoing studies are aimed at characterizing the mRNP compositions of these long 3’ UTRs and identifying cis-acting sequences and/or trans-acting factors involved in modulating Upf1 binding/association and transcript susceptibility to NMD.

Keywords: NMD, mRNA decay, 3 UTR

92. Identification of splice isoforms in T helper (Th) cells using Nanopore-based cDNA sequencing

Quoseena Mir (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Rajneesh Srivastava (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Ulrich, Benjamin Joseph (Microbiology & Immunology, Indiana University School of Medicine), Mark H kaplan (Wells Center for Pediatric Research, Indiana University School of Medicine), Janga, Sarath Chandra (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University)

Abstract not available online - please check the printed booklet.

93. Characterization of R-loop structures in budding yeast

Youssef A. Hegazy (Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA), Chrishan Fernando (Department of Chemistry, Purdue University, West Lafayette, IN 47907, ), Sara C. Cloutier (Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA), Elizabeth J. Tran (Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA)

Abstract:
R-loops are nucleic-acid structures composed of an RNA:DNA hybrid and a displaced single-stranded DNA. R-loops occur more frequently in the genome and have greater physiological importance than was previously predicted. They play vital roles in regulating gene expression, DNA replication, and DNA and histone modifications. Paradoxically, while they do play essential positive functions, they also contribute to DNA damage and genome instability. Recent evidence shows that R-loops are involved in a number of human diseases, including neurological disorders and cancer. Despite the growing interest in R-loop regulation, there are currently few techniques available to identify and characterize R-loop structures in vivo. We are developing new strategies for mapping R-loops genome wide. We will present progress on establishing these techniques to map R-loops in Saccharomyces cerevisiae. We are also using reporter assay and the budding yeast genetic system to identify factors that may promote formation of R-loops formed by lncRNAs, an abundant class of non-coding RNAs that have been implicated in gene regulation. Taken together, we hope to provide a detailed understanding of molecular interactions that promote formation of biologically beneficial R-loops.

Keywords: R-loop, LncRNAs, R-loop mapping

94. Modulation of RNA Editing Helicase 1 level regulates RNA editing profile of mitochondrial transcripts in trypanosomes

Amartya Mishra (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA), Ashutosh P. Dubey (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA.), Brianna L. Tylec (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA.), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Buffalo, New York 14203, USA. )

Abstract:
In trypanosomes, most mitochondrial encoded mRNA sequences require post-transcriptional insertion and deletion of uridine residues. This U-indel RNA editing is directed by trans-acting gRNAs and mediated by a holoenzyme comprised of the catalytic RNA editing core complexes (RECC) and non-catalytic RNA editing substrate binding complex (RESC). RNA Editing Helicase 1 (REH1), an ATP-dependent mitochondrial RNA helicase, interacts transiently with RECC and RESC. Previous evidence indicates a role for REH1 in RNA editing of at least some mRNAs, but its mechanism of action is unclear. Here, we confirm by co-IP interactions between REH1 and several subunits of both RECC and RESC. We show that depletion of REH1 causes modest decreases in the levels of a subset of edited mRNAs. In contrast, overexpression of wild type (WT) REH1 from an ectopic locus causes a significant growth defect and leads to a 75-90% decrease in fully edited versions of most pan-edited mRNAs in the absence of pre-edited mRNA increases. Minimally edited mRNAs are largely unaffected. Together, these data suggest that 3’ to 5’ editing progression is impaired in REH1 overexpressors. To define the role of REH1 ATP-binding, we overexpressed REH1 mutated in the conserved ATP-binding motif. Mutant overexpression leads to cell death, indicating a dominant negative (DN) phenotype. This is accompanied a dramatic decrease in almost all edited mRNAs with concomitant accumulation of pre-edited transcripts, including 2 of 3 minimally edited mRNAs. Thus, unlike WT REH1, the DN mutant impairs initiation of editing such that pre-edited mRNAs accumulate. WT and DN REH1 bind RESC and RECC to a similar extent, suggesting that DN REH1 binding to the editing holoenzyme impairs its productive interaction with mRNA or gRNA, thereby interfering with editing initiation. We have now generated REH1 knockout cells and are analyzing these and REH1 WT and DN overexpressors by high throughput sequencing to define at the single nucleotide level the step(s) at which editing is blocked. This genomic approach, together with additional protein-protein and RNA-protein interaction studies, will unveil the mechanism of REH1 action in U-indel editing at the molecular level.

Keywords: Trypanosoma, RNA editing, RNA helicase

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

Chaitali Misra, Sushant Bangru, Feikai Lin, Darren Parker, Auinash Kalsotra (Department of Biochemistry, University of Illinois, Urbana-Champaign), Sara N. Koenig, Ellen R. Lubbers, Nathaniel P. Murphy, Peter J. Mohler (Department of Physiology and Cell Biology, Davis Heart and Lung Research Institute, College of Medicine, Wexner Medical Center, The Ohio State University, OH), Jamila Hedhli, Lawrence W. Dobrucki (Department of Bioengineering, Centers for Macromolecular Modeling, Bioinformatics, and Experimental Molecular Imaging at Beckman Institute for Advanced Science and Technology, Urbana-Champaign, IL), Thomas A. Cooper (Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX), Kin Lam,Emad Tajkhorshid (Center for Macromolecular Modeling and Bioinformatics, Beckman Institute for Advanced Science and Technology, University of Illinois. Urbana-Champaign,IL)

Abstract:
Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disease caused by a CTG repeat expansion in 3’-UTR of DMPK gene. DM1 affects multiple tissues, but cardiac dysfunctions are the second leading cause of death. The best characterized pathogenic mechanism of DM1 is toxic gain-of-function of expanded CUG repeat (CUGexp) RNA that accumulates to form ribonuclear foci causing sequestration of MBNL and overexpression of CELF1 family of splicing factors.
Here we report that steady-state protein levels of RBFOX2, a critical splicing regulator, are drastically upregulated in DM1 heart tissue. This is accompanied by aberrant skipping of a muscle-specific 43bp exon in RBFOX2 transcript, resulting in selective up-regulation of the non-muscle RBFOX2 splice isoform in adult cardiomyocytes. We demonstrate that expression of CUGexp RNA in DM1 affects RBFOX2 in two distinct ways: reduced expression of a subset of microRNAs causes de-repression of RBFOX2 protein production; and CELF1 overexpression promotes skipping of the muscle-specific exon of RBFOX2. Remarkably, tet-inducible overexpression of the non-muscle RBFOX2 isoform, or CRISPR/Cas9 mediated deletion of the muscle-specific RBFOX2 exon in the mouse heart results in prolonged PR and QR intervals, slower conduction velocity and spontaneous cardiac arrhythmias that mirror human DM1 pathology. Integration of RBFOX2-CLIP with RNA-seq data from cardiomyocytes of mice expressing the non-muscle RBFOX2 isoform identified a core network of mRNA splicing defects in genes encoding sodium, potassium, and calcium ion channels that are similarly misspliced in hearts of DM1 patients. These aberrantly spliced ion channels alter their rate of ion diffusion and electrophysiological properties thereby induce cardiac arrhythmias in DM1 heart. Thus, we have uncovered a crucial role for RBFOX2 isoform switching in DM1 cardiac pathogenesis.

Keywords: Alternate splicing, Myotonic Dystrophy, Cardiac Arrhythmia

96. Theoretical pKa calculations of modified purine nucleobases using explicit waters and a polarizable Ccontinuum model

Alan J. Mlotkowski (Department of Chemistry, Wayne State University), Evan Jones (Department of Chemistry, Wayne State University), Sebastien Hebert (Department of Chemistry, Wayne State University), H. Bernhard Schlegel (Department of Chemistry, Wayne State University), John SantaLucia, Jr (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
There are numerous modified nucleobases, which are believed to play roles in fine-tuning structure and function of various RNA molecules. Modifications of nucleobases have a wide range of effects including participating in regulation of expression and response to stress in the cell. Despite a growing wealth of knowledge, specific details about modifications are still not known. The pKa value is a defining characteristic of nucleobases, which is used to understand base-pairing energetics of modified nucleobases. Purine nucleobases and their modifications contain a minimum of two protonation/deprotonation sites that have unique pKa values. Modifications on nucleobases affect the pKa values, resulting in a change of nucleobase behavior. In this study, we determined the pKa values of 19 modified purine nucleobases by performing calculations with the Gaussian 16 program using the B3LYP/6-31+G(d,p) method with a combination of implicit-explicit solvation. This method involved the use of explicit water molecules forming hydrogen bonds with the nucleobases surrounded by an implicit solvation field. The pKa values were calculated from the change in the free energy between protonated and deprotonated forms of the nucleobases and the energy of the solvated proton. By using this approach, we have determined that a single modification can change the pKa value of protonation/deprotonation site on a purine nucleobase by as much as 4.0 units when compared to the unmodified purine nucleobase.

Keywords: modification, pka, compuational

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

Rushdhi Rauff (Chemistry and biochemistry), Paul DAmico (Chemistry and biochemistry), Sanjaya Abeysirigunawardena (Chemistry and biochemistry)

Abstract not available online - please check the printed booklet.

98. Translation is altered by the presence of N1-methylpseudouridine and 7-methylguanosine in mRNA coding regions

Jeremy Monroe (Department of Chemistry, University of Michigan), Kristin S. Koutmou (Department of Chemistry, University of Michigan)

Abstract:
Translation is altered by the presence of N1-methylpseudouridine and 7-methylguanosine in mRNA coding regions
Jeremy Monroe and Kristin S. Koutmou
Department of Chemistry, University of Michigan, 930 N University, Ann Arbor, MI 48109

Chemical modifications to mRNAs have the potential to impact every step in the life cycle of an mRNA after transcription to modulate protein expression. Initial in vitro studies of varying resolution by ourselves and others on a limited set of mRNA modifications indicate that they can alter the overall rate and fidelity of protein synthesis. However, there is still not a broad molecular level picture of how modifications change ribosome decoding. Establishing this basic knowledge will be vital as we work to discern the biological consequences of mRNA modifications. We are working fill this key gap in understanding by systematically varying the chemical moieties on mRNA nucleobases to directly establish the contributions of individual nucleobase positions and functional groups to mRNA decoding. Towards this goal, we have tested how two unique modifications, N1-methyl-pseudouridine (m1Ψ) and N-7-methylguanosine (m7G), impact protein synthesis in a fully-reconstituted in vitro E. coli translation system. These modifications were chosen for study because of their recent discovery (m7G) in eukaryotic mRNAs coding regions, and their incorporation into mRNA-based drugs (m1Ψ). Our early work reveals that m1Ψ slows amino acid addition when inserted into the first position of a phenylalanine (Phe) codon (m1Ψ UU), and promotes the incorporation of non-cognate isoleucine and leucine amino acids on a Phe codon. In contrast, m7G modification impedes translation termination by releaser factor 2. These findings provide further evidence that mRNA modification has consequences for translation, and suggest that individual modifications are likely to have discrete effects on the mechanism of protein synthesis.

Keywords: translation, mRNA modifications

99. Identification of the methyltransferase responsible for 2’-O-methylation of tRNA residue 39 in plants

Rachel Morgeson (Chemistry and Biochemistry of Northern Kentucky University ), Holly M. Funk (Chemistry and Biochemistry of Northern Kentucky University ), Ramey Hensley (Chemistry and Biochemistry of Northern Kentucky University ), Michaela Vogel (Chemistry and Biochemistry of Northern Kentucky University ), Michael P. Guy (Chemistry and Biochemistry of Northern Kentucky University )

Abstract not available online - please check the printed booklet.

100. Computational Discovery of Riboswitches in the Human Microbiom

Matthew Mouck (Biochemistry and Molecular Biology, Pennsylvania State University), Catherine Douds (Department of Biochemistry and Molecular Biology, Pennsylvania State University), Phil Bevilacqua (Department of Chemistry, Pennsylvania State University)

Abstract not available online - please check the printed booklet.

