Rustbelt RNA Meeting 2020

Poster abstracts

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

Sudeshi M. Abedeera (Department of Chemistry and Biochemistry, Kent State University), Jiale Xie (Department of Chemistry and Biochemistry, Kent State University), Sanjaya C. Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)

Abstract not available online - please check the printed booklet.

3. Liquid-Liquid Phase Transition of Protein-RNA Complexes: Droplets, Micelles and Vesicles

Ibraheem Alshareedah (Department of Physics, University at Buffalo), Priya R. Banerjee (Department of Physics, University at Buffalo)

Abstract:
Liquid-liquid phase separation of multivalent disordered proteins with RNA is prevalent in natural and biomimetic systems. Recently, we showed that protein-RNA condensation is reentrant and sensitively depends on the RNA-to-protein mixing ratio as well as the sequence-encoded protein-protein and protein-RNA interactions¹. In this work, by quantitatively mapping the phase behavior of protein-RNA complexes at various mixture compositions, we report the formation of hollow vesicle-like protein-RNA condensates that are distinct from liquid droplets. These hollow condensates are preceded by micellar condensates and are formed at disproportionate mixture compositions of non-amphipathic oppositely charged proteins and RNAs². We further show that these hollow condensates display a striking resemblance to classical vesicles formed by amphipathic biopolymers. Specifically, protein-RNA hollow condensates display fluid-like properties, liquid-crystalline order, size-dependent permeability and selective encapsulation of biomolecules. In contrast to lipid vesicles, protein-RNA hollow condensates form robustly and spontaneously upon mixing and are highly responsive to biochemical stimuli. Our findings reveal hollow vesicle-like enclosures as a new phase in the topological spectrum of protein-RNA phase separation.

References:
1 Alshareedah, I. et al. Interplay between Short-Range Attraction and Long-Range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein–RNA Complexes. Journal of the American Chemical Society 141, 14593-14602, doi:10.1021/jacs.9b03689 (2019).
2 Alshareedah, I., Moosa, M. M., Raju, M., Potoyan, D. A. & Banerjee, P. R. Phase transition of RNA−protein complexes into ordered hollow condensates. Proceedings of the National Academy of Sciences 117, 15650-15658, doi:10.1073/pnas.1922365117 (2020).

Keywords: Liquid-Liquid phase separation, RNA vesicles, RNA micelles

4. Conformational changes in ribosomal RNA 3D Motifs correlated with ribosome functional state

Sri Devan Appasamy (Department of Biological Sciences, Bowling Green State University), Craig Zirbel (Department of Mathematics and Statistics, Bowling Green State University), Neocles Leontis (Department of Chemistry, Bowling Green State University)

Abstract not available online - please check the printed booklet.

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

Sushant Bangru (University of Illinois, Urbana-Champaign), Jackie Chen, Waqar Arif (University of Illinois, Urbana-Champaign), Frances Alencastro (University of Pittsburgh), Russ P Carstens (University of Pennsylvania), Andrew Duncan (University of Pittsburgh), Auinash Kalsotra (University of Illinois, Urbana-Champaign)

Abstract not available online - please check the printed booklet.

6. Identification of temperature responsive post-transcriptional regulatory sRNAs in Staphylococcus aureus

Raeven A. Bastock (Biological Sciences at Ohio University ), Rebecca A. Keogh (Biological Sciences at Ohio University ), Ronan K. Carroll (Biological Sciences at Ohio University ), Erin R. Murphy (Biological Sciences at Ohio University )

Abstract:
Staphylococcus aureus is a Gram-positive opportunistic pathogen that colonizes the anterior nares of approximately 30% of the global population. Colonization is most commonly asymptomatic however, it is associated with a higher risk of invasive infections. If the bacterium internalizes, it can cause potentially fatal infections. In the transition between the nares and the body, the bacterium is subject to many environmental changes, including a slight temperature difference. The average temperature of the nares is 34C while the internal body is 37C. Temperature responsive post-transcriptional RNA regulatory elements, such as RNA thermometers, are known to exist in many Gram-negative bacteria but, few examples have been described in Gram-positive pathogens to date. Given all of this previous knowledge, we investigated the influence of temperature on S. aureus gene expression. RNAseq and proteomic analyses were performed on cultures grown at three different temperatures: 34C, the temperature of the nares, 37C, internal body temperature, and 40C, representative of pyrexia. Cross-reference analysis of the data sets revealed extremely low correlation between transcript and protein levels and was highly suggestive of post-transcriptional regulation. RNAseq analysis revealed that sRNAs were the most altered transcripts with 29 in total altered between 34C V 37C and 18 altered between 37C V 40C. Interestingly higher sRNA expression values were mostly observed at the lower temperature. Fifteen sRNAs had a significant change in both paired analyses (34C V 37C and 37C V 40C) in a thermo-responsive manner. The discrepancy between transcript and protein levels is highly suggestive of post-transcriptional regulation, and we hypothesize that these 15 temperature-responsive sRNAs play a critical role in the transition of the pathogen from the nares to systemic infection. One temperature-responsive sRNA of particular interest is Teg49. Located in the 5’ UTR of sarA, Teg49 is processed into a trans-acting sRNA that has been shown to influence virulence factor expression, independent of SarA activity. We are investigating these temperature-responsive sRNAs to determine if they play a role as S. aureus, transitions from a colonizing commensal, to an invasive pathogen.

Keywords: S aureus, temperature

7. Identification of a tRNA-specific function for the tRNA methyltransferase Trm10 in Saccharomyces cerevisiae

Isobel Bowles (Ohio State Biochemistry Program), Jane Jackman (OSU Chemistry and Biochemistry)

Abstract:
tRNA methyltransferase 10 (Trm10), first discovered in Saccharomyces cerevisiae (S. cerevisiae), methylates N1 of guanosine at the 9th position of tRNA molecules using methyl donor S-adenosyl methionine (SAM). Upon the deletion of TRM10, S. cerevisiae strains experience growth defects in the presence of antitumor drug 5-fluorouracil (5FU). We hypothesized that tRNA stability decreases with the lack of m¹G₉ in trm10Δ strains and that certain tRNA species are more reliant upon the presence of the methylated G₉ nucleotide, as evident from different growth phenotypes observed upon tRNA overexpression. When Trm10 substrate tRNA^Trp is overexpressed in trm10Δ strains, growth hypersensitivity to 5FU is rescued, while overexpression of other tRNA species in S. cerevisiae do not display a growth rescue. Levels of tRNA^Trp decrease in trm10Δ strains compared to levels of substrate tRNA^Gly while overexpression of tRNA^Trp recovers levels of tRNA^Trp in trm10Δ strains. The specific role of the m¹G₉ tRNA modification is being investigated by analyzing mechanisms of tRNA^Trp breakdown in S. cerevisiae trm10Δ strains with 5FU while also analyzing tRNA abundance for multiple Trm10 substrate tRNAs in the presence and absence of 5FU. tRNA structures that correlate with active substrates are being determined with nuclease and chemical footprinting, as well as 2’ hydroxyl acylation analyzed by primer extension (SHAPE). Together these studies will provide further understanding of tRNA structural elements that promote methylation by Trm10.

Keywords: trna modification, m1G9, SHAPE

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

Audrey N. Bui (Biology Department, St. Bonaventure University), Anna R. Hu (Biochemistry Program, Biology Department, St. Bonaventure University), Arden N. Bui (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.

9. Cationic amphiphilic co-polymers as carriers of RNA nanoparticles for controlled gene silencing, immunostimulation, and biodistribution

Morgan Chandler (Department of Chemistry, University of North Carolina at Charlotte), Justin R. Halman, Lauren Rackley, M. Brittany Johnson, Ian Marriott (Department of Chemistry, Department of Biology, University of North Carolina at Charlotte), Ki-Taek Kim, So-Jung Gwak, Richard Pace (Department of Bioengineering, Clemson University), Mathias Viard (Cancer and Inflammation Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research), Jeoung Soo Lee (Department of Bioengineering, Clemson University), Kirill A. Afonin (Department of Chemistry, University of North Carolina at Charlotte)

Abstract:
The programmed assembly of RNA nanoparticles provides a means for the controlled coordination of therapeutic moieties on a biocompatible scaffold. While the delivery of nucleic acids in general remains a challenge, new candidates to serve as carriers are emerging and are continuously tested. Polymeric platforms have proven to be promising for clinical translation, as they are efficient in shielding nucleic acid cargos from nuclease degradation while still promoting their delivery and intracellular release. In this work, we combine the stable cationic poly-(lactide-co-glycolide)-graft-polyethylenimine, known as the carrier PgP, with functionalized RNA nanoparticles. We compare several representative nanostructure designs of different connectivity and dimensionality, including cubic, ring, and fibrous RNA nanoparticles, to assess how overall structure influences delivery with the same carrier. As a result, the extensive study of these formulations both in vitro and in vivo reveals nanoparticle-dependent differences in their immunostimulatory activity, gene silencing efficiency, and biodistribution.

References:
Halman, J. R.; Kim, K.-T.; Gwak, S.-J.; Pace, R.; Johnson, M. B.; Chandler, M. R.; Rackley, L.; Viard, M.; Marriott, I.; Lee, J. S.; Afonin, K. A., A cationic amphiphilic co-polymer as a carrier of nucleic acid nanoparticles (NANPs) for controlled gene silencing, immunostimulation, and biodistribution. Nanomedicine: Nanotechnology, Biology and Medicine 2020, 23, 102094.

Keywords: nanoparticle, carrier, immunostimulation

10. Funding Opportunities at the National Science Foundation

Karen Cone (Genetic Mechanisms, Division of Molecular and Cellular Biosciences, National Science Foundation)

Abstract:
The National Science Foundation's Directorate for Biological Sciences (BIO) has a mission to fund research on fundamental, basic research questions to advance understanding of life processes across scales from molecules to ecosystems.

Awards are available to support research grants for faculty. These include opportunities through: 1) Core Programs in the divisions of Molecular and Cellular Biosciences, Integrative Organismal Systems, Environmental Biology, and Biological Infrastructure, 2) Faculty early career development (CAREER), and 3) Research in primarily undergraduate institutions (RUI).

Awards to support training for individuals who are US citizens or permanent residents include Graduate Research Fellowships and Postdoctoral Research Fellowships in Biology.

Information on these and other programs will be presented.

References:
NSF BIO website: https://www.nsf.gov/BIO
BIO blogs: https://oadblog.nsfbio.com/
Virtual Office Hours: https://mcbblog.nsfbio.com/office-hours/
Contacting a program director: https://nsfmcb.files.wordpress.com/2020/02/mcb-virtual-office-hour-slides_2-12-20.pdf

Keywords: funding opportunities, biological sciences, research and education

11. New insights into mRNA regulation by the Drosophila TRIM-NHL protein Brat

Robert P. Connacher (Dept. Biochemistry, Molecular Biology, & Biophysics, University of Minnesota), Michael B. OConnor (Dept. Genetics, Cell Biology, and Development, University of Minnesota), Aaron C. Goldstrohm (Dept. Biochemistry, Molecular Biology, & Biophysics, University of Minnesota)

Abstract:
Members of the TRIM-NHL family of proteins share a conserved domain architecture and play crucial roles in stem cell biology, fertility, and development. Recently, multiple TRIM-NHLs have been shown to recognize specific RNA motifs and structures via the NHL domain. Functional and genetic analysis shows TRIM-NHLs negatively regulate protein expression of the mRNAs that they bind. However, it is unclear whether RNA-binding is utilized in the multiple developmental processes in which TRIM-NHLs are involved, and how these proteins negatively regulate mRNAs upon binding. These questions were investigated with the Drosophila TRIM-NHL protein Brat. First, RNA-binding defective mutations were introduced into the endogenous brat locus in flies via CRISPR. Alanine substitution of key residues known to abolish RNA-binding in-vitro mimics loss-of-function brat mutations; implying the primary biological function of Brat utilizes RNA-binding. Furthermore, cell culture assays identify three autonomous domains of Brat which exert RNA repression, each utilizing unique means to negatively regulate RNA stability and/or translation. Further analysis of CRISPR mutants and investigation of these autonomous domains will provide a full picture of TRIM-NHL function, at both the transcriptome-wide and molecular levels.