101. Why do Glioblastomas require the non-functional deaminase ADAR3?

Priyanka Mukherjee (Cell, Molecular and Cancer Biology; Indiana University School of Medicine Bloomington), Eimile Oakes (Genome, Cell and Developmental Biology; Indiana University Bloomington ), Heather A. Hundley (Medical Sciences Program; Indiana University School of Medicine Bloomington)

Abstract not available online - please check the printed booklet.

102. Short looped DNA as a Force Transducer: Conversion of single molecule FRET signal into force information

Golam Mustafa, William A. Roy (Department of Physics, Kent State University, Kent, OH 44242, United States), Cho-Ying Chuang, Matthew J. Comstock (Department of Physics, Michigan State University, East Lansing, MI 48824, United States), Mohamed M. Farhath, Soumitra Basu (Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, United States), Nilisha Pokhrel, Edwin Antony (Department of Biological Sciences, Marquette University, Milwaukee, WI 53201, United States), Yue Ma, Kazuo Nagasawa (Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan), Hamza Balci (Department of Physics, Kent State University, Kent, OH 44242, United States)

Abstract:
A multiplexed, high throughput single-molecule force sensor and transducer concept has been developed that converts fluorescence signal into force information via single molecule Förster resonance energy transfer (smFRET). A double-stranded DNA (dsDNA) loop has been formed by bridging the ends of a ~100 base pair (bp) long dsDNA with a nucleic acid secondary structure (NAS), such as a hairpin or a G-quadruplex (GQ). The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. As proof-of-principle measurements, 70 bp, 90 bp and 110 bp long dsDNA constructs were bridged by a DNA hairpin and KCl was titrated to change the tension across the DNA hairpin. Later, the interactions of a GQ structure formed by thrombin binding aptamer (TBA) with a destabilizing protein, Replication Protein A (RPA), and a stabilizing small molecule, an oxazole telomestatin derivative, were studied while the TBA-GQ is maintained under tension by a 110-bp long looped dsDNA. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. Since hundreds of such molecules could potentially be imaged simultaneously, it is possible to perform high-throughput force measurements with single molecule sensitivity. The proposed method enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.

References:
1. Vafabakhsh, Reza, and Taekjip Ha. "Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization." Science 337.6098 (2012): 1097-1101.
2. Jeong, Jiyoun, Tung T. Le, and Harold D. Kim. "Single-molecule fluorescence studies on DNA looping." Methods 105 (2016): 34-43.
3. Woodside, Michael T., et al. "Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins." Proceedings of the National Academy of Sciences 103.16 (2006): 6190-6195.
4. Woodside, Michael T., et al. "Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins." Proceedings of the National Academy of Sciences 103.16 (2006): 6190-6195.

Keywords: Single molecule FRET, Force sensor, Looped dsDNA

103. DNA methylation regulates alternative polyadenylation via CTCF and the cohesin complex

Vishal Nanavaty (Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Elizabeth W. Abrash (Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Changjin Hong, Sunho Park, Tae Hyun Hwang (Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Thomas J. Sweet, Jeffrey M. Bhasin, Srinidhi Singuri (Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Byron H. Lee (Cardiovascular & Metabolic Sciences, Lerner Research Institute, Glickman Urologic Institute, Cleveland Clinic, Cleveland, OH 44195, USA), Angela H. Ting (Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA, Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University)

Abstract:
Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer, but they have been studied as independent processes. While promoter hypermethylation is known to silence transcription of tumor suppressor genes, functions of DNA methylation in non-promoter regions are poorly understood, hindering a complete understanding of the biological impact of aberrant methylation in tumorigenesis and cancer progression. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation (MBD-seq), gene expression (RNA-seq) and polyadenylation (poly(A)) site usage between DNA methylation-competent HCT116 and DNA methylation-deficient DKO cells. Using RNA-seq and DNA methylation data from The Cancer Genome Atlas (TCGA) for 11 cancer types, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. Through sequence motif analysis and chromatin immunoprecipitation (ChIP), we identified and confirmed CCCTC-binding factor (CTCF), which binds DNA in a DNA methylation-sensitive manner, to be preferentially recruited to unmethylated DNA downstream of the proximal poly(A) site. Removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which in turn, promotes proximal polyadenylation site usage. In this DNA de-methylated context, depletion of the RAD21 a cohesin complex component can recover distal poly(A) site usage. Deletion of CTCF binding motifs via CRISPR/CAS9 highlights the importance of CTCF and cohesin complex proteins in chromatin loop formation and alternative polyadenylation. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.

Keywords: DNA methylation, Alternative polyadenylation, Chromatin loops

104. Do tRNA introns have a biological function?

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

Abstract:
A subset of genes encoding tRNAs contain introns. In most species, tRNA introns are located 3’ to the anticodon and are removed from pre-tRNAs by the tRNA splicing endonuclease. Pre-tRNA splicing is essential in most organisms because, for at least one tRNA family, all reiterated tRNA genes contain an intron. Thus, the genome cannot be decoded without tRNA splicing. Why has the possession of tRNA introns been conserved from archaea to vertebrates? One possibility is that the released introns serve biological roles. In support of this, we found that there are at least 5 pathways for tRNA intron turnover in budding yeast, and that although normally degraded, various stressors cause tRNA- and stress-specific tRNA intron accumulation. Further, sequence analysis reveals that some tRNA introns possess long stretches of complementarity to sequences in or around ORFs. The existence of these “tRNA intron complementarity (TIC) sequences” raises the possibility that particular tRNA introns may function as novel non-coding RNAs in the regulation of gene expression. To test this hypothesis, we are employing 3 different strategies. First, we are generating yeast strains lacking a specific species of tRNA intron. Second, we are adding stressors known to modulate tRNA intron levels. Lastly, we are incorporating synonymous codons into the TIC sequence of mRNAs. Sequence analysis of the mRNA for ORC2 reveals 14bp of perfect complementarity to the tRNA^Ile_UAU intron in its ORF. Endogenous deletion of the introns from the tRNA^Ile_UAU genes decreased Orc2-GFP protein, without a concomitant change in its mRNA, despite the fact that proteins of all non-TIC sequence containing genes examined were unaltered. These findings suggest the exciting possibility that the tRNA^Ile_UAU intron may act post-transcriptionally and sequence-specifically to positively regulate Orc2 levels. Overall, these data indicate that tRNA introns may be the newest member of non-coding regulatory RNAs.

Keywords: tRNA, introns, gene expression

105. Structural features of plastid chromatin

V. Miguel Palomar (Molecular, Cellular and Developmental Biology, University of Michigan), Sho Fujii (Molecular, Cellular and Developmental Biology, University of Michigan), M. Hafiz Rothi (Molecular, Cellular and Developmental Biology, University of Michigan), Jan Kucinski (Molecular, Cellular and Developmental Biology, University of Michigan), Masayuki Tsuzuki (Molecular, Cellular and Developmental Biology, University of Michigan), Andrzej T. Wierzbicki (Molecular, Cellular and Developmental Biology, University of Michigan)

Abstract not available online - please check the printed booklet.

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

Rahi Patel (Molecular Genetics & The Center for RNA Biology; The Ohio State University), Olivia Howard (The Center for RNA Biology; The Ohio State University ), Wayne O. Miles (Molecular Genetics & The Center for RNA Biology; The Ohio State University)

Abstract not available online - please check the printed booklet.

107. Investigating the effect of post translational modifications of LysRS on HIV-1 gRNA binding

William A. Cantara (Department of Chemistry and Biochemistry, The Ohio State University), Chathuri Pathirage (Department of Chemistry and Biochemistry, The Ohio State University), Joshua Hatterschide (Department of Chemistry and Biochemistry, The Ohio State University), Erik D. Olson (Department of Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

108. Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA

Robert D. Patton (Department of Physics, The Ohio State University), Manu Sanjeev (Department of Molecular Genetics, The Ohio State University), Lauren A. Woodward (Department of Molecular Genetics, The Ohio State University), Justin W. Mabin (Department of Molecular Genetics, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, The Ohio State University)

Abstract:
In eukaryotic cells, RNA binding proteins (RBPs) regulate RNA activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify RBPs, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing existing human RBP catalogs that were defined based on ultraviolet light (UV)-dependent and independent approaches, we classify RBPs into direct RBPs (efficiently UV crosslinkable) and indirect RBPs (poorly UV crosslinkable). As the UV cross-linking and immunoprecipitation followed by sequencing (CLIP-Seq) approach will be ill-suited to identify binding sites of indirect RBPs, we show that formaldehyde crosslinking stabilizes indirect RBPs within ribonucleoproteins to allow for their purification under stringent conditions. Using a direct RBP (CASC3) and an indirect RBP (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking of cells combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-Seq) far exceeds CLIP-Seq to identify binding sites of RNPS1. However, both xRIPiT-Seq and CLIP-Seq are comparable for enrichment of CASC3 binding sites. Overall, our work highlights the existence of a significant number of indirect RBPs in the human proteome, and shows that xRIPiT-Seq is more suitable to study RNA cargoes of these proteins.

References:
1. Brannan, K., Wenhao, J., Huelga, S., Banks, C., Gilmore, J., Florens, L., Washburn, M., Nostrand, E., Pratt G., Schwinn, M., Daniels, D., and Yeo, G. (2016). SONAR discovers RNA binding proteins from analysis of
large-scale protein-protein interactomes. Mol Cell. 64(2): 282-293.
2. Trendel, J., Schwarzl, T., Horos, R., Prakash, A., Bateman, A., Hentze, and M., Krijgsveld, J. (2019). The Human RNA-Binding Proteome and Its Dynamics during Translational Arrest. Cell. 176:391-403.

Keywords: CLIP-Seq, RIPiT-Seq, RBP

109. Perchlorate brine abrogates protein function whereas ribozymes thrive

Matthew Pawlak (Department of Genetics, Cell Biology, and Devlopment, University of Minnesota, Minneapolis, MN, Unites States), Tanner Hoog (Department of Genetics, Cell Biology, and Devlopment, University of Minnesota, Minneapolis, MN, Unites States), Nathaniel Gaut (Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, Unites States), Kate Adamala (Department of Genetics, Cell Biology, and Devlopment, University of Minnesota, Minneapolis, MN, Unites States), Aaron Engelhart (Department of Genetics, Cell Biology, and Devlopment, University of Minnesota, Minneapolis, MN, Unites States)

Abstract:
Mars is among those most attractive environments with sufficient similarity to earth Earth to potentially host life. The potential presence of liquid water and chemical similarity of the Martian regolith to that of Earth have led many to consider Mars as a potential environment that could host life. Work to date has largely focused on the presence of microbes on Mars, and less work has been done regarding Mars as a potential prebiotic planet - past or present - that could harbor biosignatures or pre-biosignatures. Reasons for optimism for Mars as both a biotic and a prebiotic planet exist. One such reason is the likely, potentially periodic, potential of liquid water enabled by the presence of hygroscopic oxychlorine salts such as perchlorate, first detected by the Phoenix mission and confirmed by the Curiosity mission. Perchorate is a unique ion. Beyond its potential role in enabling the presence of liquid water, it has been invoked as a potential oxygen source. Here, we consider the fact that it is a profoundly chaotropic Hofmeister ion and destabilizing to biopolymer secondary structure. Despite this, we have observed in a range of experiments that an RNA world in perchlorate brines is possible, and even favored over one employing protein catalysts.

Keywords: G Quadruplex, Mars, Perchlorate

110. Limited proteolysis of a DEAD-Box protein reveals small conformational changes

Katelyn R Perroz (Allegheny College, Department of Biochemistry), Michelle Raymond (Allegheny College, Department of Biochemistry), Megan Arnold (Allegheny College, Department of Biochemistry), Dr. Ivelitza Garcia (Allegheny College, Department of Biochemistry)

Abstract:
RNA unwinding proteins play an essential role in most RNA processing pathways. One ubiquitous family of RNA unwindases are DEAD-Box proteins. DEAD-box proteins are characterized by 12 conserved motifs and two Rec-A like structural domains. These proteins utilize the conformational changes that occur during ATP hydrolysis to modulations in RNA-RNA, RNA-protein, and protein-protein interactions. For example, Rok1p is a yeast DEAD-Box protein involved in rRNA processing. Rok1p’s function is dependent on ATP hydrolysis. Interestingly, this protein has both an N-terminal domain (NTD) and a C-terminal domain (CTD). The peripheral domains are hypothesized to play a role in rRNA processing and protein recruitment. Rrp5, an rRNA processing factor, binds pre-rRNAs and guides ITS1 cleavage separating the large subunit RNA and small subunit RNA during biogenesis. The interaction and regulation between Rrp5 and Rok1p are proposed to be controlled by Rok1p’s peripheral domains. Thus, the effects of Rok1p’s peripheral domains on the Rec-A structural dynamics were explored with domain truncations in combination with limited proteolysis. Since previous work suggests that Rok1p is more dynamic at higher temperatures as well as in the presence of certain ligands, a comparative analysis was also performed in the presence and absence of RNA under various conditions. Proteolysis patterns dramatically change in the absence of both peripheral domains, suggesting that both the NTD and CTD domain affect the global dynamics of Rok1p. Similar results were obtained in the presence of RNA. The structural dynamics are noticeably affected in the presence of the CTD and RNA at lower temperatures. Whereas, the NTD domain effects are prevalent at high temperatures. This study demonstrated the variable structural consequence of peripheral domains.

Keywords: RNA , DEAD-Box, Proteins

111. Determinantion of recognition elements governing binding of human selenocysteine synthase (SepSecS) to its substrate tRNA

Anupama Puppala (Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago), Jennifer Castillo (Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago), Miljan Simonovic (Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago)

Abstract not available online - please check the printed booklet.

112. Gene regulatory role of ADAR3 in Glioblastoma

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

Abstract not available online - please check the printed booklet.

113. Purification and analysis of proteins associated with nonsense codon-containing mRNA in Saccharomyces cerevisiae

Hamza Rahman (Case Western Reserve University Depertment of genome and genomic sciences ), Dajuan Whiteside (Case Western Reserve University Depertment of genome and genomic sciences ), Kristian Baker (Case Western Reserve University Depertment of genome and genomic sciences )

Abstract:
Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular RNA quality control pathway that recognizes and rapidly degrades mRNAs harboring nonsense codons. The detection and rapid elimination of these aberrant mRNAs help to promote fidelity in gene expression and prevent accumulation of truncated polypeptides that are likely non-functional or have deleterious effects in the cell. Three proteins are essential for NMD and make up the core NMD machinery - Up-frameshift 1 (Upf1), Upf2, and Upf3 – with Upf1 being the only factor with known enzymatic activity and an ability to bind directly to RNA. It remains unclear how the NMD machinery is able to discriminate between normal and aberrant mRNA and target only those harboring nonsense codons for rapid degradation. In an attempt to gain a better understanding of the mechanism of NMD substrate recognition, we have developed a protocol to isolate transcript-specific mRNA-protein complexes from normal and nonsense-containing mRNA from the model organism, Saccharomyces cerevisiae, and have identified the associated proteins by mass spectrometry. It is anticipated that differences in protein complex composition will help shed light on how nonsense-containing mRNAs are recognized by the NMD machinery.
We have identified novel proteins specifically enriched on an NMD substrate and are performing experiments to identify a potential a role for these in NMD substrate recognition. To validate and extend these initial findings, I have developing a second mRNA reporter for mRNP purification employing the endogenous PRC1 gene. I have cloned PRC1 onto a high-copy number plasmid and confirmed the construct by restriction digestion and DNA sequencing, and characterized mRNA expression by Northern blot. Northern blot results indicate that: (i) PRC1 mRNA is highly expressed from the plasmid; (ii) introduction of a nonsense codon at position 133 (i.e. PRC1PTC133) results in a dramatic reduction in the steady-state level of this reporter transcript; and, (iii) inhibition of NMD leads to an increase in the abundance of PRC1PTC133 mRNA. On-going studies with this reporter will contribute to identifying differences between NMD sensitive and insensitive mRNPs and help reveal how cells recognize and target nonsense-containing mRNA to NMD.

Keywords: NMD, RNA, Upf1

114. Pumilio-mediated post-transcriptional regulation of Dicer1

Swetha Rajasekaran (Department of Molecular Gentics, The Ohio State University), Kathleen M. Dotts (The James Comprehensive Cancer Center, The Ohio State University), Andre Gerber (Faculty of Health and Medical Sciences, University of Surrey, United Kingdom), Wayne O. Miles (The James Comprehensive Cancer Center, The Ohio State University)

Abstract:
DICER1 is an endoribonuclease involved in mature miRNAs processing. Maintaining homeostasis of DICER1 protein levels has shown to be important as changes in Dicer1 levels can affect cellular phenotypes, and drive tumor growth. Mutations in the DICER1 gene cause Dicer1 syndrome – an inherited disorder that results in the development of uncommon cancerous and benign tumors. Most DICER1 mutations occur in the coding sequence of the transcript, however around 30% of the Dicer1 syndrome patients do not have a mapped mutation within this region. By analyzing sequences from a large cohort of patients with Dicer1 syndrome, our lab has identified an A-to-G mutation in the 3’ UTR of DICER1. Motif mapping studies have shown that this mutation disrupts the binding site of the Pumilio (PUM) RNA-binding protein. The PUM family of proteins binds to a consensus PUM Regulatory Element (PRE) within the 3’UTR of the mRNA to affect the level of protein translation and mRNA turnover of the substrate. PUM proteins are generally considered to be translational repressors that bind to their targets and reduce the translation efficiency and ultimately the protein expression of the transcript. Surprisingly, on depleting PUM proteins using shRNA or CRISPR, we observed a decrease in DICER1 protein levels compared to WT, contrary to the expected model of PUM-mediated translational repression. Luciferase assays with WT and PRE-mutant DICER1 3’UTR fragments showed that this regulation is PRE-mediated and caused by direct binding. To test the importance of PUM-mediated regulation of Dicer1, we made knock-in of PRE-mutations in the endogenous DICER1 3’UTR in human cell lines using CRISPR. We then measured how this mutation affected growth rate, invasion and migration and compared with WT cells. These experiments showed that an isolated mutation in the DICER1-PRE resulted in slower growth, lesser invasion and decreased rate of migration in these cells. DICER1 expression is a double-edged sword in cancer and it is important for the cells to maintain the right amount of DICER1 making it hard to develop therapeutic strategies directly targeting DICER1. Understanding PUM-mediated post-transcriptional regulation of DICER1 and its role in promoting DICER1 protein production may provide opportunities to target DICER1 activity in tumors.

Keywords: Dicer1, Pumilio

115. Molecular Mechanisms to Regulate RNA Editing in nervous system

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

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. 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. We determined that ADR-2, the editing enzyme has reduced affinity for RNA. ADR-2 interacts with ADR-1, an editing-deficient member of the ADAR family, and substrate recognition is determined by this ADR-1/ADR-2 complex. 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 To begin to elucidate the neural specific regulation of editing in development, we performed high-throughput sequencing of RNA isolated neural cells from two different life stages of C. elegans (first stage larval (L1) and young adults). Differential gene expression analysis indicates that ~10,000 genes are developmentally regulated and 35% of them are dependent on the expression of ADR-2. De novo identification of editing sites indicates that L1 stage has ~ 10-fold higher number of sites than young adults, that are enriched for intronic where sites from young adults enriched for 3’-UTRs. Majority of the sites (62%) identified in young adults show increased level of editing than L1. We identify a group of genes, have only few editing sites (<5) and exhibit increased editing during development. This group includes genes where ADR-1 was previously shown to be not required foe editing. Interestingly, the ratio of ADR-2/ADR-1 is increased during development at the RNA level. We hypothesize that this regulation of ADR-2/ADR-1 ratio determines the substrate recognition mechanism and editing efficiency during development. Elucidating how ADR-2 binds to substrate mRNAs in specific tissues during development will result in identification of molecular mechanisms that regulate RNA editing.

Keywords: RNA editing, Nervous system

116. Branchpoint identity and branch length effects on lariat debranching enzyme activity

Timothy Chad Ratterman (Chemistry, Carnegie Mellon University, Center for Nucleic Acids Science and Technology), Subha R. Das (Chemistry, Carnegie Mellon University, Center for Nucleic Acids Science and Technology)

Abstract:
Lariat introns are formed during splicing when the 5ʹ-end of the intron is attacked by the 2ʹ-hydroxyl of an internal adenosine residue. Consequently, the canonical branchpoint residue in an excised lariat intron is an adenosine that contains a 2ʹ,5ʹ-phosphodiester linkage. These lariat introns must subsequently be opened at the 2ʹ,5ʹ-linkage by the lariat debranching enzyme (Dbr1p) before they can continue on in other regulatory processes. Hence the mechanism of Dbr1p binding and cleavage of the 2',5'-linkage at the branchpoint is of significant interest. Previous work from our lab has shown that S. cerevisiae and E. histolytica Dbr1p are capable of cleaving backbone branched RNAs (bbRNAs) containing non-adenosine branchpoint residues. Further, a recently published crystal structure of Dbr1p shows the nucleotide arms bound by the enzyme differ in the number of bound residues. Here we detail our analysis of the kinetics of binding and cleavage activity of E. histolytica Dbr1p using both bbRNAs substrates that contain non-canonical branchpoint residues and branched RNAs with a different number of residues in the nucleotide arms. These data provide new details on Dbr1p interactions at and close to the branchpoint of lariat introns.

Keywords: Lariat Debranching Enzyme, Branched RNAs, Lariat Introns

117. Structural characterization of DEAD-Box protein peripheral domains

Michelle Raymond (Biochemistry Department Allegheny College), Katie Perroz (Biochemistry Department Allegheny College), Megan Arnold (Biochemistry Department Allegheny College), Dr. Ivelitza Garcia (Biochemistry Department Allegheny College)

Abstract:
Ribosomal RNA processing and folding require many trans-acting factors to achieve mature ribosomes. DEAD-box proteins are essential for both small and large subunit formation. These proteins couple ATP hydrolysis and protein conformational changes to RNA folding and/or RNP complex modulation. For example, Rok1p, a yeast DEAD-Box protein, guides the cleavage of ITS1 in pre-rRNA to separate the large and small subunit RNAs. Structurally, Rok1p contains two Rec-A like domains the form an ATP active site and an RNA binding surface. Two peripheral domains [the N-terminal domain (NTD) and C-terminal domain (CTD)] flank the Rec-A domains and are proposed to aid in RNA and protein recruitment. However, recent studies have suggested a role in ATP binding. Thus, limited proteolysis was utilized to examination global structural changes in Rok1p and Rok1p domain truncation in the presence of ADP or AMPPNP as well as under varying temperatures. ADP binding to Rok1p and Rok1p variants yielded two predominate proteolytic fragments. The rate of cleavage was greatly affected by the binding of ADP. However, AMPPNP (nonhydrolyzable analog) binding limited proteolysis under various temperatures and peptidase concentrations. The presence of CTD affects the extent and rate of cleavage at all temperatures. In the presence of both peripheral domains, the structural stability caused by the NTD lessens the CTD median effects. This dampening of proteolysis affects ADP bound protein more than the AMPPNP bound complex. Although minor structural changes are suggested, this study shows that peripheral domains have variable structural consequences in the presence of nucleotide-binding.