Keywords: Drosophila, CRISPR, development

12. Semi-empirical in vivo RNA structure prediction by motif constraining

Catherine Douds (Department of Biochemistry and Molecular Biology, Penn State University), Paul Babitzke (Department of Biochemistry and Molecular Biology, Penn State University), Philip C. Bevilacqua (Chemistry Department, Penn State University)

Abstract not available online - please check the printed booklet.

13. Trypanosome RNA Editing Substrate Binding Complex integrity and function depends on the upstream action of RESC10

Ashutosh P. Dubey (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Brianna L. Tylec (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Natalie M. McAdams (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Katherine Sortino (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA), Laurie K. Read (Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14203, USA)

Abstract not available online - please check the printed booklet.

14. RNA - protein coupled alignment families (RPfam)

Quinn E. Eberhard (Bowling Green State University), Neocles B. Leontis (Bowling Green State University), Craig L. Zirbel (Bowling Green State University)

Abstract:
RNA - Protein Coupled Alignment Families (RPfam) is an algorithm which formulates tandem RNA and protein sequence alignments given specified Pfam and Rfam families. Additional sequences are sourced from RNAcentral and UniProt to complete the alignments so that variations in the sequences can be studied in a coevolutionary fashion across hundreds of organisms at once. RPfam can also be utilized for RNA - protein interaction studies as the user can provide the IDs of specific ribosomal PDB 3D structures which then allows for “anchor” sequences to be implemented into the alignment. Analyzing the sequence variation in relation to the anchor PDB structure can provide insight to how specific RNA - protein interactions may also vary in organisms in the alignment with unresolved structures.

Keywords: Alignments, 3D structure, RNA-protein interactions

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

Emily A Erdmann (Department of Biology, Indiana University, Bloomington IN, 47405), Maggie Holdeman, Olivia Abraham, Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine-Bloomington, Bloomington IN, 47405)

Abstract not available online - please check the printed booklet.

17. Computational discovery and validation of novel functional RNAs in SARS-CoV-2

Peter C. Forstmeier (Pennsylvania State University, Department of Biochemistry and Molecular Biology), McCauley O. Meyer (Pennsylvania State University, Department of Biochemistry and Molecular Biology), Philip C. Bevilacqua (Pennsylvania State University, Department of Biochemistry and Molecular Biology, Department of Chemistry)

Abstract:
The COVID-19 pandemic continues to have a profound impact on the world. With millions affected in the United States alone, it is critical to quickly develop therapeutics against the virus. The causal virus of COVID-19, SARS-CoV-2, is an RNA virus known to contain functional RNA structures¹, which are good therapeutic targets. Finding and characterizing them in a rapid and accurate manner is imperative. Regions with putative RNA structures were identified via a novel computational pipeline that we describe here for the first time, called ScanFilter. In phase one of ScanFilter, the program ScanFold² is used to find structured regions, while two other programs are used to filter out known structures. In phase two of the pipeline, SPOT-RNA, a pseudoknot-predicting program, selects for structured regions that contain pseudoknots³. We chose pseudoknots as an indicator of functionality because most functional RNA elements contain at least one pseudoknot⁴. In order to assess the functionality of these regions, a series of programs was added to the third phase of the pipeline. These programs find the SNPs from the ~24,000 (as of 9/24/2020) sequenced genomes of SARS-CoV-2 in the putatively functional regions and determine if a SNP causes a change in the ensemble structure of the RNA or disrupts a pseudoknot. Disruptions in the functional region could change its ability to fold and prevent its function. Regions that do not contain many changes or disruptions may be functional because their invariant structure serves a vital biological function. The presented pipeline is a powerful tool that can computationally find and validate novel therapeutic targets for SARS-CoV-2 using existing data.

References:
(1) Kim, D.; Lee, J. Y.; Yang, J. S.; Kim, J. W.; Kim, V. N.; Chang, H. The Architecture of SARS-CoV-2 Transcriptome. Cell 2020, 181 (4), 914-921.e10. https://doi.org/10.1016/j.cell.2020.04.011.
(2) Andrews, R. J.; Roche, J.; Moss, W. N. ScanFold: An Approach for Genome-Wide Discovery of Local RNA Structural Elements—Applications to Zika Virus and HIV. PeerJ 2018, 6, e6136. https://doi.org/10.7717/peerj.6136.
(3) Singh, J.; Hanson, J.; Paliwal, K.; Zhou, Y. RNA Secondary Structure Prediction Using an Ensemble of Two-Dimensional Deep Neural Networks and Transfer Learning. Nat. Commun. 2019, 10 (1), 5407.
(4) Staple, D. W.; Butcher, S. E. Pseudoknots: RNA Structures with Diverse Functions. PLoS Biol. 2005, 3 (6), e213. https://doi.org/10.1371/journal.pbio.0030213.

Keywords: SARS-CoV-2, RNA, Pseudoknots

18. Methyltransferase enzyme RsmC act as an RNA chaperone during bacterial ribosome biogenesis

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

Abstract:
A ribosome is the ribonucleoprotein molecular machine that biosynthesizes proteins in all living cells. Biogenesis of ribosomes is crucial for the survival of all living organisms. The presence of various transacting factors including modification enzymes is important to synchronize various processes that happens during ribosome biogenesis. We have identified the ability of modification enzyme RsmC to function as an RNA chaperone protein during ribosome assembly in addition to its methyltransferase activity. The rate of helix 34 (h34) strand annealing is increased by 25-fold in the presence of RsmC. Furthermore, RsmC denatures non-native secondary structures observed in RsmC substrate strand and the protein also destabilizes the secondary structure of the substrate strand. Furthermore, smFRET experiments confirmed the annealing and chaperone activity of protein RsmC. Various mutant rRNAs were also used in this study to investigate the nature of various intermediates observed during RsmC-dependent h34 strand annealing. These observed chaperone activity of RsmC will facilitate the fast formation of 30S head domain during ribosome biogenesis.

Keywords: Ribosome biogenesis, RNA chaperone proteins, rRNA methyltransferase

19. RAZOR: a Unified Database for Respiratory Virus PCR Primers

Hunter Mathias Gill (BioHealth Informatics, Indiana University School of Informatics & Computing at IUPUI)

Abstract:
The current COVID-19 pandemic highlights the significance of human respiratory RNA viruses. PCR-based technologies have been indispensable tools in the global response to COVID-19 by providing platforms for both biological research and molecular diagnostic testing [1]. The RAZOR database was developed to help provide high-quality primers for the detection of the causative COVID-19 agent, SARS-CoV-2, as well as similar RNA-genome respiratory viruses. All RAZOR primers sets are designed with the Primer3 algorithm and cover amplicon size ranges for both conventional and RT PCR protocols. Primer sets are also binned into distinct conservation categories according to BLAST hits against a database of all known viruses. The primer sets can be accessed and downloaded from highly interactive results pages powered by igv.js [2].

RAZOR Database: https://sysbio.informatics.iupui.edu/primer_project/website/

References:
[1] Bustin SA, T Nolan. (2020). RT-qPCR testing of SARS-CoV-2: A primer. Int. J. Mol. Sci.2020 Apr; 21(8); 3004. doi: 10.3390/ijms21083004
[2] https://github.com/igvteam/igv.js/

Keywords: database, respiratory virus, PCR

20. The effect of the small RNA RyhB on the structure of its mRNA targets

Samantha M. Grecco (Department of Biochemistry and Molecular Biology, Beckman Scholar, Schreyer Honors College, Pennsylvania State University, University Park, Pennsylvania, USA), Janie K. Frandsen (Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA), Philip C. Bevilacqua (Department of Biochemistry and Molecular Biology, Department of Chemistry, Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA)

Abstract:
There are many ways that Escherichia coli combats environmental stressors, one such mechanism is gene regulation by small non-coding RNAs (sRNA). sRNAs are post-transcriptional regulators that base pair with mRNA targets to change the expression of the encoded gene. RyhB is a sRNA that is expressed under iron-limiting conditions and regulates the iron sparing response by shifting the limited iron present to essential proteins and downregulating nonessential iron utilizing proteins.¹ However, the majority of RyhB targets have not been characterized and the role of RNA structure in the sRNA-mRNA interactions is unknown. It is hypothesized that the target mRNA secondary structure influences the ability of RhyB to interact and that the interaction may induce a structural rearrangement that alters gene expression. In this study, we used data from six-genome-wide analyses¹ to select putative RyhB targets for in vivo RNA structure probing analysis to determine the effect of RyhB on the structure of its target mRNAs. The uncharacterized mRNA targets of RyhB that were identified in four or more of the genome-wide studies were then further analyzed using the IntaRNA² and RNAstructure³ programs. Target mRNAs that showed the potential for both a strong interaction with RyhB and a relatively unstructured binding site with a high probability of structural rearrangement upon RyhB binding were chosen for further analysis. Four putative RyhB targets were selected: the positively regulated genes, cirA and fliA, and the negatively regulated genes, sufAB and frdA. This research will provide a deeper understanding of the regulatory system in E. coli, which could help combat pathogenic infections and antibiotic resistance.

References:
1. Chareyre, S., Mandin, P. (2018) Bacterial Iron Homeostasis Regulation by sRNAs, Microbiol. Spectr. 6(2), 267-281.
2. Mann, M., Wright, P. R., and Backofen, R. (2017) IntaRNA 2.0: enhanced and customizable prediction of RNA–RNA interactions, Nucleic Acids Research, 45(W1), W435–W439
3. Reuter, J. S., and Mathews, D. H. (2010) RNAstructure: software for RNA secondary structure prediction and analysis, BMC Bioinformatics 11,129.

Keywords: sRNA, Ecoli

21. SARS-CoV-2 Diversity and Phylogeny in Indiana COVID-19 Patients

Dwight William Hall (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Quoseena Mir, Rajneesh Srivastava (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Guang-Sheng Lei (Department of Pathology and Laboratory Medicine, IUSM, Indianapolis, Indiana), David James Tauriainen (School of Informatics and Computing, Indiana University Purdue University), Ryan F. Relich, John-Paul Lavik (Department of Pathology and Laboratory Medicine, IUSM, Indianapolis, Indiana), Sarath Chandra Janga (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University)

Abstract:
The novel coronavirus (COVID-19) outbreak, which initially began in China, has spread to many countries around the globe. The number of confirmed COVID-19 cases increase daily with a death toll exceeding the SARS (Severe Acute Respiratory Syndrome) outbreak in 2002 and 2003. SARS-CoV-2 has led to a pandemic, which put all health organizations on high alert. In this study we aimed to sequence, genotype and map the diversity of the positive COVID-19 Indiana samples onto a global phylogenetic tree of COVID19 strains to understand the evolution and transmission dynamics of Indiana isolates. The phylogenetic analysis for positive COVID-19 samples could lead to insights on which mutations are causing more transmissible and virulent strains of SARS-CoV-2.

Nasal swabs from 40 positive COVID-19 samples were employed for SARS-CoV2 isolation and sequenced using Oxford MinIon Sequencer and processed using Artic Network (https://artic.network/ncov-2019). Consensus sequences of the complete viral genome were assembled using a pipeline proposed by Artic Network. The phylogenetic tree, geographical map, and genotype diversity were built using Nextstrain’s open source software. Resulting phylogenetic trees were analyzed for genomic diversity and geographical origin of the collected samples. Genomic diversity was used to infer mutation sites among the samples. Geographical location was used to determine which countries had the most similar sequences to the samples from Indiana. The phylogenetic tree and transmission map can be viewed at https://covid19-indiana.soic.iupui.edu/.

The global phylogenetic tree indicated that 39 of the Indiana samples belonged to the G (glycine) group, while 1 Indiana sample corresponded to the D (aspartic acid) group classified based on the mutations in the Spike Protein Codon 614. Based on previous studies and our own observations, this modification is attributing to a more transmissible type of SARS-CoV-2 [1-3]. Geographical analysis of the Indiana samples revealed that within the United States, the SARS-CoV-2 sequences were most similar to sequences from Virginia and Michigan while outside the United States, most similar to sequences from Victoria, Australia.