Keywords: RNA, DEAD-Box, Limited Proteolysis

118. Optimization and Quantification of Biofilm Produced by Escherichia coli K12 for Selection of DNA Aptamers

Tristan Sanford (Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL 62026), Elliot Clerc (Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL 62026), Mina Sumita (Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL 62026)

Abstract:
Our main research interest is the development of a biosensor that detects foodborne pathogens by using DNA aptamers for food safety. Bacteria, when stationary, can adhere to a surface and begin to form a biofilm. A biofilm is a thin, slimy layer of aggregated bacteria that is utilized as a networking system to transfer nutrients and act as a defense mechanism. The substance encompassing the bacteria and responsible for these actions is the extracellular polymeric substance (EPS). The biofilm is also a threat to food safety because the biofilm makes the bacterial colony stronger and more resistant to sanitization. The focus of my research project is to identify a DNA aptamer for the biofilm and compare with the recently identified DNA aptamer for E. coli K12 in the lab by SPR-cell-SELEX. The production of biofilm formation using Escherichia coli K12 is also optimized at different growth periods.

Keywords: Biofilm, Aptamer, SELEX

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

Tracy M. Roach (Department of Chemistry and Biochemistry, OSU), Marie-Theres Poehler (Institute for Biochemistry, Leipzig University), Brandon W.J. Iwaniec (Department of Chemistry and Biochemistry, OSU), Mario Moerl (Institute for Biochemistry, Leipzig University), Jane E. Jackman (Department of Chemistry and Biochemistry, OSU)

Abstract not available online - please check the printed booklet.

120. Noncoding RNA-mediated DNA methylation controls nucleosome positioning

M Hafiz Rothi (Department of Molecular, Cellular, and Developmental Biology, University of Michigan), Shriya Sethuraman (Department of Computational Medicine and Bioinformatics, University of Michigan), Jakub Dolata (Department of Gene Expression, Adam Mickiewicz University Poznan), Andrzej T Wierzbicki (Department of Molecular, Cellular, and Developmental Biology, University of Michigan)

Abstract:
In Arabidopsis thaliana, noncoding RNAs (ncRNAs) are produced in the RNA-directed DNA methylation (RdDM) pathway to target transposable elements for silencing. Previously, we had identified the SWI/SNF chromatin remodeling complex to possibly function downstream of RNA Polymerase V (Pol V) transcription. Here, we show that Pol V positions nucleosomes through the SWI/SNF chromatin remodeling complex. The positioned nucleosomes are enriched with DNA methylation and histone 3 lysine 9 di-methylation (H3K9me2). IDN2, an RNA-binding protein functioning downstream of Pol V, connects short interfering RNA (siRNA) and long noncoding (lncRNA) to nucleosome positioning. We established that DNA methylation is preferentially enriched in linker regions genome-wide and that SWI/SNF is not required for DNA methylation in Pol V positioned nucleosomes. However, in DNA methyltransferase mutants, we observed a loss of nucleosome signal in differentially methylated regions. We propose a model where RdDM pathway directs nucleosome positioning through DNA methylation.

Keywords: noncoding RNA, chromatin, gene regulation

121. Global analysis of protein synthesis in Flavobacterium johnsoniae reveals the use of Kozak-like sequences in diverse bacteria

Bappaditya Roy (Department of Microbiology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), William D. Baez (Department of Physics, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Zakkary A. McNutt (Department of Microbiology, Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Elan A. Shatoff (Department of Physics, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Ralf Bundschuh (Department of Physics, Department of Chemistry and Biochemistry, Division of Hematology, Department of Internal Medicine, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Kurt Fredrick (Department of Microbiology, Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in F. johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine-Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position -3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of E. coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved beta-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.

Keywords: Kozak sequences, Initiation

122. Structure of bPNA triplex hybrid determined by x-ray crystallography

Sarah Rundell (Chemistry and Biochemistry, The Ohio State University), Prof. Dennis Bong (Chemistry and Biochemistry, The Ohio State University)

Abstract:
Our lab has identified oligo T/U recognition with melamine in triplex structures, however, we still lack detailed structural information. Our goal is to crystallize DNA and RNA triplex hybrids with small molecules and peptides and elucidate the structures via X-ray analysis. The structures will be used in rational design of DNA/RNA binding molecules which may extend into nucleotide-based therapeutics. Following established literature methods, we designed DNA/RNA templates with oligo T/U stretches as binding sites and incorporated known crystal packing sequences. Large scale synthesis of the peptide (bPNA) and the small molecule (t4M) was accomplished. The binding between the nucleic acids and melamine based molecules was confirmed by electrophoretic mobility shift assay (EMSA) and the annealed molecules were screened for crystals using sitting drop vapor diffusion. Potential crystals were found in both DNA and RNA screens which were confirmed by denaturing PAGE gels against their original templates. We describe herein our efforts and progress to date in screening crystallization conditions for X-ray diffraction.

Keywords: Crystallization, Triplex hybrids, bPNA

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

Alexander T Runyon (Department of Microbiology, Ohio State University), Tina M Henkin (Department of Microbiology, Ohio State University)

Abstract not available online - please check the printed booklet.

124. Functions of telomerase RNA (TR) core domains in its biogenesis and telomere maintenance in a medically relevant parasite, Trypanosoma brucei

Arpita Saha (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Sciences, College of Sciences and Health Professions, Cleveland State University, Cleveland), Abhishek Dey (Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223), Kausik Chakrabarti (Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223), Bibo Li (Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Sciences, College of Sciences and Health Professions, Cleveland State University, Cleveland)

Abstract not available online - please check the printed booklet.

125. Dynamic phosphorylation of yeast eIF4A drives changes in gene expression that couple protein synthesis with cell division

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

Abstract:
The eukaryotic translation initiation factor 4A (eIF4A) supports mRNA recruitment to the ribosomal preinitiation complex through the activity of its conserved DEAD-box RNA helicase motif. Interestingly, in plants eIF4A is differentially phosphorylated near the DEAD motif during various phases of the cell cycle by CDKA, lowering its translational activity during mitosis (Bush et al., 2016). The same Threonine residue of yeast eIF4A (T146) is present in a consensus CDK1/CDKA/Cdc28 motif, and is highly phosphorylated in large-scale studies (Soulard et al., 2010). To further dissect the molecular function of eIF4A, we are analyzing specific in vitro and in vivo changes that take place due to phosphorylation of eIF4A under normal conditions and in response to stress. We have mutated eIF4A residue T146 to phosphodeficient (Ala) and phosphomimetic (Glu, Asp) forms, and analyzed the effects of these mutations on growth and translation. Our data indicate that the phosphorylation status of eIF4A sensitizes yeast cells to membrane stressors in a manner affected by eIF4A•eIF4B coupling, and that constitutive phosphorylation of this residue is lethal due to arrest prior to cell division. These studies illustrate phenotypic changes conferred by modification of a general translation initiation factor in response to environmental conditions.

References:
1. Soulard, A., Cremonesi, A., Moes, S., Schütz, F., Jenö, P., & Hall, M. N. (2010) Molecular biology of the cell, 21(19), 3475-3486.
2. Albuquerque, C. P. & Zhou, H. et al. (2008) Molecular & Cellular Proteomics, 7(7), 1389-1396.
3. Helbig, A. O., Rosati, S., Pijnappel, P. W., van Breukelen, B., Timmers, M. H., Mohammed, S., ... & Heck, A. J. (2010) BMC genomics, 11(1), 685.
4. Bush, M. S., Pierrat, O., Nibau, C., Mikitova, V., Zheng, T., Corke, F. M., ... & Doonan, J. H. (2016) Plant physiology, 172(1), 128-140.
5. Zhou, F., Walker, S. E., Mitchell, S. F., Lorsch, J. R., & Hinnebusch, A. G. (2014)JBC, 289(3), 1704-1722.

Keywords: Translation initiation, eIF4A, Phosphorylation

126. Role of SR proteins in mRNP packaging and Nonsense Mediated Decay

Manu Sanjeev (Department of Molecular Genetics, The Ohio state University), Guramrit Singh (Department of Molecular Genetics, The Ohio state University)

Abstract:
After their transcription, mRNAs in cells are packaged into large rod-shaped ribonucleoprotein (RNP) complexes with sizes comparable to the small ribosome subunit [1]–[3]. This packaging is crucial for genomic stability, and presumably for mRNA export and translation. The exon junction complex (EJC) is a major mRNP component that plays important roles in multiple levels of gene regulation [4]. The EJC interacts with a variety
of peripheral proteins including several Serine-Arginine-rich (SR) proteins in an RNAindependent manner to form mRNPs that protect RNA from nuclease digestion [5]. Recent work from our lab has shown that EJCs are dynamic and undergo remodeling in the cytoplasm into smaller complexes that contain the EJC core but lack SR proteins [6]. The removal of SR proteins from mRNPs also reduces the Nonsense Mediated Decay (NMD) efficiency of target mRNAs [6], [7]. My overarching goal is to understand how different SR and SR-like proteins assemble on the periphery of the EJC core to form the mRNP in the nucleus, to identify the mechanism by which SR proteins are evicted from mRNPs in the cytoplasm to cause its remodeling, and to determine the effect of mRNP packaging on EJC dependent processes like mRNA export and NMD. Here, I show preliminary results that a major SR protein SRSF1 can be chemically crosslinked with the peripheral EJC factor RNPS1, suggesting a mode of SR protein interaction with the EJC. Further, overexpression of SR protein kinase SRPK1, but not its kinase-dead mutant version, slows down the NMD of a reporter RNA. This suggests that SRPK1 potentially plays a role in the remodeling of mRNPs in the cytoplasm, presumably by phosphorylation and eviction of SR proteins from mRNPs. To understand how SRSF1-RNPS1-EJC nexus
affects NMD efficiency of mRNPs, I have developed a triose phosphate isomerase (TPI)-based reporter mRNAs as described before [8], which contain enhanced SR protein recruitment signals. These reporters undergo NMD with increased efficiency as compared to a normal NMD reporter RNA. I am currently testing if knockdown of SR proteins and RNPS1 slows down the NMD of this reporter. Overall, this study provides insights into three important aspects of gene regulation: (i) how EJC and SR proteins interact to form
mRNPs in the nucleus, (ii) how mRNP remodeling occurs in the cytoplasm, and (iii) how changes in mRNP composition affect NMD efficiency.

References:
[1] G. Singh, G. Pratt, G. W. Yeo, and M. J. Moore, “The Clothes Make the mRNA: Past and Present Trends in mRNP Fashion,” Annu Rev Biochem, vol. 84, pp. 325–354, 2015.
[2] S. Adivarahan et al., “Spatial Organization of Single mRNPs at Different Stages of the Gene Expression Pathway,” Molecular Cell, vol. 72, no. 4, pp. 727-738.e5, Nov. 2018.
[3] M. Metkar et al., “Higher-Order Organization Principles of Pre-translational mRNPs,” Molecular Cell, vol. 72, no. 4, pp. 715-726.e3, Nov. 2018.
[4] L. A. Woodward, J. W. Mabin, P. Gangras, and G. Singh, “The exon junction complex: a lifelong guardian of mRNA fate: EJC: assembly, structure, and function,” WIREs RNA, vol. 8, no. 3, p. e1411, May 2017.
[5] G. Singh et al., “The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus,” Cell, vol. 151, no. 4, pp. 750–764, Nov. 2012.
[6] J. W. Mabin et al., “The Exon Junction Complex Undergoes a Compositional Switch that Alters mRNP Structure and Nonsense-Mediated mRNA Decay Activity,” Cell Reports, vol. 25, no. 9, pp. 2431-2446.e7, Nov. 2018.
[7] I. Aznarez et al., “Mechanism of Nonsense-Mediated mRNA Decay Stimulation by Splicing Factor SRSF1,” Cell Reports, vol. 23, no. 7, pp. 2186–2198, May 2018.
[8] J. P. Gudikote, J. S. Imam, R. F. Garcia, and M. F. Wilkinson, “RNA splicing promotes translation and RNA surveillance,” Nat Struct Mol Biol, vol. 12, no. 9, pp. 801–809, Sep. 2005.

Keywords: Nonsense Mediated Decay, SR proteins, mRNP packaging

127. Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

Eric M. Patrick (Institute for Quantitative Health Sciences and Engineering, Michigan State University, East Lansing, MI), Joseph Slivka, Bramyn Payne, Matthew J. Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing MI), Jens C. Schmidt (Institute for Quantitative Health Sciences and Engineering, Department of Physics and Astronomy, Michigan State University, East Lansing MI)

Abstract:
Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. After release of multiple telomeric repeats from telomerase, we observed folding of product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

Keywords: Telomerase catalysis, optical tweezers

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

Stefani Schmocker (Department of Chemistry and Biochemistry, Kent State University), Kumudie Jayalath (Department of Chemistry and Biochemistry, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)

Abstract not available online - please check the printed booklet.

129. Identification of plant TRMT1 genes using fluorescent primer extension in yeast

Nichlas Sebree (Chemistry and Biochemistry, Northern Kentucky University), Sarah Spigelmyer (Chemistry and Biochemistry, Northern Kentucky University), Holly M. Funk (Chemistry and Biochemistry, Northern Kentucky University), Maggie Thomas (Chemistry and Biochemistry, Northern Kentucky University), Ross Skeens (Chemistry and Biochemistry, Northern Kentucky University), Michael P. Guy (Chemistry and Biochemistry, Northern Kentucky University)

Abstract not available online - please check the printed booklet.

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

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

Abstract not available online - please check the printed booklet.

131. LC-MS identification of riboswitch ligands

Jacob Sieg (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802), Philp Smith (Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802), Andrew Patterson (Department of Biochemistry and Molecular Biology, Huck Institutes of the Life Sciences Pennsylvania State University, University Park, PA, 16802), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, 16802)

Abstract not available online - please check the printed booklet.

132. 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.

133. Modeling microcephaly in patient iPS cell-derived brain organoids with U4atac snRNA mutations

Jagjit Singh (Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195), Filomena Pirozzi (Seattle Childrens Research Institute, Center for Integrative Brain Research, Seattle, WA 98101), Luke A. Bury (Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106), Anthony Wynshaw-Boris (Department of Genetics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106), Richard A. Padgett (Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195)

Abstract not available online - please check the printed booklet.

134. Mechanistic framework for understanding ribosome sliding on homopolymeric A stretches

Tyler J. Smith (Department of Chemistry - University of Michigan ), Kristin S. Koutmou (Department of Chemistry - University of Michigan )

Abstract:
Translation speed and fidelity can be modulated by a variety of factors including mRNA structure, codon usage, and peptide identity. The synthesis of positively charged peptides is thought to cause the ribosome to stall because of interactions between the cationic nascent peptide chain and the negatively charged ribosome exit tunnel. However, emerging evidence suggests that strong charge-charge interactions between the ribosome and nascent chain may be insufficient to explain reduced protein output during the translation of positively charged proteins. Recent work demonstrated that the ribosome translates mRNAs containing iterative lysine AAA and AAG codons differently, losing frame and undergoing non-canonical ribosome movement (sliding) on homopolymeric AAA, but not AAG, sequences. Here, we investigate the contributions of peptide charge, mRNA sequence, mRNA modification, and EF-G to ribosome sliding events using a reconstituted in vitro E. coli translation system. We demonstrate that the ribosome slides into the -2, -1 and +1 frames on poly(A) sequences, and are working to characterize the kinetics of each of these shifts. Our data indicate that ribosome sliding depends on EF-G, much as in -1 programmed frameshifting events. Furthermore, we find that codon modification status contributes to the degree of sliding; the incorporation of a single m6A into AAA codons modulates sliding. Moreover, neither ribosome stalling and sliding on homopolymeric (A) sequences appear to strictly require the translation of a positively charged peptide sequence – sliding and slow translation still occur when tRNALys is mischarged with Val (Val-tRNALys). Our data provide a mechanistic framework for understanding how protein synthesis is modulated by mRNA sequences encoding positively charged amino acids.

Keywords: Translation, Frame-loss, Sliding

135. Two distinct mechanisms of CYb and COIII mRNA editing regulation in Trypanosoma brucei

Joseph T. Smith (Department of Microbiology and Immunology, University at Buffalo), Brianna Tylec (Department of Microbiology and Immunology, University at Buffalo), Eva Dolezelova (Institute of Parasitology, Biology Centre Czech Academy of Science), Alena Zikova (Institute of Parasitology, Biology Centre Czech Academy of Science), Laurie K. Read (Department of Microbiology and Immunology, University at Buffalo)

Abstract not available online - please check the printed booklet.

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

Sarah M. Spigelmyer (Chemistry and Biochemistry, Northern Kentucky University), Maggie Thomas (Chemistry and Biochemistry, Northern Kentucky University), Holly M. Funk (Chemistry and Biochemistry, Northern Kentucky University), Michael P. Guy (Chemistry and Biochemistry, Northern Kentucky University)

Abstract not available online - please check the printed booklet.

137. 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), Zhonghua Liu (Department of Pathology), Tsan Sam Xiao (Department of pathology), Eckhard Jankowsky (Center for RNA Science and Therapeutics)

Abstract:
The DEAH/RHA helicase DHX36 is linked to DNA and RNA G-quadruplex structures and AU-rich element (ARE) - mediated mRNA deadenylation and decay. In vitro, DHX36 resolves DNA and RNA G-quadruplex structures and unwinds duplexes. However, the molecular basis for RNA structure remodeling 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 the non-hydrolysable ATP analog AMP-PNP and in complex with ADP. The overall structures of mDHX36 closely resemble structures for previously reported bovine and fly DHX36. However, we observe differences to the previous structures, consistent with conformational changes that accompany the different stages of the ATP binding and hydrolysis and the RNA remodeling cycle.
Biochemical experiments demonstrate that at saturating DHX36 concentration, unwinding rate constants are four-fold higher for quadruplexes than for duplex structures. Besides, 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. Besides, we demonstrate that two previously uncharacterized DHX36 regions, a 5’-β-hairpin in the helicase core, and the OB-fold domain are essential for the ability of DHX36 to bind and remodel both, quadruplex and duplex structures. We also show that DHX36 utilizes all nucleotides, but not indiscriminately. Remarkably, ATP at physiological levels negatively regulates DHX36 unwinding of duplexes and positively regulates DHX36 remodeling of quadruplexes. This indicates that the nucleotide selectivity of DHX36 influences its RNA substrate selectivity and vice versa. Collectively, our data reveal that the auxiliary domains of DHX36 work in conjunction with the helicase core to remodel RNA structures.

Keywords: Helicase, G-quadruplexes, RNA

138. Transcriptome-wide high-throughput mapping of protein-RNA occupancy profiles using an integrated phase separation and crosslinking approach

Mansi Srivastava (BioHealth Informatics,School of Informatics and Computing, IUPUI), Rajneesh Srivastava (BioHealth Informatics,School of Informatics and Computing, IUPUI), Sarath Chandra Janga (BioHealth Informatics,School of Informatics and Computing, IUPUI)

Abstract:
RNA-binding proteins (RBPs) associate physically with RNA to regulate multiple biological processes such as capping, splicing, polyadenylation, export and localization in a dynamic manner (1). Several transcriptome- wide approaches that determine the binding pockets of RBPs on its target RNA rely largely on the capture of polyadenylated RNAs (2,3). Recently a phase separation strategy using Trizol has emerged as a robust technology that has expanded the identification of RBP binding sites independent of the poly-A capture (4,5). Here, we present POP-seq (Protein-occupancy profile-sequencing) that employs UV and formaldehyde crosslinking to enhance the cellular RBP-RNA interactions followed by multi-step phase separation strategy using Trizol to yield RBP bound protected RNA regions of 30-50 bases. POP-seq libraries (in replicates) were sequenced to generate ~20 million of reads each, in Non cross-linked (NCL), formaldehyde cross-linked (FCL) and UV cross-linked (UCL) K562 cells respectively. We implemented a Next Generation Sequencing (NGS) pipeline to facilitate the analysis of the POP-seq data which included quality control, read alignment followed by peak calling, resulting in the identification of 321039, 289025 and 321541 unique peaks in NCL, FCL and UCL samples respectively, with ~35% peak overlap across the three protocols and ~85% of total peaks had length below 50 bp. We annotated these peaks onto human reference genome (hg38) and found that ~93.6% of the peaks were mapped to protein-coding genes followed by ~2.8% to lincRNA, ~1.7% to miRNA and ~1.9 to other RNA genes. Comparison of POP-seq peaks with the ENCODE eCLIP profile of RBPs in K562 cells revealed a high confident protein-RNA interaction capture with ~70% precision. Overall, this study illustrates a novel and cost-effective methodology to precisely capture the RNA-protein interaction sites with increased signal to noise ratio. Thus, application of POP-seq to additional cell lines originating from different tissue types would provide the first comprehensive understanding of the global occupancy profiles of RBPs across tissue types.

References:
1. Hentze, M. W., Castello, A., Schwarzl, T. & Preiss, T. A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell Biol. 19, 327–341 (2018).
2. Baltz, A. G. et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol. Cell 46, 674–690 (2012).
3. Schueler, M. et al. Differential protein occupancy profiling of the mRNA transcriptome. Genome Biol. 15, R15 (2014).
4. Queiroz, R. M. L. et al. Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS). Nat. Biotechnol. 37, 169–178 (2019).
5. Trendel, J. et al. The Human RNA-Binding Proteome and Its Dynamics during Translational Arrest. Cell 176, 391-403.e19 (2019).

Keywords: RNA binding proteins, Protein occupancy profile, Phase separation

139. Functional annotation of the human protein-RNA interaction network via transcriptome-wide pooled CRISPR/Cas9 screens to edit the RBP binding sites

Rajneesh Srivastava (Department of BioHealth Informatics, School of Informatics and Computing, IUPUI), Aarthi Ramakrishnan (Department of BioHealth Informatics, School of Informatics and Computing, IUPUI), Seyedsasan Hashemikhabir (Department of BioHealth Informatics, School of Informatics and Computing, IUPUI), Sasank Vemuri (Department of BioHealth Informatics, School of Informatics and Computing, IUPUI), Quoseena Mir (Department of BioHealth Informatics, School of Informatics and Computing, IUPUI), Sarath Chandra Janga (Department of BioHealth Informatics, School of Informatics and Computing, IUPUI)

Abstract:
RNA-binding proteins (RBPs) bind to RNAs at specific recognition sites and decide the post-transcriptional fate of the targets within the cell. Although existing resources such as ENCODE, have documented millions of binding sites (BS) of 123 RBPs across cell types, the biological significance of these BS, is still lacking. Recently, we built SliceIt (https://sliceit.soic.iupui.edu/), an in silico library of ~4.8 million unique sgRNA (or single guide RNA) targeting all possible BS of 123 RBPs from eCLIP experiments in HepG2 and K562 cell lines from ENCODE project. It facilitates the easy navigation of RBP binding sites along with sgRNAs, SNP and GWAS annotations enabling researchers to conduct CRISPR experiments for studying the phenotypes associated with RBP binding sites. We designed a massive CRISPR/ Cas9 pooled screen for transcriptome-wide perturbation of RBP binding locations in HepG2 and K562 cells. We selected 23013 sgRNAs targeting 14256 unique binding (~52% exonic and ~48% intronic) sites of 108 RBPs in 4111 genes (including 254 lncRNA genes) with 89% of the BS targeted by atleast 2-8 sgRNAs. We also included ~134 control non-targeting sgRNA library in the screen. Pooled screening experiments were accomplished in HepG2 and K562 cell lines in replicates, resulting in the generation of ~50 million sgRNA reads (with ~99.8 correct sgRNAs representation) per replicate per cell line using Illumina Hi-seq followed by deep RNA-sequencing to generate 200 million reads per cell line of control and edited cells respectively. In a preliminary analysis of this dataset, we observed 68,323 and 87,319 exons were found exhibiting ±1.5 fold change in expression between control and edited HepG2 and K562 cells respectively. We anticipate this resource to be the first genome-wide CRISPR screen that will facilitate understanding and mapping the functional protein RNA interaction networks across human cell types.