References:
1. Brufsky, A. (2020), Distinct viral clades of SARS‐CoV‐2: Implications for modeling of viral spread. J Med Virol, 92: 1386-1390. doi:10.1002/jmv.25902
2. Bette Korber et al.: Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell 2020, Volume 182(Issue 4):Pages 812-827.e819.
3. Muthukrishnan Eaaswarkhanth, Al Madhoun A, Fahd Al-Mulla: Could the D614G substitution in the SARS-CoV-2 spike (S) protein be associated with higher COVID-19 mortality? International Journal of Infectious Diseases 2020, Volume 96:Pages 459-460.

Keywords: Phylogenetic , SARS-CoV-2 , COVID19

22. Penguin: Predicting RNA Pseudouridine Sites in Nanopore Sequencing Data

Doaa Hassan (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Daniel Acevedo (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Swapna Vidhur Daulatabad (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Quoseena Mir (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Sarath Chandra Janga (Department of BioHealth Informatics, School of Informatics and Computing, Indiana University Purdue University)

Abstract:
Pseudouridine is one of the abundant RNA modification that occurs at the uridine site and catalyzed by Pseudouridine synthase. It plays an important role in many biological processes and also has an importance in drug development. Recently, the single-molecule sequencing techniques such the direct RNA sequencing platform offered by Oxford Nanopore Technologies enable direct detection of RNA modifications on the molecule that is being sequenced, but to our knowledge this technology has not been used to identify RNA Pseudouridine sites. To this end, in this paper, we address this limitation by introducing a tool called Penguin that integrates several developed ML learning models (i.e., predictors) to identify RNA Pseudouridine sites in Nanopore direct RNA sequencing reads. Penguin extracts a set of features from the raw signal measured by the Oxford Nanopore and the corresponding basecalled kmer. Those features will be used to train the predictors included in penguin platform which will be in turn able to predict whether the signal is modified by the presence of Pseudouridine sites or not in the testing phase. We have included various predictors in Penguin including Support vector machine (SVM), Random Forest (RF), and Neural network (NN). In comparison with the state-of-the-art predictors presented in the literature for identifying Pseudouridine sites, the results on the two benchmark data sets show that Penguin is able to identify Pseudouridine sites with higher accuracy of 93.38% and 92.61% using SVM in random split testing and independent validation testing respectively. A github of the tool is accessible at https://github.com/daniel235/Penguin.

References:
[1] Zhibin Lv, Jun Zhang, Hui Ding and Quan Zou. RF-PseU: A Random Forest Predictor for RNA Pseudouridine Sites. Frontiers in Bioengineering and Biotechnology, Volume 8, Article 134, February 2020.
[2] Kewei Liu, Wei Chen, and Hao Lin. XG-PseU: an eXtreme Gradient Boosting based method for identifying pseudouridine sites. Molecular Genetics and Genomics,295, 13-21 (2020).
[3] M. Tahir, H. Tayara, and K.T. Chong iPseU-CNN: identifying RNA pseudouridine sites using convolutional neural networks. Molecular Therapy—Nucleic Acids, 16, 463-470.
[4] J. J., Fang, T., Zhang, Z. Z., Huang, B., Zhu, X. L., and Xiong, Y. PseUI:pseudouridine sites identification based on RNA sequence information. BMC Bioinformatics, 19:11.

Keywords: RNA modification , Pseudouridine, Nanopore

23. The Piwi/piRNA pathway promotes fertility by regulating expression of protein-coding genes

Hannah L. Hertz (Department of Biological Chemistry and Pharmacology, The Ohio State University), Ian F. Price (The Ohio State Biochemistry Program, The Ohio State University), Benjamin Pastore (The Ohio State Biochemistry Program, The Ohio State University), Wen Tang (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract:
The PIWI/piRNA pathway plays an essential role in fertility across metazoa. In the nematode Caenorhobitis elegans, PIWI ortholog PRG-1 and its small RNA cofactors (piRNAs) silence transposons and mRNAs via complementary base pairing. Loss of PRG-1 leads to infertility. Our goal is to identify germline transcripts responsible for reduced fertility in the absence of PRG-1/piRNA targeting. To this end, we conducted germline-specific RNAi to systematically knock down PRG-1/piRNA targets in prg-1 mutant animals and assessed their fertility. Although depletion of many PRG-1/piRNA targets did not have an effect, knockdown of some somatic transcripts in the germline may rescue fertility. Altogether, our study suggests a role for the PRG-1/piRNA pathway in maintaining germ-cell identity by repressing somatic transcripts.

Keywords: PIWI-interacting small RNAs, Caenorhabditis elegans

24. Looking into the internal coordination within the ASAP complex for gene regulation in Arabidopsis

Anna Hu (Biochemistry Program, Department of Biology), Xiao-Ning Zhang (Biochemistry Program, Department of Biology)

Abstract:
An important event in gene expression is the covalent modification of histone proteins. In eukaryotic cells, the Sin3-associated protein 18 (SAP18) can recruit histone deacetylase to repress target gene transcription. In Arabidopsis thaliana, an existing model suggests that SAP18 interacts with other RNA binding proteins, RNPS1 and apoptotic chromatin condensation inducer in the nucleus (Acinus), to form the conserved apoptosis and splicing-associated protein (ASAP) complex at the promoter of the FLOWER LOCUS C (FLC) gene for silencing. Besides transcriptional regulation, the ASAP complex also affects splicing and nonsense mediated decay by binding to exon junction complexes. In Arabidopsis, a null mutation in the RNPS1 ortholog, Serine/Arginine-rich 45 (SR45), has been found to cause an increased expression in a set of genes including FLC and a substantial decrease in nuclear SAP18 protein. Questions remain as to whether some of these more actively transcribed genes in the sr45-1 mutant are targets for SAP18-mediated gene silencing. To answer this question, the activity of SAP18 at the chromatin level was investigated by ChIP-seq assay using translational fusion SAP18pro::SAP18-GFP transgenic plants in the wild type background. A comparison between SAP18-associated DNA regions and SR45-differentially regulated genes revealed that some SAP18-associated sequences were in the promoter region of SR45-differentially expressed genes indicating a possible role of SAP18 in regulating the transcription of these genes, which may help explain some of the observed phenotypes of the sr45-1 mutant. More interestingly, SAP18-associated sequences were also identified in exonic and/or intronic regions. In order to understand the effects of targeting different parts of the gene body, further investigation will be conducted.

References:
Zhang, X., Shi, Y., Powers, J. J., Gowda, N. B., Zhang, C., Ibrahim, H. M., . . . Mount, S. M. (2017). Transcriptome analyses reveal SR45 to be a neutral splicing regulator and a suppressor of innate immunity in Arabidopsis thaliana. BMC Genomics, 18(1). doi:10.1186/s12864-017-4183-7
Questa, J. I., Song, J., Geraldo, N., An, H., Dean, C. (2016). Arabidopsis transcriptional repressor VAL1 triggers Polycomb silencing at FLC during vernalization. Science, 353(6298), 485-488. doi:10.1126/science.aaf7354

Keywords: SAP18, ASAP complex , transcriptional regulation

25. Expression and in vivo characterization of a proline-rich antimicrobial peptide that targets 23S ribosomal RNA

Rabiul Islam (Department of Chemistry Wayne State University), Christine Chow (Department of Chemistry Wayne State University)

Abstract:
The development of bacterial resistance to antibiotics is a major challenge for clinicians. Naturally occurring proline-rich antimicrobial peptides (PrAMPs) have shown significant activity against pathogenic bacteria. One example is the 19-mer peptide oncocin, which has promising antibiotic action against multidrug-resistant species. Synthesis of oncocin using standard solid-phase synthesis protocols and in vitro testing are both time and resource intensive. In this study, we used an inducible expression system to generate oncocin and measure its antibiotic activity. We employed non-template PCR, cloning, and transformation, then the transformed cells were isolated and screened for antimicrobial activity upon induction of peptide expression with arabinose. In a similar manner, truncated versions of oncocin were produced to determine the contributions of the N- and C-termini to antibiotic activity upon in vivo expression. Dimethyl sulfate (DMS) was used to map the oncocin binding sites on the ribosome under cellular conditions. Total RNA was isolated from DMS-treated induced cells and reverse transcription was performed with radiolabeled primers. The DMS stop sites on rRNA were visualized by gel electrophoresis and quantified by densitometry. Our results reveal that oncocin binds at the peptidyl transferase center of 23S ribosomal RNA, consistent with previous high resolution structure studies. Overall, these studies show the promise of using in vivo expression to fine-tune antimicrobial peptides for improved activity along with mapping of the target rRNA binding sites in cellular milieu.

References:
Nisansala S. Muthunayake, Rabiul Islam, Ellen D. Inutan, Wesley Colangelo, Sarah Trimpin, Philip R. Cunningham, and Christine S. Chow, Biochemistry 2020, 59(36): 3380-3391

Keywords: PrAMPs, DMS probing, bacterial resistance

27. The role of Gis2 in fine tuning gene regulation under hydrogen peroxide and fluconazole stress in C. neoformans

Jan Naseer Kaur (Department of Microbiology and Immunology, University at Buffalo), Jay Leipheimer (Department of Microbiology and Immunology, University at Buffalo), John C. Panepinto (Department of Microbiology and Immunology, University at Buffalo)

Abstract not available online - please check the printed booklet.

28. RNA Regulates the Morphology of Multiphasic Biomolecular Condensates

Taranpreet Kaur (Department of Physics, University at Buffalo, Buffalo, NY, USA), Muralikrishna Raju (Department of Chemistry, Iowa State University, Ames IA, USA), Ibraheem Alshareedah (Department of Physics, University at Buffalo, Buffalo, NY, USA), Richoo B. Davis (Department of Physics, University at Buffalo, Buffalo, NY, USA), Davit A. Potoyan (Department of Chemistry, Iowa State University, Ames IA, USA), Priya R. Banerjee (Department of Physics, University at Buffalo, Buffalo, NY, USA)

Abstract:
Biomolecular condensates formed through liquid-liquid phase separation (LLPS) such as the nucleolus, para-speckles and stress granules are known to organize into multi-layered structures in vivo with distinct sub-compartments. This multilayered structuring is a result of LLPS of constituents into multiple coexisting phases driven by many-body interactions within a dense network of proteins and RNA. Utilizing a model multicomponent system comprising of a Prion-like polypeptide (PLP), an Arginine-rich polypeptide (RLP), and RNA, we investigate the role of competitive RNA-protein and protein-protein interactions in regulating the composition and spatial organization of biomolecular condensates. We show that in absence of RNA, RLP enhances the phase separation of PLP, forming associative PLP-RLP condensates. Contrastingly, in the presence of RNA, competition between PLP and RNA for the shared binding partner (RLP) leads to PLP-RLP de-mixing into bi-phasic condensates with distinct compositions. Employing a combination of biophysical experiments and computer simulation, we reveal a rich variety of multiphasic pattering of these co-existing condensates ranging from completely engulfed to partially engulfed to completely separated as well as Janus droplets. We show that these diverse patterns of bi-phasic condensates can be controlled via changing the amount of RNA as well as sequence perturbations within the RNA-protein interaction network. These results establish a hitherto unknown link between relative interfacial energies of coexisting fluids at the mesoscale and the protein-RNA interactions at the molecular level. In essence, our results shed light on the phase-behavior of a multi-component RNA-protein system and uncover a regulatory role of competitive RNA-protein interactions in dictating the spatial organization of multiphasic bio-condensates.