References:
1. Van Nostrand, E. L. et al. Nature methods, doi:10.1038/nmeth.3810 (2016).
2. Dixit, A. et al. Cell. doi:10.1016/j.cell.2016.11.038 (2016).
3. Vemuri S et al. Methods. doi: 10.1016/j.ymeth.2019.09.004 (2019).
4. Datlinger, P. et al. Nature methods. doi:10.1038/nmeth.4177 (2017).
5. Rauscher, B. et al. Nucleic Acids Research. doi:10.1093/nar/gkw997 (2017).
6. Davis, C. A. et al. Nucleic Acids Research. doi:10.1093/nar/gkx1081 (2018).

Keywords: RNA binding proteins, in silico guideRNA library, pooled CRISPR screening

140. A ligand gated strand displacement mechanism for ZTP riboswitch transcription control

Eric J. Strobel (Department of Chemical and Biological Engineering, Northwestern University), Luyi Cheng (Department of Chemical and Biological Engineering, Northwestern University), Katherine E. Berman (Department of Chemical and Biological Engineering, Northwestern University), Paul D. Carlson (Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University), Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University)

Abstract not available online - please check the printed booklet.

141. Exploring the role of adr-2 in the innate immune response of C. elegans

Halle D. Stump (Biotechnology Professional Science Masters Program, Indiana University), Pranathi Vadlamani (Medical Sciences Program, Indiana University School of Medicine-Bloomington), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine-Bloomington)

Abstract not available online - please check the printed booklet.

142. Structural Dynamics of HIV ESS2p RNA Element

Andrew Sugarman (Chemistry, Case Western Reserve University), Liang-Yuan Chiu (Chemistry, Case Western Reserve University), NaShea Kendrick (Chemistry, Oglethorpe University), William Ford (Chemistry, Case Western Reserve University), Alexander Hansen (CCIC NMR Facility, The Ohio State University), Blanton Tolbert (Chemistry, Case Western Reserve University)

Abstract:
ESS2p is an exonic splicing silencer significant for its role in HIV replication and the virus’s ability
to produce the vital Tat protein. The 43 nucleotide RNA molecule is located downstream of the 3’
splice site A3 on the HIV-1 transcript, where it is typically known to interact with hnRNP H.
Recruitment of this protein is necessary for inhibition of the spliceosome1, and the structure of
ESS2p is responsible for its binding interaction with hnRNP H. In this investigation we report the
solution structure of ESS2p and an assessment of its binding interactions with hnRNP H, and another
vital protein hnRNP A1. NMR techniques including NOESY and RDC experiments were conducted
to evaluate hydrogen bonding, base pairing, and sugar pucker data for ESS2p. Small Angle X-ray
Scattering (SAXS) was also performed to provide a molecular density map of the molecule. Data
from these experiments were combined in XPLOR and AMBER, where a series of molecular
dynamics simulations were carried out to obtain a robust solution structure. In addition, we report
the results of ITC experiments that reveal ESS2p is capable of strong binding interactions with
hnRNP A1 through the apical loop AG sequence. On the other hand, the G-tract sequence of ESS2p
is buried in the base-paired region, as a result, hnRNP H did not show canonical binding profile in
the ITC experiment, indicating that in vivo, hnRNP H likely needs need assistance of other proteins
to unwind the G-tract base pair region of ESS2p to allow recognition.

Keywords: Dynamics, NMR , Simulations

143. Comparative Analysis of ATP Dissociation from Rok1p Variants of in the presence of RNA

Kathryn Sutter (Biochemistry Allegheny College), Amanda DiLoreto, Lisa Yoder, Zachary Iezzi, and Ivelitza Garci (Biochemistry Allegheny College)

Abstract:
Superfamily 2 helicases (SF2) are ubiquitous and often associated with RNA metabolism. For example, 14 DEAD-box proteins, members of the SF2 family, play a vital role in Saccharomyces cerevisiae ribosome biogenesis. These ATP-dependent unwindases use conformational changes to guide and regulate RNA-RNA, RNA-protein, and/or protein-protein interactions during rRNA processing. Structurally, DEAD-box proteins have 2 Rec-A like domains as well as an N-terminal (NTD) and/or C-terminal (CTD) domains. Peripheral domains are hypothesized to facilitate RNA binding and/or protein scaffolding. To investigate the role of the peripheral domains on ATP binding, variants of Rok1p (Rok1p-ΔCTD, Rok1p-ΔNTD, Rok1p-ΔNTD-ΔCTD) were constructed and biochemically analyzed. Specifically, ATP dissociation rates were measured in the presence of RNA using stopped-flow fluorescence spectroscopy. The dissociation of ATP yielded a bi-exponential regression indicating a two-step mechanism (k_fast and k_slow). The proposed mechanism involves a conformational change (k_slow) before TNP-ATP release (k_fast). Measured rates were comparatively analyzed as a function of temperature. This study illustrates that RNA binding, in the presence of NTD, generally increases the enthalpic contribution while decreasing the entropic contribution for k_fast. This observation is similar for constructs that lack the NTD when comparing k_slow. Interestingly, the presence of the NTD affects the conformation change needed to release TNP-ATP resulting in an enthalpically driven process.

Keywords: DEAD-box protein

144. mRNA splicing is a marker of molecular pathology and response to therapeutics in myotonic muscular dystrophy patients and mouse models

Matthew Tanner (Medical Scientist Training Program, University of Rochester Medical Center), Johanna Hamel (Department of Neurology, University of Rochester Medical Center), Katy Eichinger (Department of Neurology, University of Rochester Medical Center), Jeanne Dekdebrun (Department of Neurology, University of Rochester Medical Center), Charles Thornton (Department of Neurology, University of Rochester Medical Center)

Abstract not available online - please check the printed booklet.

145. High-resolution integrative analysis to define putative cytoplasmic RNA recapping sites in mammalian cells

Trinh T Tat (Department of Cardiovascular Sciences - Houston Methodist Research Institute), Dongyu Zhao (Department of Cardiovascular Sciences - Houston Methodist Research Institute), Kaifu Chen (Department of Cardiovascular Sciences - Houston Methodist Research Institute), Daniel L Kiss (Department of Cardiovascular Sciences - Houston Methodist Research Institute)

Abstract:
Multiple lines of evidence showed cytoplasmically-recapped transcripts undergo N7 methylation to generate the same m7G caps found on nuclear-capped RNAs. This mechanism remains poorly understood although several studies show that these recapped targets are involved in certain key biological processes, such as cell cycle, cellular stress response, and development. Many questions regarding cytoplasmic capping remain unanswered including how the cell identifies and selects cytoplasmic capping sites. By integrating public CAGE and ChIP-Seq datasets that mark transcription start sites (TSSs) (RNA polymerase II and 8 chromatin marks) and gene bodies (2 chromatin marks) we have developed a high-resolution integrative analysis to precisely define the locations of non-TSS-linked caps. These m7G caps are candidate cytoplasmic RNA capping sites (non-traditional 5’ m7G caps along gene bodies) in mammalian cells. 5’ RACE will be using to experimentally validate some of these putative cytoplasmic capping sites predicted by this bioinformatic approach. In addition, nanopore long read direct RNA sequencing is expected to elucidate the targets of cytoplasmic recapping and may greatly aid in defining the site selection rules for cytoplasmic capping.

Keywords: cytoplasmic recapping, CAGE, TSS

146. S4-like domain regulates binding of RsuA to 16S rRNA

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

Abstract:
The Ribosomes are ribonucleoprotein complexes that are involved in protein biosynthesis in all living organisms. Bacterial ribosome biogenesis is a complex process that is dependent on the integration of several cellular events, such as ribosomal RNA transcription, ribosome assembly, RNA processing and post-transcriptional and post-translational modification of ribosomal RNA and proteins, respectively. Even though the ribosome assembly is well studied, the impact of post-transcriptional rRNA modification enzymes and their respective modifications on ribosomal assembly is not completely understood. Pseudouridine synthase, RsuA, is responsible for the formation of pseudouridine at position 516. Protein RsuA is composed of two domains including the C-terminal catalytic domain and the N-terminal S4-like domain. My study focuses on understanding the importance of the N-terminal S4 like domain for RsuA binding and its function. Our filter binding assays show decrease in binding affinity in the absence of the S4-like domain. However, unlike the full length protein, the truncated RsuA protein is capable of binding to 16S rRNA at much higher protein concentrations suggesting an auto inhibitory role for the S4-like domain.

Keywords: Ribosome biogenesis, RNA modification enzyme, Pseudouridine

147. Stem-loop antisense RNA and DNA exclude mismatched target sequences

Samuel D. Stimple (Department of Chemical and Biomolecular Engineering, The Ohio State University), Elizabeth Traverse (Department of Chemistry and Biochemistry, The Ohio State University), Si Yu Tan (Department of Chemistry and Biochemistry, The Ohio State University), Valerie Zaino (Department of Chemistry and Biochemistry, The Ohio State University), Richard A. Lease (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

148. Single-cell transcriptomics reveal distinct fetal like cellular states during murine liver regeneration

Ullas Valiya Chembazhi (Department of Biochemistry, University of Illinois at Urbana-Champaign), Sushant Bangru (Department of Biochemistry, Cancer Center@Illinois, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, Cancer Center@Illinois, 3Carl R. Woese Institute of Genomic Biology, University of Illinois at Urbana-Champaign)

Abstract not available online - please check the printed booklet.

149. Towards understanding the role of circRNAs in intracellular signaling pathways

Megan Van Horn (Carnegie Mellon University Department of Chemistry), Anna M. Kietrys (Carnegie Mellon University Department of Chemistry)

Abstract:
Non-coding RNAs like microRNAs (miRNAs) and circular RNAs (circRNAs) have recently been shown to play important roles in gene expression and regulation through their interactions with proteins and other RNAs. CircRNAs have been described as encoding functional proteins in cells and undergoing translation, and only recently were proven to influence neuron sensitivity and brain functionality. Interestingly, many circRNAs have been found in exosomes. These small extracellular vesicles are present in eukaryotic fluids and contain various bioactive cargoes, like proteins and RNAs, that are unique to the phenotype of their parent cell. Exosomes are already being studied as a potential RNA drug delivery system due to their modular nature, ability to pass through the blood-brain barrier, bioavailability, and biocompatibility.

Consequently, coupled with evidence that exosomes are involved in cell-cell transduction of biologically active molecules, exosomes become a prime vehicle by which to investigate the role of circRNAs in the intracellular signaling pathway. We will analyze published sets of RNAseq data to identify circRNAs enriched in both exosomal and neuronal RNAs pools. These selected circRNAs of interest will be isolated from exosomes enriched from mouse blood and from mouse brains and investigated in order to compare their sequence, structure, and epitranscriptomic profile. Finally, we will infuse exosomes with labeled circRNAs of interest and monitor their biological potential as information carriers. Our work will help to understand mechanisms of intracellular signaling and role of circRNA in this process.