Keywords: Liquid-Liquid phase separation, Multi-layered bio-condensates

29. Impact of transcription start site heterogeneity on HIV-1 5′UTR structure and conformational dynamics

Jonathan P. Kitzrow (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Shuohui Liu (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Kevin Jamison (Department of Physics, The Ohio State University, Columbus, OH 43210), Dennis Bong (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Michael Poirier (Department of Chemistry and Biochemistry, Center for RNA Biology, Department of Physics, The Ohio State University, Columbus, OH 43210), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)

Abstract:
The highly conserved ~350-nt 5′UTR of the HIV-1 RNA genome (gRNA) is central to the regulation of virus replication. Interactions between the 5′UTR and viral and host factors are critical to the production of infectious virions. Previous biochemical and NMR experiments support a model in which the 5′UTR can adopt at least two mutually exclusive conformational states. In one state, the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed and the genome is competent for dimerization and packaging into assembling virions. We have previously characterized the intrinsic conformational dynamics of a 238-nt 5′UTR lacking the 5′ TAR/PolyA hairpin domains using single-molecule Förster resonance energy transfer (FRET). We showed that viral and host factor binding modulates the RNA conformation and dynamics. Recently, heterogenous transcription start site selection has been implicated in the localization of HIV-1 gRNA; transcripts with three 5′ guanosines (3G) are abundant in the cytoplasm while 1G transcripts are packaged into virions. Here, we investigate the structure and conformational dynamics of the full 1G and 3G 5′UTRs using a new bifacial peptide nucleic acid-labeling strategy to position internal fluorophores. In-gel selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) and FRET support the conclusion that 1G and 3G 5′UTRs adopt different conformations. Single-molecule FRET studies to probe the conformational dynamics of 1G/3G RNAs are underway. This work shows how a 2-nt sequence difference within a 9.4 kb HIV-1 gRNA can shift the global 5′UTR conformation, and supports the development of RNA-binding therapeutics that can shift the equilibrium to a non-functional state.

Keywords: HIV-1, RNA Structure, FRET

30. Ccr4 is required for translational repression during stress

Corey Knowles (Department of Microbiology and Immunology Jacobs School of Medicine and Biomedical Sciences University at Buffalo), Amanda L. M. Bloom (Department of Microbiology and Immunology Jacobs School of Medicine and Biomedical Sciences University at Buffalo), John C. Panepinto (Department of Microbiology and Immunology Jacobs School of Medicine and Biomedical Sciences University at Buffalo)

Abstract not available online - please check the printed booklet.

31. Divalent metal-ion tunable catalytic flexibility of the lariat-debranching enzyme, Dbr1

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

Abstract not available online - please check the printed booklet.

32. Understanding the role of CLP1 in mammalian mRNA transcription and cleavage

Geneva LaForce (Department of Genetics and Genome Sciences, Case Western Reserve University), Jordan S. Farr (Department of Genetics and Genome Sciences, Case Western Reserve University), Cydni Akesson (Department of Genetics and Genome Sciences, Case Western Reserve University), Evren Gumus (Department of Medical Genetics, Faculty of Medicine, University of Harran), 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 not available online - please check the printed booklet.

33. Human spliceosomal snRNA sequence variants generate variant spliceosomes

Justin W Mabin (Biomolecular Chemistry, University of Wisconsin-Madison), Peter Lewis (Biomolecular Chemistry, University of Wisconsin-Madison), David Brow (Biomolecular Chemistry, University of Wisconsin-Madison), Heidi Dvinge (Biomolecular Chemistry, University of Wisconsin-Madison)

Abstract not available online - please check the printed booklet.

34. Deducing the role of ADR-2 in neural immune gene regulation in C. elegans

Ananya Mahapatra (Genome, Cellular and Developmental Biology, Indiana University), Heather A. Hundley (Medical Sciences, Indiana University)

Abstract not available online - please check the printed booklet.

35. Kinetoplast RNA Editing Helicase 1 plays a distinct role in RNA editing profile of mitochondrial transcripts in Trypanosoma brucei

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 mRNAs require post-transcriptional uridine (U) insertion/deletion (U-indel) editing, which is directed by trans-acting gRNAs and mediated by a holoenzyme comprised of the catalytic RECC, the REH2C helicase complex, and non-catalytic RNA editing substrate binding complex (RESC). Another ATP-dependent RNA helicase, Kinetoplast RNA Editing Helicase 1 (KREH1), interacts transiently with RECC and RESC. Previous evidence suggested a role for KREH1 gRNA removal during editing progression. Here, we show that a KREH1 null mutant (KO) in procyclic form T. brucei exhibits a modest decrease in a subset of edited mRNAs compared to wild type (WT), in particular ATPase (A6) mRNA. High throughput sequencing followed by TREAT analysis of A6 mRNA in KREH1 KO cells revealed no evidence for KREH1 function in gRNA removal. Rather, we identified multiple sequences in the KREH1 KO with a long stretch of pre-edited sequence followed by one or two modified editing sites outside of the first gRNA. These data suggest a role of KREH1 in modulation of RNA structure. Overexpression (OE) of WT KREH1 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 while pre-edited mRNAs were unaffected. Together, these data suggest that 3’ to 5’ editing progression is impaired in KREH1 OE. To define the role of KREH1 ATP-binding, we overexpressed KREH1 mutated in the conserved ATP-binding motif. Mutant OE leads to cell death, indicating a dominant negative (DN) phenotype. This is accompanied by a dramatic decrease in almost all edited mRNAs with concomitant accumulation of pre-edited transcripts, including 2 of 3 moderately edited mRNAs. Thus, unlike WT KREH1, the DN mutant impairs initiation of editing. Overexpression of DN KREH1 alters RESC homeostasis by interfering with the interaction between its GRBC module and a subset of additional RESC proteins. Together, these data are consistent with a model in which GRBC and RECC assemble normally on mRNA, while KREH1-mediated ATP hydrolysis is necessary for subsequent assembly of a functional RESC, capable of promoting editing initiation. Thus, KREH1 is important for both initiation and progression of U-indel editing.

Keywords: Trypanosoma, RNA editing, RNA helicase

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

Chaitali Misra (Departments of Biochemistry, University of Illinois, Urbana-Champaign), Ullas V. Chembazhi, Sarah N. Matatov, Sushant Bangru, and Auinash Kalsotra (Departments of Biochemistry, University of Illinois, Urbana-Champaign)

Abstract not available online - please check the printed booklet.

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

Rushdhi Rauff (Department of Chemistry and Biochemistry, Kent State University), Paul DAmico (Department of Chemistry and Biochemistry, Kent State University), Stefani Schmocker (Department of Chemistry, University of Michigan), Jiale Xie (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.

38. Therapeutic potential of splicing: SRSF2 binding modulation in MDM2 alternative splicing

Matias Montes (Molecular, Cellular and developmental biology program, The Ohio State University), Dawn Chandler (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital)

Abstract not available online - please check the printed booklet.

39. Understanding the cellular and molecular functions of ADAR3

Priyanka Mukherjee (Cell, Molecular and Cancer Biology Graduate Program, Indiana University School of Medicine Bloomington), Eimile Oakes (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.

40. The use of RNA secondary and tertiary structure prediction software for remote undergraduate laboratory and research during pandemic distance learning

Sanjana Nallagatla (Adlai E. Stevenson High School), Joshua E. Sokoloski (Chemistry Department, Salisbury University)

Abstract:
The covid-19 pandemic has forced many undergraduate institutions to move their STEM instruction to online or remote modalities. Upper level, research based courses have a particular challenge to provide meaningful experiential learning opportunities for students. Here, we present a summary of free web-based RNA secondary and tertiary structure prediction software and how they can be used for both remote upper level courses and undergraduate/high school remote research. We evaluated several programs for their reliability and ease of use including Mfold 1, SPOT-RNA, RNAComposer, Vsfold5 4, and RNAfold 5. We also used these free web-based programs to analyze the structure of SARS-CoV-2 viral RNA as an example of upper level experiential learning during times of remote instruction. Using several structure prediction programs, we have found that a conserved coronavirus eight nucleotide sequence is involved in significant secondary and tertiary structure that differs from the structural context of the eight nucleotide sequence in other coronaviruses. In the MERS viral RNA, this octet is not significantly involved in secondary structures; however, when examining the secondary structure of SARS-CoV-2, this octet almost always forms a stem of 3 base pairs with a 5 nucleotide loop. In addition, the octet participates in a lot of hydrogen bonding when looking at the tertiary structure of SARS-CoV-2, but in MERS’s tertiary structure, the octet did not form any hydrogen bonds with the other nucleotides. These results show that the structural bioinformatics work of the past decade has allowed for meaningful research and teaching experiences to continue in a remote environment.

References:
1. M. Zuker. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 (13), 3406-3415, 2003.
2. Singh, J., Hanson, J., Paliwal, K., Zhou, Y. RNA secondary structure prediction using an ensemble of two-dimensional deep neural networks and transfer learning. Nat Commun 10, 5407 (2019)
3. Popenda, M., Szachniuk, M., Antczak, M., Purzycka, K.J., Lukasiak, P., Bartol, N., Blazewicz, J., Adamiak, R.W. Automated 3D structure composition for large RNAs, Nucleic Acids Research, 2012, 40(14):e112.
4. Dawson, W., Fujiwara, K., Futamura, Y., Yamamoto, K., and Kawai, G. (2006) A method for finding optimal RNA secondary structures using a new entropy model (vsfold). Nucleosides, Nucleotides, and Nucleic Acids 25, 171-189.
5.Vienna RNA webservices: rna.tbi.univie.ac.at/

Keywords: RNA Secondary Structure, SAR-CoV-2, RNA Folding

41. Repressing Ago2 mRNA translation by Trim71 maintains pluripotency through inhibiting let-7 microRNAs

Mariah Novak (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester), Qiuying Liu (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester), Wenqian Hu (Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester)

Abstract not available online - please check the printed booklet.

42. Eukaryotic translation initiation factor 4B levels are negatively associated with metastatic potential and immune evasion in murine breast cancer models

Aravind Srinivasan (Department of Immunology, Roswell Park Comprehensive Cancer Center), Leila Zabihi Diba (Department of Biological Sciences, State University of New York at Buffalo), Thomas Ossevoort (Department of Biological Sciences, State University of New York at Buffalo), Joseph Barbi (Department of Immunology, Roswell Park Comprehensive Cancer Center), Sarah E. Walker (Department of Biological Sciences, State University of New York at Buffalo)

Abstract:
The primary factor responsible for most cancer mortality is the metastatic spread of malignant cells. However, the molecular determinants of metastatic growth and disease outcome in breast cancer, especially those active at the protein level, remain incompletely understood. While cancer-related over-expression of specific translation initiation factors, including the eukaryotic translation initiation factor 4 (eIF4) group of proteins, were previously implicated in as tumor-promoting, a more recent analysis of patient survival data in TCGA suggested a significant survival benefit for relatively high expression of eIF4B, an RNA-binding protein that activates the eIF4F (the cytoplasmic mRNA cap-binding-complex of eIF4A, eIF4G, and eIF4E). In line with this observation, we found that eIF4B levels were inversely related to metastatic potential in two widely used murine breast cancer models (i.e., EMT6 and 4T1). While eIF4F levels were consistent across both cell lines, eIF4B levels were much lower in the more metastatic 4T1 cells relative to the less invasive EMT6 line that displayed robust eIF4B protein levels. Interestingly, while shRNA-mediated knockdown of eIF4B failed to significantly affect the development of primary tumors after implantation in mouse mammary fat pads, both cell lines displayed increased metastatic spread upon eIF4B silencing. Preliminary findings link naturally low and experimentally suppressed eIF4B levels to a greater ability to upregulate the immune checkpoint receptor ligand PD-L1 - a well characterized mechanism of immune evasion known to be controlled by at the level of translation by the activity of other eIF4 factors. Together, these data suggest that high eIF4B levels promote a translation program that undercuts the ability of tumor cells to evade the host immune system and thrive at secondary sites. Our findings have implications for predicting the development of metastatic disease in breast cancer patients and the treatment decision making process. Further study of the translational signature associated with high and low eIF4B levels will likely inform future characterization of novel treatment targets and biomarkers.