Keywords: circRNA, exosome, intracellular signaling

150. 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), McKenzie Clapp (Center for RNA Science and Therapeutics, Case Western Reserve University), Andrea Putnam (Center for RNA Science and Therapeutics, Case Western Reserve University), Eckhard Jankowsky (Center for RNA Science and Therapeutics, Case Western Reserve University)

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 DDX3X association with translation initiation factors and stress granules. While interactions between K7 and DDX3X are thought to disrupt immune signaling pathways, the impact of K7 on DDX3X’s biochemical activities and roles in RNA metabolism is unknown. We show that K7 inhibits both the ATP-dependent RNA duplex unwinding and ATP hydrolysis activities of DDX3X. We further show that K7 impairs the formation of recombinant DDX3X phase-separated droplets in vitro, suggesting that K7 may impact the formation of DDX3X-containing stress granules within cells. As both DDX3X’s ability to resolve RNA secondary structure and its association with stress granules influence DDX3X’s role in translation initiation, characterizing the interaction between K7 and DDX3X provides mechanistic insight into strategies by which viruses target DDX3X to alter RNA metabolism in the host.

Keywords: DEAD-box RNA helicase , DDX3X

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

Hongzhi Wang (College of Pharmacy, The Ohio State University), Zhefeng Li (College of Pharmacy, The Ohio State University), Hongran Yin (College of Pharmacy, The Ohio State University), Congcong Xu (College of Pharmacy, The Ohio State University), Daniel W.Binzel (College of Pharmacy, The Ohio State University), Peixuan Guo (College of Pharmacy, The Ohio State University)

Abstract not available online - please check the printed booklet.

152. Nuclear speckle protein SON's role in transcription regulation at an inducible reporter gene array

Melissa J. Ward (Department of Biological Sciences, Wright State University), Paula A. Bubulya (Department of Biological Sciences, Wright State University)

Abstract not available online - please check the printed booklet.

153. Dissecting the role of protein arginine methylation on translation initiation factor eIF1A

Rebecca L. Wegman (Department of Biological Sciences, University at Buffalo ), Skyler Kelly (Department of Biological Sciences, University at Buffalo ), Michael Langberg (Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center), Min-kui Luo (Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center), Sarah E. Walker (Department of Biological Sciences, University at Buffalo ), Michael C. Yu (Department of Biological Sciences, University at Buffalo )

Abstract:
Eukaryotic translation initiation is a vital process for proper expression of proteins within a cell. At the start of initiation, a pre-initiation complex, (PIC) binds to the capped 5’-end of the mRNA, and moves along the untranslated region using the anti-codon of the initiator tRNA to identify a proper AUG start codon. Previous work has shown that both N- and C- terminal tails of one eukaryotic initiation factor, eIF1A, provide critical interactions that stabilize the PIC in either an open conformation (conducive to scanning) or a closed conformation (inhibitory of scanning and supportive of initiation) (1,2). The balance between the open and closed conformations of the PIC is critical to proper start codon recognition and thus it is important to gain a better understanding of how this conformational change is regulated.

It is known that translation factors can be regulated through post-translation modifications, such as phosphorylation and methylation. Recently, we carried out bioorthogonal profiling to comprehensively identify substrates of yeast protein arginine methyltransferase Hmt1 in vivo, and this approach yielded eIF1A as a candidate. To validate our proteomic identification, we carried out an in vitro methylation assay to show that eIF1A can act as an Hmt1 substrate. Structural analysis of eIF1A suggests two potential sites of arginine methylation – amino acids 13 and 14 in the N-terminal tail. These two arginines reside within or adjacent to the signature RG/RGG box that is commonly found in Hmt1 substrates. To better understand how methylation of eIF1A can potentially affect its function, we generated a combination of substitution mutants of eIF1A (R to K or R to A) at these positions and determined their effects on fidelity of translation start codon usage relative to wild-type. Our data demonstrate that methylation of eIF1A decreases fidelity of start codon recognition as these mutations severely decrease usage of near-cognate start codons. Overall, our data suggest that eIF1A methylation stabilizes interactions of the N-terminal tail in a closed conformation of the PIC that is not conducive to scanning allowing arrest at near-cognate codons. This allows Hmt1 to fine tune the fidelity of start codon recognition for proper translation initiation.

References:
1) Fekete et al. (2007). N- and C-terminal residues of eIF1A have opposing effect on the fidelity of start codon selection. The EMBO Journal, 26(6), 106-1614.
2) Saini et al. (2010). Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNA_i^Met binding to the ribosome. Genes & Development, 24, 97-110.

Keywords: eIF1A, Methylation , Translation

154. RNA structure and dynamics act as a gate keeper of 3’ alternative splicing

Robb Welty (University of Michigan, Department of Chemistry), Nils Walter (University of Michigan, Department of Chemistry)

Abstract not available online - please check the printed booklet.

155. Maintenance of the polypeptide exit tunnel marks a nucleoplasmic checkpoint in large ribosomal subunit assembly

Daniel Wilson (Biological Sciences, Carnegie Mellon University), Amber LaPeruta (Biological Sciences, Carnegie Mellon University), Yu Li (Peking University), Ning Gao (Peking University), John L. Woolford Jr. (Biological Sciences, Carnegie Mellon University)

Abstract not available online - please check the printed booklet.

156. Synthesis of a fluorescent cytidine analogue

Ashlynn Winkler (Department of Chemistry at SIUE), Mina Sumita (Department of Chemistry at SIUE)

Abstract not available online - please check the printed booklet.

157. Using deep learning networks to solve the RNA design problem

Ciara M. Witt (Chemistry, University of Michigan), Aaron T. Frank (Chemistry & Biophysics, University of Michigan)

Abstract not available online - please check the printed booklet.

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

Austin Woodard (Department of Chemistry, Southern Illinois University Edwardsville), Sun Jeong Im (Department of Chemistry, Southern Illinois University Edwardsville), Andrew Riley (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) outbreaks pose serious health threats in the United States and across the globe today. These outbreaks are caused by pathogenic E. coli strains but could be prevented by detection of the harmful E. coli via a 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, and selection progress cannot be monitored in real time 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. Furthermore, a consensus sequence generated from multiple DNA aptamer sequences that bind strongly to E. coli can be utilized in future studies of related biosensors.

Keywords: SPR-SELEX, DNA Aptamer, Biosensor

159. EPRS Is a Critical Translational Control Factor in Cardiac Fibrosis

Jiangbin Wu (Aab Cardiovascular Research Institute, URMC), Kadiam C. Venkata Subbaiah (Aab Cardiovascular Research Institute, URMC), Feng Jiang (Department of Biochemistry and Biophysics, URMC), Peng Yao (Aab Cardiovascular Research Institute & Department of Biochemistry and Biophysics. URMC)

Abstract not available online - please check the printed booklet.

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

Congcong Xu (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; College of Medicine, Dorothy M. Davis Heart and Lung Research Institute ), Kaiming Zhang, Shanshan Li (Departments of Bioengineering, Microbiology and Immunology, and James H. Clark Center, Stanford University, Stanford, CA 94305 USA), Hongran Yin, Zhefeng Li, Zhen Zheng, Zhouxiang Ji, Sijin Guo, (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; College of Medicine, Dorothy M. Davis Heart and Lung Research Institute ), Alexey Krasnoslobodtsev (Department of Physics, University of Nebraska at Omaha, Omaha, NE 68182 USA; Nanoimaging Core Facility, Office of Vice-Chancellor for Research, University of Nebraska Medical Center, Omaha, NE 68198), Wah Chiu (SLAC National Accelerator Laboratory, Stanford University, Menlo Park, CA 94025 USA), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry; College of Medicine, Dorothy M. Davis Heart and Lung Research Institute )

Abstract not available online - please check the printed booklet.

161. De Novo Nucleic Acid Design Using Variational Autoencoder

Ziqiao Xu (Department of Chemistry and Biophysics at University of Michigan), Aaron Frank (Department of Chemistry and Biophysics at University of Michigan)

Abstract not available online - please check the printed booklet.

162. Tetrahelical monomolecular architecture of nucleic acids probed by NMR

Mengkun Yang (Department of Chemsitry and Biochemistry, The Ohio State University), Levan Lomidze (Institute of Biophysics, Ilia State University, Tbilisi 0162, Republic of Georgia), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Besik Kankia (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
We recently described a tetrahelical monomolecular architecture of nucleic acids that employs G (guanine)-quartets as a structural element. The monomeric units of the architecture, GGGTGGGTGGGTGGG (G3T) or its RNA analog, g3u, are folded in an intramolecular quadruplex with three G-quartets connected to each other by chain-reversal or propeller loops. In this architecture, the G3T or g3u monomers are stacked on each other forming an extraordinarily stable uninterrupted polymer. For instance, whereas the G3T monomer in 0.1 mM KCl melts at 55 °C, the molecule (G3T)2, consisting of six G-tetrads, melts at ~85 °C as a single cooperative unit. Based on thermodynamic and melting studies, we propose that the tetrahelical architecture consists of n G3T domains, (G3T)n, wherein the terminal G3 segments of adjacent G3T domains form G6-segments. Each G3T domain of the architecture is formed by zigzagging of G3-segments and T-loops, while the G6 segment is shared by adjacent domains and serves as a bridge between them. Here, we employ proton NMR to probe the solution structure of (G3T)2. Preliminary NMR data are consistent with formation of an uninterrupted homopolymer consisting of repetitive G-tetrads with strong structural symmetry. Specific guanine-to-inosine and DNA-to-RNA substitutions are underway, allowing detailed analysis of the (G3T)2 structure. The tetrahelical architecture has strong potential in DNA nanotechnology as a programmable alternative to a DNA duplex.

Keywords: tetrahelical, quadruplex, NMR

163. Creating Super-Aptamers with Modified Nucleobases for Biosensor

Adam Litterst (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:
Our main research interest is the development of a food safety biosensor that can detect foodborne pathogens by using DNA and RNA aptamers. These aptamers are evolved to have a high affinity and specificity to the target pathogen and are developed by a new SPR-SELEX, Surface Plasmon Resonance-Systematic Evolution of Ligands by Exponential enrichment, method. To do this, the aptamers were evolved to target Exchericha coli K12 and were tested for specificity with Bacillus subtilis. Using SPR-SELEX, we have developed a super aptamer that utilizes deoxyuridine, the DNA form of the RNA base uridine, along with the 3 other common dNTPs. Deoxyuridine was chosen to be used in this super aptamer for its role in structural conformations in RNA and is used in deoxy-form for its increased stability of DNA. The success of this project allows for more possibilities of super aptamers such as Pseudouridine in RNA aptamers.

Keywords: super-aptamers, E. coli, biosensor

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

Angela M Yu (Tri-Institutional Program in Computational Biology and Medicine, Weill Cornell Medicine; Department of Chemical and Biological Engineering, Northwestern University), Paul M. Gasper, Simi Kaur, Alan A. Chen (Department of Chemistry and the RNA Institute, University at Albany), Luyi Cheng (Interdisciplinary Biological Sciences Graduate Program, Northwestern University), Lien B. Lai, Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University), Kyle E. Watters (Department of Molecular and Cell Biology, University of California Berkeley), Eric J. Strobel, Julius B. Lucks (Department of Chemical and Biological Engineering, Northwestern University)

Abstract not available online - please check the printed booklet.