Keywords: eIF4B

43. Quality control of piRNAs mediated by nontemplated nucleotide addition in C. elegans

Benjamin Pastore (Department of Biological Chemistry and Pharmacology, The Ohio State University), Hannah L. Hertz (Department of Biological Chemistry and Pharmacology, The Ohio State University), Ian F. Price (Department of Biological Chemistry and Pharmacology, The Ohio State University), Wen Tang (Department of Biological Chemistry and Pharmacology, The Ohio State University)

Abstract not available online - please check the printed booklet.

44. SHAPE probing of the HIV-1 reverse transcriptase initiation complex reveals RNA flexibility changes adjacent to the primer binding site

Chathuri Pathirage (Department of Chemistry and Biochemistry, The Ohio State University), William Cantara (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.

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

Ian F. Price (Biological Chemistry and Pharmacology, The Ohio State University), Hannah L. Hertz (Biological Chemistry and Pharmacology, The Ohio State University), Benjamin Pastore (Biological Chemistry and Pharmacology, The Ohio State University), Wen Tang (Biological Chemistry and Pharmacology, The Ohio State University)

Abstract not available online - please check the printed booklet.

46. Amino acid composition in cytosolic and mitochondrial ribosomal proteins

Seyoung Ree (Notre Dame Academy/High School), Craig L. Zirbel (Math and Statistics Department, Bowling Green State University), Neocles Leontis (Chemistry Department, Bowling Green State University)

Abstract:
Animals have two ribosomes, cytosolic and mitochondrial, and they are under different types of evolutionary pressure. Mitochondria are the site of cellular respiration, so mitochondrial ribosomes, like other components of the matrix, are more exposed to reactive oxygen species (ROS), the by-products of cellular respiration. In previous work [1], we have provided evidence that mitochondrial ribosomal RNAs have evolved to protect themselves by reducing the number of guanine nucleotides, which are more easily oxidized than the other nucleotides. Mitochondrial ribosomes also acquired more and longer proteins since their divergence from bacterial origins, so that almost all of the surface of the RNA is protected by solvent from proteins. Another mechanism through which mitochondrial ribosomes may have evolved to protect themselves from ROS is by populating their ribosomal proteins with amino acids that carry electrons from glutathione in the solvent [2] to the RNA. These amino acids include CYS, TRP, TYR, and MET. We analyzed the CYS, TRP, TYR, and MET composition of mitochondrial and cytosolic ribosomal protein sequences of different groups of animals and plants. We find that the mitochondrial ribosomes have a higher percentage of CYS, TRP, TYR, and MET amino acids than the cytosolic ribosomes. Also, animals with high metabolisms, which have a higher rate of cellular respiration in the mitochondria, or larger problems with ROS, may have a higher composition of CYS, TRP, TYR, and MET amino acids in their proteins.

References:
[1] How to fold and protect mitochondrial ribosomal RNA with fewer guanines, by Maryam Hosseini, Poorna Roy, Marie Sissler, Craig L. Zirbel, Eric Westhof and Neocles Leontis. Published September 12, 2018 in Nucleic Acids Research. https://doi.org/10.1093/nar/gky762
[2] Ribas V, García-Ruiz C, Fernández-Checa JC. Glutathione and mitochondria. Front Pharmacol. 2014 Jul 1;5:151. PMID: 25024695; PMCID: PMC4079069. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079069/

Keywords: mitochondrial ribosomes, reactive oxygen species, amino acids

47. Human Lysyl tRNA Synthetase Interaction with Nucleic Acid Hairpins

Mary Anne Refaei (Dept of Chemistry, University of Cincinnati), Sheng Liu (Dept of Chemistry, University of Cincinnati), Pearl Tsang (Dept of Chemistry, University of Cincinnati)

Abstract not available online - please check the printed booklet.

48. Hidden Breaks in Rice Chloroplast rRNA Revealed by Structure-seq2

Kaylin M. Gaudette (Chemistry Department, University of Pittsburgh at Johnstown), Colby K. Hillegass (Chemistry Department, University of Pittsburgh at Johnstown), Cooper J. Halliday (Biology Department, University of Pittsburgh at Johnstown), Laura E. Ritchey (Chemistry Department, University of Pittsburgh at Johnstown)

Abstract:
Within plants, chloroplasts are light-harvesting organelles that retain their own limited genomes. Part of this genome encodes chloroplast ribosomal RNA (rRNA) that is used to translate chloroplast-specific genes. Interestingly, the large subunit (23S) of the chloroplast rRNA harbors hidden breaks. (1-3) These covalent breaks in the backbone of the RNA are thought to be caused by endonucleases during or after assembly of the ribosome such that the ribosome structure remains intact. It has been proposed that hidden breaks are necessary for efficient translation in the chloroplast, and they have been identified in rRNAs of a select few organisms. By analyzing control Structure-seq2 data, (4-6) we were able to identify the presence of these hidden breaks in rice chloroplast rRNA and bacterial rRNA. We have also found evidence that the extent that the break occurs may be affected by stress conditions. Future plans are to study the extent of these hidden breaks in the chloroplast, mitochondria, and/or bacterial rRNA of various organisms to identify any trends in either sequence or structure. We will also study how stress affects the extent of breakage to begin to elucidate how these breaks affect translation. Currently, initial results in stress-treated rice, coffee, and spinach using reverse transcription and PCR spanning the hidden break will be presented. Preliminary results indicate that these hidden breaks are not found in every rRNA molecule. Advice on additional appropriate assays to perform at a primarily undergraduate institute with limited resources would be much appreciated.

References:
1. Nishimura, K., et al. Plant J 63 (5), 766-777. (2010)
2. Liu, J., et al. Plant Physiol 168 (1), 205-221, (2015).
3. Bieri, P., et al. EMBO J 36 (4), 475-486, (2017).
4. Ritchey, L.E. et al. Nucleic Acids Res 45 (14), e135 (2017).
5. Su, Z,... Ritchey, L.E. et al. PNAS.115 (48), 12170-12175, (2018)
6. Ritchey, L.E., et al. RNA 26 (10), 1431-1447, (2020)

Keywords: RNA structure, rRNA, chloroplast

49. Using RNA nanoring scaffolds to modulate the fluorescent properties of silver nanoclusters

Lewis Rolband (Department of Chemistry, University of North Carolina at Charlotte), Liam Yourston (Department of Physics, University of Nebraska Omaha), Caroline West (Department of Chemistry, University of North Carolina at Charlotte), Alexander Lushnikov (Nanoimaging Core Facility at the University of Nebraska Medical Center), Kirill Afonin (Department of Chemistry, University of North Carolina at Charlotte), Alexey Krasnoslobodtsev (Department of Physics, University of Nebraska Omaha)

Abstract:
Nucleic acid nanoparticles (NANPs) are self-assembling which reliably fold into sequence determined architectures. By using NANPs as a scaffold, functional moieties can be spatially oriented with defined structural relationships to one another. Upon combining DNA oligonucleotides, which templated the formation of silver nanoclusters (AgNCs), with an RNA based nanoring, the resultant inorganically modified biopolymer exhibited unexpected tunable fluorescent properties. The AgNC formation around the nanoring was found to be localized to the templating DNA strand, which resulted in the formation of 6 individual AgNCs around the hexameric RNA nanoring scaffold. The optical properties of the fluorescent AgNC-NANP complexes were characterized thoroughly using 2D analysis of excitation-emission matrices including their evolution with time of storage. Two distinct fluorescent species were observed with the emitted color being either ‘red’ (λExc/λEm = 565/623 nm) or ‘green’ (λExc/λEm = 440/523 nm). The relative stability of each of these species was reliant upon the orientation of the AgNC in respect to the NANP scaffold. ‘Red’ fluorescent species began to emit ‘green’ over a storage period, due to oxidation of the AgNC. The rate of decay from ‘red’ to ‘green’ was dramatically slower for AgNCs which were oriented towards the inside of the ring as opposed to those oriented towards the outside. The ‘red’ fluorescence of the oxidized AgNCs was restored by re-reduction of the Ag+ to Ag0. The findings presented herein display the complexity of AgNC-NANP fluorescence properties and add to the fluorescent hybrid biomaterials toolkit.

References:
E. Hong, J. R. Halman, A. B. Shah, E. F. Khisamutdinov, M. A. Dobrovolskaia and K. A. Afonin, Nano Lett., 2018, 18, 4309–4321.
J. R. Halman, K.-T. Kim, S.-J. Gwak, R. Pace, M. B. Johnson, M. R. Chandler, L. Rackley, M. Viard, I. Marriott, J. S. Lee and K. A. Afonin, Nanomedicine, 2020, 23, 102094.
K. A. Afonin, D. Schultz, L. Jaeger, E. Gwinn and B. A. Shapiro, Methods Mol. Biol., 2015, 1297, 59–66.
L. E. Yourston, A. Y. Lushnikov, O. A. Shevchenko, K. A. Afonin and A. V. Krasnoslobodtsev, Nanomaterials, 2019, 9, 613.
D. Schultz and E. Gwinn, Chem. Commun., 2011, 47, 4715– 4717.
E. Gwinn, D. Schultz, S. M. Copp and S. Swasey, Nanomaterials, 2015, 5, 180–207.
E. Thyrhaug, S. A. Bogh, M. R. Carro-Temboury, C. S. Madsen, T. Vosch and D. Zigmantas, Nat. Commun., 2017, 8, 15577.
H. Ramsay, D. Simon, E. Steele, A. Hebert, R. D. Oleschuk and K. G. Stamplecoskie, RSC Adv., 2018, 8, 42080–42086.

Keywords: Silver Nanoclusters, Fluorescence, Nucleic Acid Nanoparticle

50. Using the Bacillus subtilis thrS T-box riboswitch to determine the importance of conserved T-box elements

Alexander T Runyon (Microbiology Department, The Ohio State University), Tina Henkin (Microbiology Department, The Ohio State University), Frank Grundy (Microbiology Department, The Ohio State University)

Abstract not available online - please check the printed booklet.

51. The effect of DHX36/G4R1 on the transcription of an abnormal DNA sequence linked to ALS

Siara N. Sandwith (Department of Biology, Ball State University), Yi-Ju Tseng, A. Krans, K. Green, Stephen Goutman, Eva Feldman, Peter Todd (Department of Neurology, University of Michigan), Eric Routh, Y.H. Wang, James Vaughn (Department of Cancer Biology, Wake Forest School of Medicine), H. Raimer, J. Wang (Department of Biochemistry and Molecular Biology, John Hopkins School of Medicine), Adam Richardson, Antonio Chambers, Melissa Smaldino, Philip Smaldino (Department of Biology, Ball State University), Peter Beerbower (Department of Plant Sciences, North Dakota State University)

Abstract:
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results in the breakdown of motor neurons, leading to severe deficiencies in eating, breathing, and moving; 80% of all ALS patients succumb to the disease within 5 years of diagnosis. The most common ALS-linked mutation occurs in the C9ORF72 gene (C9), consisting of a GGGGCC-sequence which becomes repeated hundreds to thousands of times, compared to healthy individuals who have <30 repeats. The mutated C9 sequence folds into extensive G-quadruplex (G4) DNA structures, which form within guanine-rich DNA and RNA sequences. G4s generally provide negative regulation on transcription and translation. G4-helicases such as DHX36/G4R1 (aliases: RHAU), potentially provide positive regulation on these processes via unwinding G4s. C9-repeat G4-DNA structures are partially unwound and transcribed, and the resulting RNAs form toxic RNA foci that sequester RNA binding proteins. It is unknown if G4R1 binds to the C9-repeat expansion or if it affects C9-repeat transcription. We hypothesized that G4R1 directly binds to and unwinds C9-G4 DNA and increases the transcription of toxic C9-repeat RNAs. To test this, we expressed and isolated recombinant DHX36/G4R1 and perform gel mobility shift assays with C9-G4 DNA. Furthermore, we used a C9-repeat in vitro transcription assay to determine if DHX36/G4R1 affects C9-repeat transcription. We found that DHX36/G4R1 directly and preferentially binds to C9 G4 DNA and facilitates the transcription of toxic C9 RNA in vitro. These data suggest that DHX36/G4R1 has the biochemical capabilities to facilitate the production of toxic C9 repeat RNAs, which are observed in C9 patient cells.

Keywords: G-quadruplexes, DHX36G4R1, ALS

52. Enzymatic synthesis of pseudouridine

Tristan Sanford (Chemsitry; SIUE), Andrew Riley (Chemsitry; SIUE), Austin Woodard (Chemistry; SIUE), Mina Sumita (Chemistry; SIUE)

Abstract not available online - please check the printed booklet.

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

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

Abstract not available online - please check the printed booklet.

54. Characterization of a putative toxin-antitoxin locus in Shigella flexneri

David D. Sarpong (Biology, Ohio University), Peter W. Coschigano (Biomedical Sciences, Ohio University College of Osetopathic Medicine), Erin R. Murphy (Biomedical Sciences, Ohio University College of Osetopathic Medicine)

Abstract not available online - please check the printed booklet.

55. Biochemical dissection of translational control RBPs that are mutated in neurological disorders

MaKenzie R. Scarpitti (The Biomedical Sciences GraduateProgram, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University), Michael G. Kearse (The Biomedical Sciences GraduateProgram, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University)

Abstract:
Defects in translational control are common in many neurological disorders. In some cases, mutations in RNA-binding proteins (RBPs) that are highly enriched in the brain cause these defects and disorders. For example, loss of function of the RBP encoded by the fragile X gene, FMRP, causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders (ASD). FMRP is thought to bind target mRNAs and then inhibit translation by sterically blocking the A site of the elongating ribosome. However, it remains highly debated how FMRP binds to mRNAs. Here, we developed a specialized reporter system using a split nanoLuciferase strategy to determine the requirements of FMRP to inhibit translation elongation. We have further adapted this approach to investigate other brain-enriched RBPs mutated in schizophrenia and ASD. Together, this work elucidates translation regulatory mechanisms that are critical to maintain homeostatic levels of protein synthesis in healthy cells and points to the mechanisms that underly neurological disease.

Keywords: translation, ribosomes, RBP

56. : Discovering the role of SAP18 in transcriptional regulation by creating a SAP18 knockdown line.

Claire Schaef (Biochemistry Program, Department of Biology, St. Bonaventure University), Xiao-Ning Zhang (Biochemistry Program, Department of Biology, St. Bonaventure University)

Abstract:
SAP18 is a highly conserved gene in eukaryotic systems, shown to control stress-related responses through transcriptional regulation. The highly conserved nature of SAP18 suggests that this gene is important for proper life functioning. As one of the three subunits of the apoptosis and splicing associated complex (ASAP), SAP18functions in regulating RNA metabolism in various ways, recruiting histone deacetylases and controlling polyadenylation for example. While known to function in transcriptional silencing, it is largely unknown which specific genes are regulated by SAP18 as part of the ASAP complex. To study the processes that SAP18 regulates, a SAP18-specific amiRNA was cloned under the control of the native SAP18 promoter. To create SAP18 knockdown lines, this construct was introduced into the flowering pSAP18::SAP18-GFP qrt homozygote plants through transformation with Agrobacteria. The first generation was screened with kanamycin. This gave 42 candidate transformants. Then the GFP signal strength was visualized in their pollen tetrads with confocal imaging to determine the knockdown effect. 19 plants which tetrads had two dim and two bright pollen grains were kept and allowed to seed. They were reexamined in second generation, looking for plants homozygous for the SAP18knockdown. In both rounds of screening, several abnormal pollen phenotypes were repeatedly observed, including multiple plants with closed anthers or shriveled pollen. Expression levels of SAP18 in 7 promising lines were then analyzed with RT-qPCR, comparing SAP18 expression to Col-0 wild type. Preliminary results suggest potential success in several silencing lines, with the best knockdown of SAP18 expression to 0.46 and 0.53 of wild type. Once these results are confirmed, the plant lines with the least SAP18 expression will be used to explore how a decrease in SAP18 alters the expression of other genes. If SAP18 is as important as its highly conserved nature indicates, we expect to see abnormalities in the knockdown lines, such as in abiotic and biotic stress responses, and fertility. Based on the prevalence of shriveled pollen seen in mutant lines, a hypothesis is that SAP18 may regulate gamete formation.

References:
Chen, S. L., Rooney, T. J., Hu, A. R., Beard, H. S., Garrett, W. M., Mangalath, L. M., . . . Zhang, X. (2019). Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana. Frontiers in Plant Science, 10. doi:10.3389/fpls.2019.01116

Song, C., & Galbraith, D. W. (2006). AtSAP18, An Orthologue of Human SAP18, is Involved in the Regulation of Salt Stress and Mediates Transcriptional Repression in Arabidopsis. Plant Molecular Biology, 60(2), 241-257. doi:10.1007/s11103-005-3880-9

Zhang, Y., Iratni, R., Erdjument-Bromage, H., Tempst, P., & Reinberg, D. (1997). Histone Deacetylases and SAP18, a Novel Polypeptide, Are Components of a Human Sin3 Complex. Cell, 89(3), 357-364. doi:10.1016/s0092-8674(00)80216-0

Keywords: SAP18, artificial microRNA, transcriptional regulation

58. Evidence for autoregulation of bS21 in Flavobacteria.

Elan Shatoff (Department of Physics, The Ohio State University), Bappaditya Roy, Zakkary A. McNutt, Bethany L. Boleratz, Dean E. Watkins (Department of Microbiology, Ohio State Biochemistry Program, The Ohio State University), Vikash Jha, Dushyant Jahagirdar, Kaustuv Basu (Department of Anatomy and Cell Biology, McGill University), Joaquin Ortega (Department of Anatomy and Cell Biology, McGill University), Ralf Bundschuh (Department of Physics, The Ohio State University), Kurt Fredrick (Department of Microbiology, The Ohio State University)

Abstract not available online - please check the printed booklet.

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

Jalal K. Siddiqui (Comprehensive Cancer Center, The Ohio State University), Wayne O. Miles (Comprehensive Cancer Center, The Ohio State University)

Abstract not available online - please check the printed booklet.

60. RNA Deamination as a Mechanism to Regulate Gene Expression in Staphylococcus aureus

Hailee M. Sorensen (Biological Sciences, Ohio University), Donald L. Holzschu (Biological Sciences, Ohio University), Richard E. Wiemels (Biological Sciences, Ohio University), Ronan K. Carroll (Biological Sciences, Ohio University)

Abstract not available online - please check the printed booklet.

61. Transcriptome-wide high-throughput mapping of protein-RNA occupancy profiles using POP-seq

Mansi Srivastava (BioHealth Informatics, School of Informatics and Computing, IUPUI), Gayathri Panangipalli (BioHealth Informatics, School of Informatics and Computing, IUPUI), Rajneesh Srivastava (BioHealth Informatics, School of Informatics and Computing, IUPUI), Neel Sangani (BioHealth Informatics, School of Informatics and Computing, IUPUI), Hunter Gill (BioHealth Informatics, School of Informatics and Computing, IUPUI), Sarath Chandra Janga (BioHealth Informatics, School of Informatics and Computing, IUPUI)

Abstract:
Interaction between RNA-binding proteins (RBPs) and RNA is critical for post-transcriptional regulatory processes1. Existing high throughput methods based on crosslinking of the protein-RNA are reported to contribute to biases in the resulting protein occupancy profiles2–6. We present Protein Occupancy Profile-Sequencing (POP-seq), a phase separation-based method that does not require crosslinking thus providing unbiased protein occupancy profiles on whole cell transcriptomes. In order to compare the robustness of identified protein occupied sites, POP-seq was implemented in two phases: the first phase comprised of UV crosslinking and no-crosslinking approaches on K562 and HepG2 cells, resulting ~200,000 peaks detected across the protocols. The results were further cross-validated with the publicly available ENCODE eCLIP profile for scores of RBPs in the two cell lines. Our analysis reveals that majority of detected genes (>70%) overlap between the two approaches indicating the reproducible nature of the generated interaction maps in the two cell lines. We observed an abundance of binding sites on the intronic region of the genomic location (~40%) for both the cell lines with maximum number of POP-seq peaks detected on protein-coding genes (>85%). Cross-validation with the ENCODE eCLIP profile of RBPs demonstrated relatively higher recall for UV-crosslinking (~24% and 17%) compared to no-crosslinking approach in K562 and HepG2 cells respectively, indicating the robustness of both approaches. In the second phase, we expanded POP-seq on multiple cell lines (MCF7, A549, Jurkat and HEK293) using the no-crosslinking approach resulting in ~60,000 reproducible peaks between replicates and are currently investigating their significance across cell types. Altogether, our data supports POP-seq as a robust and cost-effective method enabling comprehensive mapping and understanding of post-transcriptional regulatory networks.

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.Silverman, I. M. et al. RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome. Genome Biol. 15, R3 (2014).
5.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).
6.Trendel, J. et al. The Human RNA-Binding Proteome and Its Dynamics during Translational Arrest. Cell 176, 391-403.e19 (2019).

Keywords: transcriptome, phase-separation, POP-seq

62. Role of SARS-CoV-2 in Altering the RNA-Binding Protein and miRNA-Directed Post-Transcriptional Regulatory Networks in Humans

Rajneesh Srivastava (Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Swapna Vidhur Daulatabad (Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Mansi Srivastava (Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University), Sarath Chandra Janga (Department of Biohealth Informatics, School of Informatics and Computing, Indiana University Purdue University)

Abstract:
The outbreak of a novel coronavirus SARS-CoV-2 responsible for the COVID-19 pandemic has caused a worldwide public health emergency. Due to the constantly evolving nature of the coronaviruses, SARS-CoV-2-mediated alterations on post-transcriptional gene regulations across human tissues remain elusive. In this study, we analyzed publicly available genomic datasets to systematically dissect the crosstalk and dysregulation of the human post-transcriptional regulatory networks governed by RNA-binding proteins (RBPs) and micro-RNAs (miRs) due to SARS-CoV-2 infection. We uncovered that 13 out of 29 SARS-CoV-2-encoded proteins directly interacted with 51 human RBPs, of which the majority of them were abundantly expressed in gonadal tissues and immune cells. We further performed a functional analysis of differentially expressed genes in mock-treated versus SARS-CoV-2-infected lung cells that revealed enrichment for the immune response, cytokine-mediated signaling, and metabolism-associated genes. This study also characterized the alternative splicing events in SARS-CoV-2-infected cells compared to the control, demonstrating that skipped exons and mutually exclusive exons were the most abundant events that potentially contributed to differential outcomes in response to the viral infection. A motif enrichment analysis on the RNA genomic sequence of SARS-CoV-2 clearly revealed the enrichment for RBPs such as SRSFs, PCBPs, ELAVs, and HNRNPs, suggesting the sponging of RBPs by the SARS-CoV-2 genome. A similar analysis to study the interactions of miRs with SARS-CoV-2 revealed functionally important miRs that were highly expressed in immune cells, suggesting that these interactions may contribute to the progression of the viral infection and modulate the host immune response across other human tissues. Given the need to understand the interactions of SARS-CoV-2 with key post-transcriptional regulators in the human genome, this study provided a systematic computational analysis to dissect the role of dysregulated post-transcriptional regulatory networks controlled by RBPs and miRs across tissue types during a SARS-CoV-2 infection.

References:
Srivastava, R. et al. Role of SARS-CoV-2 in Altering the RNA-Binding Protein and miRNA-Directed Post-Transcriptional Regulatory Networks in Humans. Int. J. Mol. Sci. 2020, 21, 7090.
Gordon, D.E. et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 2020, 583, 459–468.
Blanco-Melo et al. Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. Cell 2020, 181, 1036–1045.e9.

Keywords: SARS-CoV-2, post-transcriptional regulation, RBPs and miRs

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

Halle D. Stump (Medical Sciences at Indiana University), Pranathi Vadlamani (Medical Sciences at Indiana University), Heather Hundley (Medical Sciences at Indiana University)

Abstract not available online - please check the printed booklet.

64. Investigating the regulatory consequences of maternal RNA clearance during early embryonic development

Jayesh Kumar Sundaram (Department of Biological Sciences, University of Pittsburgh), Moira Crawshaw (Mount Holyoke College), Anelisse Dominicci-Maura (University of Puerto Rico), Miler T. Lee (Department of Biological Sciences, University of Pittsburgh)

Abstract not available online - please check the printed booklet.