165. Use of single-molecule fluorescence co-localization spectroscopy to study archaeal RNase P assembly

Walter J. Zahurancik (Chemistry and Biochemistry, The Ohio State University), Khan L. Cox (Physics, The Ohio State University), Ila A. Marathe (Microbiology, The Ohio State University), Stella M. Lai, Lien B. Lai, Xiao Ma, Vaishnavi Sidharthan, Mark P. Foster (Chemistry and Biochemistry, The Ohio State University), Michael G. Poirier (Physics, The Ohio State University), Venkat Gopalan (Chemistry and Biochemistry, The Ohio State University)

Abstract:
RNase P is an essential enzyme that catalyzes the 5′ maturation of precursor (pre)-tRNAs in all three domains of life. The ribonucleoprotein (RNP) form of RNase P is comprised of a single catalytic RNase P RNA (RPR) and a variable number of RNase P proteins (RPPs): one in Bacteria, up to five in Archaea, and up to ten in Eukarya. Given its biochemical tractability in vitro and its homology to the eukaryotic enzyme, archaeal RNase P is a useful model for studying the role of proteins in multi-subunit RNPs. While a recent cryo-electron microscopy structure has provided critical insights into the subunit organization of a functional archaeal RNase P holoenzyme¹, the enzyme’s assembly pathway and its subunit composition en route to the final, higher-order RNP remain unclear. We recently reported that there are three kink-turns in the Pyrococcus furiosus (Pfu) RPR and used native mass spectrometry to show that up to three copies of L7Ae bind to the RNA². Here, we use single-molecule fluorescence (SMF) co-localization spectroscopy to validate the number of copies of L7Ae that bind to the Pfu RPR by monitoring the binding of Cy5-labeled Pfu L7Ae to immobilized, Cy3-labeled Pfu RPR. Step-wise changes in Cy5 fluorescence reflect the sequential association and dissociation of multiple L7Ae copies to a single RPR molecule. Our goal is to use this SMF co-localization spectroscopy approach to study the binding kinetics of the other RPPs and to gain insights into the hierarchy of archaeal RNase P assembly.

References:
1. Wan F, Wang Q, Tan J, Tan M, Chen J, Shi S, Lan P, Wu J, Lei M. (2019) Cryo-electron microscopy structure of an archaeal ribonuclease P holoenzyme. Nat. Commun., 10: 2617.
2. Lai LB, Tanimoto A*, Lai SM*, Chen W-Y, Marathe IA, Westhof E, Wysocki VH, Gopalan V. (2017) A novel double kink-turn module in euryarchaeal RNase P RNAs. Nucleic Acids Res., 45: 7432-7440. *joint second authors

Keywords: RNase P, Ribozyme, Single-molecule fluorescence

166. Folding Mechanism of An RNA Thermosensor from Listeria Monocytogenes

Huaqun Zhang (Biophysics Program, University of Michigan), Amos Nissley (Department of Chemistry, University of Michigan), Ian Hall (Department of Chemistry, University of Michigan), Sarah C. Keane (Biophysics Program and Department of Chemistry, University of Michigan)

Abstract not available online - please check the printed booklet.

167. Deciphering the biological function of tRNA methyltransferase 1 (TRMT1) using mouse models

Kejia Zhang (Department of Biology, Center for RNA Biology, University of Rochester), Prevost, Christopher (Department of Biology, Center for RNA Biology, University of Rochester), Fu, Dragony (Department of Biology, Center for RNA Biology, University of Rochester)

Abstract:
In all organisms, transfer RNAs (tRNA) undergo a series of post-transcriptional modifications by enzymes. One of the first discovered tRNA modification enzymes is tRNA methyltransferase 1(TRMT1), who catalyzes the formation of the dimethylguanosine (m2,2G) in numerous tRNAs. Mutations in the TRMT1 gene are the cause of autosomal-recessive intellectual disability (ARID) in the human population. However, the link between TRMT1 and neurodevelopment is still unclear. To understand the biological function of TRMT1 in mammals, we analyzed the functional consequences and phenotype by using TRMT1 knockout mice. We are able to knockout the expression of TRMT1 in all the organs and observe the loss of tRNA m2,2G modification. We observe that the TRMT1 KO mice exhibit the reduced body weight postweaning, smaller brain size and abnormal grip strength. Notably, we find that TRMT1 exhibits ubiquitous expression in the brain with enrichment in the hippocampus and neocortex. Moreover, compared to the wildtype mice, the TRMT1 KO mice show significantly reduced learning and spatial memory in Repeated Acquisition and Performance Chamber (RAPC) test. These results indicate that TRMT1 exhibits expression in brain areas associated with learning and memory, and TRMT1 plays a role in maintaining normal brain morphology and function during development, which is coincident with the syndrome observed in the patients. In the future, we plan to use neuronal staining and immunofluorescence to check the morphological changes of dendrites and molecular pathway affected by TRMT1.

References:
Dewe JM, Fuller BL, Lentini JM, Kellner SM, Fu D. TRMT1-catalyzed tRNA modifications are required for redox homeostasis to ensure proper cellular proliferation and oxidative stress survival. Molecular and Cellular Biology. 2017;37(21).
Davarniya B, Hu H, Kahrizi K, Musante L, Fattahi Z, Hosseini M, et al. The role of a novel TRMT1 gene mutation and rare GRM1 gene defect in intellectual disability in two azeri families. PLoS ONE. 2015;10(8):e0129631-.

Keywords: tRNA modification, TRMT1

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

Hongran Yin (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Long Zhang (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Zhefeng Li (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Dan Shu (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Peixuan Guo (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University)

Abstract not available online - please check the printed booklet.

169. Deinococcus radiodurans Transfer RNA Modified Nucleosides are Minimally Impacted UV Radiation

Ruoxia Zhao (University of Cincinnati), Spencer Parrish (University of Cincinnati), Robert Ross (University of Cincinnati), Manasses Jora (University of Cincinnati), Balasubrahmanyam Addepalli (University of Cincinnati), Patrick A Limbach (University of Cincinnati)

Abstract not available online - please check the printed booklet.

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

Dan Shu (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Lixia Zhou (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Tae J. Lee, Carlo M. Croce (Department of Cancer Biology and Genetics, The Ohio State University), Hongran Yin, Hui Li, Peixuan Guo (Department of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University), Yi Shu, Ren Xu (Markey Cancer Center, College of Pharmacy, University of Kentucky), Bin Guo (Department of Pharmaceutical Sciences, North Dakota State University)

Abstract not available online - please check the printed booklet.

171. Helical bPNA targeting U-rich RNA

Oliver Munyaradzi (Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH), Dennis Bong (Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH)

Abstract:
This project seeks to design RNA sequence-specific binders that take advantage of defined secondary structure. Previous work produced bifacial peptide nucleic acid (bPNA) in which melamine displayed on a modified lysine side-chain formed T-M-T/U-M-U base triples with thymine or uracil. We hypothesized that pre-organization of the bPNA peptide backbone into a helical structure would enable biomimetic peptide recognition of a U-rich major groove of A-form RNA. We hypothesize that the combination of helical peptide display with UU targeting by the melamine base, will enable a general method for targeting U-rich A-form RNA. We have designed and synthesized peptides that incorporate lysine derivatives doubly functionalized with melamine (K2M)onto one face of a putative helical fold, using an i,i+4 pattern on an alanine-rich sequence, every third position of a PPII helix, a and d positions of a heptad repeat. Secondary structure was probed by CD spectroscopy which indicated helical propensity in all three peptides. DNA and RNA binding properties were elucidated using Tm measurements, EMSA, ITC and CD studies. For ssDNA (dT6C4T6) peptide complexes , similar thermal stability was observed for all peptides, including prior bPNA designs, though a modest increase in affinity (3-4 fold) was observed using EMSA. In contrast, the helical peptides helped unstructured U6C4U6 ssRNA to fold, yielding complexes with an 8-10 oC higher Tm and with sufficient stability for EMSA and ITC studies compared to those formed by prior bPNA with display of bases at alternate residues. Thus, early results indicate support for the notion that defining backbone secondary structure can impact bPNA recognition of nucleic acids.

Keywords: Sequence-specific, RNA binding, PNA

172. Quantitative analysis of post-transcriptional modifications from bacterial systems

Taylor A. Dodson (Department of Medicinal and Biological Chemistry, University of Toledo), Eric A. Carlson (Department of Medicinal and Biological Chemistry, University of Toledo), Nathan C. Wamer (Department of Medicinal and Biological Chemistry, University of Toledo), Sadalla Y. Jouejati (Department of Biological Sciences, University of Toledo), Erin G. Prestwich (Department of Medicinal and Biological Chemistry, University of Toledo)

Abstract not available online - please check the printed booklet.

173. Identification of the mechanism for PABPN1 translational repression during cardiac development and hypertrophy

Bo Zhang (Department of Biochemistry, University of Illinois at Urbana-Champaign), Joseph Seimetz, Chaitali Misra, Emelia Smith (Department of Biochemistry, University of Illinois at Urbana-Champaign), Qinyu Hao, Kannanganattu V. Prasanth (Department of Biochemistry, University of Illinois at Urbana-Champaign), Auinash Kalsotra (Department of Biochemistry, University of Illinois at Urbana-Champaign)

Abstract:
Cardiomyocytes are the muscle cells that make up the heart muscle. These contractile cells occupy ~70-85% the volume of the mammalian heart and enable the heart to pump blood throughout the body. The majority of cardiomyocytes become post-mitotic soon after birth and since then the heart increases in size through the expansion of the volume of existing cardiomyocytes, namely postnatal hypertrophy or maturational hypertrophy. As the animal reaches adulthood, cardiomyocytes stopped expansion and matured; at the same time, protein synthesis in these cells dramatically decreases. However, upon particular stimuli, protein synthesis surges in the mature cardiomyocytes, accompanied with physiological or pathological hypertrophy of these cells and changed heart physiology. Recently, our lab has identified the poly(A)-binding protein nuclear 1 (PABPN1) as an important regulator of cardiac hypertrophy. PABPN1 is responsible for the elongation of poly(A) tail and is involved in multiple aspects of mRNA metabolism including nucleocytoplasmic trafficking and nonsense-mediated decay (NMD). The cardiac PABPN1 levels dramatically dropped upon neonatal to adult transition. Elevated expression of PABPN1 was not only observed in mouse cardiac hypertrophy models, but also found in failing human hearts, suggesting a tight connection between postnatal re-expression of PABPN1 and pathological cardiac hypertrophy. Forced expression of this protein in adult cardiomyocytes induced severe cardiac hypertrophy, impaired contractile force and sudden death of the experimental animals. Interestingly, despite of the dramatic reduction of PABPN1 protein in the adult animals, the RNA level of Pabpn1 was hardly changed. However, little is known about the underlined mechanism for this translational regulation of Pabpn1 expression.

We hypothesize that nuclear sequestration caused by insufficient splicing of intron results into translational arrest of PABPN1 in mature striated muscle cells. We found that the repression of PABPN1 translation was achieved by the sequestration of Pabpn1 mRNA in the nucleus. RNA fluorescent in situ hybridyzation (RNA-FISH) and nucleus-cytoplasm fractionation suggested that Pabpn1 transcripts were preferably localized to the nucleus in the adult cardiomyocytes and differentiated myotubes. We have identified the intron 6 of Pabpn1 as a potential cis-acting element that is responsible for the translational regulation. We observed increased intron 6 detention in adult heart and differentiated myotubes. These intron 6-detained transcripts preferably localized to the nucleus. We have also identified the splicing factor Rbfox2 as a potential trans-acting factor for this regulation. A crosslinking and immunoprecipitation (CLIP) assay revealed that RBFOX2 bound to the intron 6 regain of Pabpn1 transcript. Interestingly, both PABPN1 protein levels and Pabpn1 mRNA splicing increased when RBFOX2 was overexpressed. Currently, we are in the process of using non-biased methods like RNA immunoprecipitation (RIP) and RNA antisense puarification-mass spectrometry (RAP-MS) RAP to verify this finding and to further exploit other potential candidates for this regulation. The identification of the mechanism for hypertrophic growth of cardiomyocyte is important for our understanding of cardiac development and hypertrophic pathology and potentially will benefit the prevention and treatment of heart disease.

Keywords: PABPB1, translational repression, cardiac development and hypertrophy