65. Identification of the amino acids associated with the novel ribonuclease activity of Cusativin via protein engineering and LC-MS

Priti Thakur (Rieveschl Laboratories for Mass spectrometry, University of Cincinnati, Cincinnati OH), Patrick A. Limbach (Rieveschl Laboratories for Mass spectrometry, University of Cincinnati, Cincinnati OH), Balasubrahmanyam Addepalli (Rieveschl Laboratories for Mass spectrometry, University of Cincinnati, Cincinnati OH)

Abstract:
Cytidine specific ribonuclease (RNase), cusativin, is a useful biochemical tool in Mass spectrometry (MS)-based RNA modification mapping [1-2]. This RNase has a novel characteristic of not cleaving bonds between two adjacent cytidines [3]. This characteristic makes it even more useful since it does not generate single nucleotides, instead, generate longer digestion products that would help match to genomic sequence and increase the sequence coverage. This unique feature of cusativin has encouraged us to identify the key amino acid residues associated with this unique property. Through protein engineering involving site-directed mutagenesis, and liquid chromatography coupled with tandem mass-spectrometry (LC-MS) analysis, we are identifying the key amino acid residues responsible for this behavior. We observed changes in base recognition behavior of mutants following analysis of digestion products of synthetic RNA and yeast tRNAPhe substrates. Our preliminary experimental data shows that more than two amino acid residues are responsible for absence of CpC bond cleavage by cusativin. Implications of these findings and utility for RNA modification mapping will be discussed.

References:
1. Kowalak J, et al. (1993) Nucleic Acid Research, 21(19): 4577–85.
2. Thakur P et al (2020) Analyst. 145(3):816-827.
2. Addepalli B, et al. (2017) Anal Bioanal Chem, 409:5645–5654.

Keywords: Ribonuclease, Cusativin, mass-spectrometry

66. Investigation of RNA-ligand interaction using a multidisciplinary approach

Elizabeth D Tidwell (Biophysics University of Michigan), Varun Gadkardi (Chemistry University of Michigan), Aaron Frank (Chemistry and Biophysics University of Michigan ), Markos Koutmos (Chemistry and Biophysics University of Michigan )

Abstract not available online - please check the printed booklet.

67. Polypyrimidine tract-binding protein 1 coordinates the proliferative and inflammatory responses of hepatocytes during toxin-induced liver injury and regeneration

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

Abstract:
Dynamic remodeling of mRNA splicing and translation within hepatocytes is pivotal for proper
liver regeneration1. The regenerative process in the liver is associated with a reversible adult-toneonatal switch in splicing patterns of multiple proliferation-related genes, orchestrated by a set of developmentally regulated RNA binding proteins (RBPs). Polypyrimidine tract-binding protein
1 (PTBP1) is one of these RBPs, which functions as an alternative splicing factor, apart from its
roles in regulating mRNA stability, localization, and translation (2). Although PTBP1 is abundantly
expressed in mature hepatocytes, its protein levels are significantly increased in regenerating
hepatocytes (1). Here, we demonstrate that while no basal phenotypes are apparent following
hepatocyte-specific PTBP1 knockout (PTBP1-HKO) during normal mouse liver development,
the lack of PTBP1 is detrimental to the adult liver when exposed to hepatotoxins. We find that
toxin-induced chronic liver damage by diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in
widespread porphyrin accumulation in the PTBP1-HKO livers. Intriguingly, PTBP1 HKOs exhibit
resistance to liver fibrosis and show reduced hepatocyte proliferation in response to DDCmediated hepatocellular injury. PTBP1 HKO mice also present significantly lower levels of serum
bilirubin, bile acids, and other biochemical markers of liver injury after chronic exposure to DDC.
Finally, we demonstrate that the impaired regenerative response in the absence of PTBP1 is
associated with reduced expression of inflammatory signals, but is independent of alternative
splicing and accumulation of PTBP2, a neuronal paralog of PTBP1 that is normally suppressed
in non-neuronal tissues due to its unproductive splicing by PTBP1. Thus, our findings reveal a
crucial role for PTBP1 in determining the regenerative response of hepatocytes to toxin-induced
liver injury and damage.

References:
1 Bangru, S. et al. Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote
liver regeneration. Nat Struct Mol Biol 25, 928-939, doi:10.1038/s41594-018-0129-2 (2018).
2 Romanelli, M. G., Diani, E. & Lievens, P. M. New insights into functional roles of the
polypyrimidine tract-binding protein. Int J Mol Sci 14, 22906-22932, doi:10.3390/ijms141122906
(2013).

Keywords: Polypyrimidine tract-binding protein

68. Putting the gene in genomes: long read transcriptomics from gene annotation to single cell biology

Jason G. Underwood (Pacific Biosciences), Elizabeth Tseng (Pacific Biosciences), Ting Hon (Pacific Biosciences), Vijay Ramani (Biochem and Biophysics, UCSF), Evan Eichler (Genome Sciences, University of Washington)

Abstract:
Long read mRNA sequencing methods such as PacBio’s Iso-Seq method offers high-throughput transcriptome profiling in prokaryotic and eukaryotic cells. By avoiding the transcript assembly problem and instead sequencing full-length cDNA, Iso-Seq has emerged as the most accurate technology for annotating isoforms and, in turn, improving proteome predictions in a wide variety of organisms. Improvements in library preparation, sequencing throughput, and bioinformatics has enabled the Iso-Seq method to be complete solution to apply to a variety of difficult biological questions.

We demonstrate how highly accurate transcript reads can annotate human genomes and distinguish paralogs that are over 99% identical. We show recent cases where the Iso-Seq data was instrumental in generating quality annotations for tricky genomic regions including some that for the last 20 years have remained gaps in human reference assemblies. (1-4)

We further show recent data from multiple single cell platforms where the workflows have been adapted for the PacBio platform with cell barcode and UMI information directly accessible in the long read data. This enables isoform-resolution at the single cell level and we use this to explore isoforms expressed during the human and mouse cell cycle.

References:
1. Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes.
Hsieh P et al.
Science. 2019 Oct 18;366(6463) doi: 10.1126/science.aax2083.

2. Transcriptional fates of human-specific segmental duplications in brain.
Dougherty ML, Underwood JG et al.
Genome Res. 2018 Sep 18. doi: 10.1101/gr.237610.118.

3. High-resolution comparative analysis of great ape genomes
Kronenberg ZN et al.
Science 2018, DOI: 10.1126/science.aar6343

4. The structure, function, and evolution of a complete human chromosome 8
Logsdon et al.
biorxiv DOI:10.1101/2020.09.08.285395

Keywords: transcriptomics, isoforms, single cell

69. Cellular plasticity balances the metabolic and proliferation dynamics of a regenerating liver

Ullas Valiya Chembazhi (Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL), Sushant Bangru (Department of Biochemistry, Cancer Center@Illinois), Mikel Hernaez (Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, IL, United States. 4 Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Navarra, Spain.), Auinash Kalsotra (Department of Biochemistry, Cancer Center@Illinois)

Abstract:
The adult liver has exceptional ability to regenerate and can replenish up to 70% of the lost tissue
mass and functionality within weeks after surgical resection. Accumulating evidence suggest that
injury provokes adult livers to re-acquire fetal-like characteristics that facilitate regeneration (1-3).
Recently, we showed that chronic liver injury induces major expression changes in RNA binding
proteins like ESRP2, to re-activate the neonatal splicing and transcriptional programs in adult
hepatocytes (2,4). However, mechanisms that enable sustenance of normal metabolic activities
during regeneration remains unclear. Here, we use partial hepatectomy (PHx) in tandem with
single-cell transcriptomics to track cellular transitions and heterogeneities of ~22,000 liver cells
through the initiation, progression, and termination phases of mouse liver regeneration. Our
results reveal that following PHx, a subset of hepatocytes transiently reactivates an earlypostnatal-like gene expression program to proliferate, while a distinct population of metabolically hyperactive cells appears to compensate for any temporary deficits in liver function. Importantly, through combined analysis of gene regulatory networks and cell-cell interaction maps, we find that regenerating hepatocytes redeploy key developmental gene regulons, which are guided by
extensive ligand-receptor mediated signaling events between hepatocytes and non-parenchymal
cells. Altogether, our study offers a detailed blueprint of the intercellular crosstalk and cellular
reprogramming that balances the metabolic and proliferation requirements of a regenerating
liver (5).

References:
1 Hyun, J. et al. Epithelial splicing regulatory protein 2-mediated alternative splicing reprograms
hepatocytes in severe alcoholic hepatitis. J Clin Invest 130, 2129-2145, doi:10.1172/JCI132691
(2020).
2 Bangru, S. et al. Alternative splicing rewires Hippo signaling pathway in hepatocytes to promote
liver regeneration. Nat Struct Mol Biol 25, 928-939, doi:10.1038/s41594-018-0129-2 (2018).
3 Hyun, J. et al. Dysregulated activation of fetal liver programme in acute liver failure. Gut 68, 1076-
1087, doi:10.1136/gutjnl-2018-317603 (2019).
4 Bhate, A. et al. ESRP2 controls an adult splicing programme in hepatocytes to support postnatal
liver maturation. Nat Commun 6, 8768, doi:10.1038/ncomms9768 (2015).
5 Chembazhi, U. V., Bangru, S., Hernaez, M. & Kalsotra, A. Cellular plasticity balances the metabolic
and proliferation dynamics of a regenerating liver. 2020.2005.2029.124263,
doi:10.1101/2020.05.29.124263 %J bioRxiv (2020).

Keywords: Liver regeneration

70. Inhibition of eIF5A hypusination attenuates fibroblast activation and cardiac fibrosis

Kadiam C Venkata Subbaiah (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry), Jiangbin Wu (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry), Peng Yao (Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine & Dentistry)

Abstract:
Cardiac fibrosis is a primary contributor to heart failure (HF) and sudden death, and is considered to be an important target for HF therapy1. Cardiac fibroblast (CF) activation, including proliferation, survival, and extracellular matrix (ECM) production, is central to the initiation and maintenance of fibrotic scaring in cardiac fibrosis. However, drug molecules that target CF activation remain limited in treating cardiac fibrosis. Eukaryotic translation initiation factor 5A (eIF5A)2,3, the only protein to contain hypusine, has been found to be a modulator of cell proliferation and apoptosis4. Indeed, depletion of eIF5A or inhibition of eIF5A hypusination antagonizes diabetes-induced inflammation5 and modulates mitochondrial function6. In this study we found that increased hypusinated eIF5A protein levels were associated with cardiac fibrosis and heart dysfunction in experimental myocardial infarction (MI) mouse models. Ciclopirox (CPX), an FDA approved antifungal drug, inhibits the deoxyhypusine hydroxylase (DOHH) enzyme that is required for the hypusine formation in eIF5A. Results from both preventive and reversal mouse models suggested that CPX treatment significantly reduced MI-driven cardiac fibrosis and improved cardiac function. In vitro studies of isolated mouse primary CFs and cultured human CFs revealed that knockdown (KD) of eIF5A using lentiviral shRNA significantly abolished TGF- induced CF activation, proliferation, and collagen protein expression compared to scrambled shRNA treated cells. Proteomic analyses from mouse and human CFs suggested that KD of eIF5A significantly down-regulates the expression of proline-proline dipeptidyl motif containing proteins. Gene ontology analysis revealed that these target proteins are enriched in extracellular matrix, extracellular exosome, and cell adhesion pathways. Our findings are relevant to human heart disease as increased hypusinated eIF5A levels were observed in heart samples of dilated cardiomyopathy and ischemic heart failure patients in comparison to healthy subjects. Together, these results suggest that eIF5A could be a novel and promising therapeutic target in treating cardiac fibrosis and human heart failure.

References:
1 Gulati, A. et al.. JAMA 309, 896-908, doi:10.1001/jama.2013.1363 (2013).
2 Schuller, A. P., Wu, C. C., Dever, T. E., Buskirk, A. R. & Green, R.. Mol Cell 66, 194-205 e195, doi:10.1016/j.molcel.2017.03.003 (2017).
3 Manjunath, H. et al.. Cell Rep 29, 3134-3146 e3136, doi:10.1016/j.celrep.2019.10.129 (2019).
4 Mathews, M. B. & Hershey, J. W.. Biochim Biophys Acta 1849, 836-844, doi:10.1016/j.bbagrm.2015.05.002 (2015).

Keywords: eIF5A, Ciclopirox, Cardiac fibrosis

71. A hepatocellular Myotonic Dystrophy Type 1 model demonstrates an increased susceptibility towards anesthetics, drug induced liver injury and fatty liver disease in mice

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

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). Their sequestration in DM1 severely inhibits this maturation process in many tissues. Recent studies show that DM1 patients have increased susceptibility toward glucose intolerance, 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 gone uninvestigated.
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. Using these mice, we have demonstrated the disruptive effect of DM1 on the hepatocellular transcriptome and have characterized the transcriptomic changes driven by DM1 in the liver and have shown these lead to changes in liver morphology, inflammation, and necrosis. These mice also demonstrate increased lipid accumulation and the earlier onset of fatty liver disease, both on basal and high fat/high sugar diets, much like DM1 patients. Additionally, symptomology becomes worth with age, as also seen in patients with DM1. Finally using a variety of drug models, we have shown that these mice are particularly susceptible to drug induced liver injury and have difficulty clearing and recovering from anesthetics. These results support the idea that the liver plays a pivotal role in DM1 symptomology.

Keywords: Myotonic Dystrophy Type 1, Alternative splicing, RNA

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

Akila S. Venkataramany (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital / The Ohio State University College of Medicine), Safiya Khurshid, PhD (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital ), Pin-Yi Wang, PhD (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital ), Timothy P. Cripe, MD PhD (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital ), Dawn Chandler, PhD (Center for Childhood Cancer and Blood Diseases, Nationwide Childrens Hospital )

Abstract not available online - please check the printed booklet.

73. Evolution of Ribozyme Ligase Sequence Space Under Selection Pressures

Zoe Weiss (Department of Molecular Biology, Center for Computational and Integrative Biology, Massachusetts General Hospital), Saurja DasGupta (Department of Molecular Biology, Center for Computational and Integrative Biology, Massachusetts General Hospital), Travis Walton (Department of Molecular Biology, Center for Computational and Integrative Biology, Massachusetts General Hospital), Jack Szostak (Department of Molecular Biology, Center for Computational and Integrative Biology, Massachusetts General Hospital)

Abstract not available online - please check the printed booklet.

74. RNA binding proteins, upstream open reading frames, and translational control of human gene expression

Cassia Williams-Rogers (Department of Biological Sciences, Carnegie Mellon University), Matthew Agar-Johnson (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)

Abstract:
Current research in the area of translational control of gene expression includes examination of the presence of upstream open reading frames (uORFs) that present potential start sites for translation of an mRNA transcript in the 5’transcript leader ahead of the main coding DNA sequence (CDS). While the presence of uORFs is generally thought to suppress translation of downstream genes, the mechanisms by which uORFs impact translation of the CDS are not well defined. Previous work in Drosophila has proposed a translational control mechanism in which the binding of the Sex-lethal RNA binding protein (RBP) downstream of a uORF increases preinitiation complex (PIC) recognition of the uORF start codon, thereby decreasing translation of the CDS (Medenbach, et al., 2011). Our project investigated the extent to which RBPs might regulate uORF usage in human mRNA. We performed computational analyses of human uORF and RBP site conservation and frequency using GENCODE transcript annotations and RBP sites obtained from the Encyclopedia of DNA Elements (ENCODE) project (Van Nostrand, et al., 2020). Genes with highly conserved AUG uORF start codons were enriched for functions and processes related to control of transcription and neurons/neurotransmitter activity. uORFs with start codons conserved among primates were also more likely to have RBP sites downstream than non-conserved uORFs. The RBPs that most often bound downstream of these conserved uORFs include ZNF622, RBM15, CSTF2T, DROSHA, and FTO. Interestingly, separate analysis of genes that have both highly conserved uORFs and downstream RBP sites showed even greater enrichment in RNA polymerase II related activities, serine/threonine kinase activity, and chromatin modification functions. These results provide compelling evidence that RBPs may regulate initiation at some human uORFs. However, further computational and experimental investigation is needed to quantify the potential interactions of the RBPs and uORFs identified here.

Keywords: RNA binding proteins (RBPs), upstream open reading frames (uORFs)

75. Biased Pol II fidelity contributes to conservation of functional domains in the Potato spindle tuber viroid genome

Jian Wu (Department of Molecular Genetics, The Ohio State University), David M. Bisaro (Department of Molecular Genetics, The Ohio State University)

Abstract not available online - please check the printed booklet.

76. FAM210A Deficiency Causes Dilated Cardiomyopathy through Persistent Activation of Integrated Stress Response

Jiangbin Wu (Aab Cardiovascular Research Institute, University of Rochester Medical Center), Kadiam C. Venkata Subbaiah (Aab Cardiovascular Research Institute, University of Rochester Medical Center), Omar Hedaya (Aab Cardiovascular Research Institute & Department of Biochemistry and Biophysics. University of Rochester Medical Center), Si Chen (Aab Cardiovascular Research Institute & Department of Pharmacology and Physiology. University of Rochester Medical Center), Chen Yan (Aab Cardiovascular Research Institute & Department of Pharmacology and Physiology. University of Rochester Medical Center), Peng Yao (Aab Cardiovascular Research Institute & Department of Biochemistry and Biophysics. University of Rochester Medical Center)

Abstract:
Heart is one of the most mitochondria enriched organ in mammals. Disturbed cardiac mitochondrial homeostasis causes mitochondrial cardiomyopathy. Integrated stress response (ISR), a major pathway downstream of multiple mitochondrial stresses, regulates translation initiation and reprograms global cellular translatome. FAM210A (family with sequence similarity 210 member A) is a mitochondria localized protein and essential for embryonic development. Prior research indicates that FAM210A is most highly expressed in the heart and FAM210A is a critical determinant of skeletal muscle function. However, the function of FAM210A in the heart is still unexplored. Here we discover that FAM210A is critical for maintaining cardiac mitochondrial function and homeostasis. Cardiomyocyte (CM) specific knockout (KO) of Fam210a in adult mice leads to progressive heart failure with enlarged left ventricle chamber, and ultimately causes mortality at ~70 days after Fam210a KO. The FAM210A deficient CMs exhibit severe myofilament disarray at ~9 weeks post Fam210a KO at late stage. Furthermore, Fam210a KO results in a remarkably elevated mitochondrial ROS production, dramatically compromised mitochondrial membrane potential, and reduced expression of mitochondrial electron transport chain (ETC) complex genes. As a result, the mitochondrial respiratory activity is significantly reduced and the mitochondrial cristae are disrupted in Fam210a KO CMs. However, at early stage of ~5 weeks post tamoxifen induced Fam210a KO, we only observed increased mitochondrial ROS production, disturbed mitochondrial membrane potential, and reduced respiratory activity while the heart function keeps normal. Transcriptomic and proteomic analyses from Fam210a KO hearts indicate that majority of genes upregulated at both early and late stages are downstream targets of ATF4 transcription factor. Phosphorylation of eIF2&alpha is greatly enhanced at the early stage, suggesting that FAM210A deficiency caused mitochondrial dysfunctions and chronic ISR activation and translational reprogramming, ultimately leading to heart failure. Altogether, we discover a novel function of FAM210A in maintaining the cardiac mitochondrial function, and deficiency of FAM210A causes persistent activation of ISR and leads to heart failure.

References:
1. Tahmasebi, S., et al. Nat Rev Mol Cell Biol 19, 791-807, doi:10.1038/s41580-018-0034-x (2018).
2. Costa-Mattioli, M. & Walter, P. Science 368, doi:10.1126/science.aat5314 (2020).
3. Rozman, J. et al. Nature communications 9, 288, doi:10.1038/s41467-017-01995-2 (2018).
4. Dickinson, M. E. et al. Nature 537, 508-514, doi:10.1038/nature19356 (2016).
5. Estrada, K. et al. Nat Genet 44, 491-501, doi:10.1038/ng.2249 (2012).
6 Tanaka, K. I. et al. Proc Natl Acad Sci U S A 115, E3759-E3768, doi:10.1073/pnas.1719089115 (2018).

Keywords: FAM210A, mitochondrial dysfunction, integrated stress response

77. The czcD (NiCo) riboswitch responds to iron(II)

Jiansong Xu (Department of Chemistry, Penn State University), Joseph Cotruvo (Department of Chemistry, Penn State University)

Abstract:
Iron is essential for nearly every organism, and mismanagement of its intracellular concentrations (either deficiency or excess) contributes to diminished virulence in human pathogens, necessitating intricate metalloregulatory mechanisms. To date, although several metal-responsive riboswitches have been identified in bacteria, none has been shown to respond to Fe^II. The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to Ni^II and Co^II but claimed not to respond to Fe^II, on the basis of aerobic, in vitro assays. Its function in vivo is not well understood. We constructed a fluorescent sensor using this riboswitch fused to the RNA aptamer, Spinach2. When assayed anaerobically, the resulting sensor responds in vitro to Fe^II, as well as to Mn^II, Co^II, Ni^II, and Zn^II, but only in the cases of Fe^II and Mn^II do the apparent K_ds (0.4 µM and 11 µM, respectively) fall within the range of labile metal concentrations maintained by known metalloregulators. We also show that the sensor – which is, to the best of our knowledge, the first reversible genetically encoded fluorescent sensor for Fe^II – responds to iron in E. coli cells. Finally, we demonstrate that the putative metal exporters directly downstream of two czcD riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results suggest that iron should be considered as a plausible physiological ligand for czcD riboswitches, although response to general metal stress cannot be ruled out at present.

Keywords: riboswitch, iron, NiCo

78. Molecular mechanisms that regulate A-to-I RNA Editing in vivo

Boyoon Yang (Molecular and Cellular Biochemistry, Indiana University Bloomington), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN)

Abstract not available online - please check the printed booklet.

79. The small RNA Teg41 is a multifunctional regulator of virulence and stress response in Staphylococcus aureus

Rachel L. Zapf (Department of Biological Sciences, Ohio University), Paul Briaud (Department of Biological Sciences, Ohio University), Richard E. Wiemels (Department of Biological Sciences, Ohio University), Rebecca A. Keogh (Department of Biological Sciences, Ohio University), Ronan K. Carroll (Department of Biological Sciences, Ohio University)

Abstract not available online - please check the printed booklet.

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

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

Abstract not available online - please check the printed booklet.

81. Artificial mirror-image RNA in Basic Research and Therapeutic Design

Yuliya Dantsu (Biochemistry and Molecular Biology, Indiana University School of Medicine), Ying Zhang (Biochemistry and Molecular Biology, Indiana University School of Medicine), Wen Zhang (Biochemistry and Molecular Biology, Indiana University School of Medicine)

Abstract not available online - please check the printed booklet.

82. Spatiotemporal control of CRISPR/Cas9 function in cells and zebrafish using light‐activated guide RNA

Wenyuan Zhou (Department of Chemistry, University of Pittsburgh), Wes Brown, Anirban Bardhan (Department of Chemistry, University of Pittsburgh), Michael Delaney, Amber S. Ilk, Randy R. Rauen, Shoeb I. Kahn (Horizon Discovery), Michael Tsang (Department of Developmental Biology, School of Medicine, University of Pittsburgh), Alexander Deiters (Department of Chemistry, University of Pittsburgh)

Abstract:
We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′‐protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA‐target hybridization and rapid restoration of CRISPR/Cas9 binding to target DNA upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off‐to‐on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing and transcriptional regulation, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

References:
Zhou, Wenyuan, et al. "Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA." Angewandte Chemie 132.23 (2020): 9083-9088.

Keywords: CRISPRCas9, optical control, RNA

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