Rustbelt RNA Meeting 2021

Keywords: Trypansoma, RNA Helicase, RNA editing

32. Investigating the role of EFTUD2 in pre-mRNA splicing and EJC deposition

Caleb M. Embree (Department of Molecular Genetics, Center for RNA Biology), Guramrit Singh (Department of Molecular Genetics, Center for RNA Biology)

Abstract:
During the gene expression pathway, pre-mRNA splicing is one of the earliest and most important RNA processing steps, which occurs co-transcriptionally. The end result of pre-mRNA splicing is an mRNP particle comprised of mRNA and RNA binding proteins deposited during splicing. One complex of RNA binding proteins, the exon junction complex (EJC), plays important roles in steps ranging from pre-mRNA splicing in the nucleus to mRNA degradation in the cytoplasm. Though the EJC is deposited by the spliceosome, the responsible spliceosomal proteins and their precise function in EJC deposition is not fully known. EFTUD2, a splicing regulator and member of the U5 snRNP that sits adjacent to the EJC within the activated spliceosome, may have a role in EJC deposition. Mutations in EFTUD2 and the EJC core factor EIF4A3 cause similar cranio-facial disorders, hinting that they function in a similar pathway. Using clinically relevant C-terminally truncated EFTUD2 proteins we are investigating the relationship between EFTUD2 and the EJC. Our work shows that the C-terminal truncations of EFTUD2 destabilize the protein, which suggests that these mutations likely cause EFTUD2 haploinsufficiency in patients. Our hypothesis is that such a haploinsufficiency leads to defects in spliceosome and/or EJC assembly resulting in splicing errors. Using EFTUD2 haploinsufficient (or knockdown) HEK293 cells, we plan to identify the splicing errors via RNA-Seq and assess transcriptome-wide spliceosome assembly via spliceosome profiling. Together these data will allow us to determine how reduced levels of EFTUD2 impairs spliceosome and/or EJC assembly leading to splicing defects.

Keywords: spliceosome, EFTUD2

33. Investigating an ADAR-dependent germline phenotype in 𝘊𝘢𝘦𝘯𝘰𝘳𝘩𝘢𝘣𝘥𝘪𝘵𝘪𝘴 𝘦𝘭𝘦𝘨𝘢𝘯𝘴

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

Abstract not available online - please check the booklet.

34. Computational discovery of novel functional RNAs in SARS-CoV-2

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

Abstract:
The global COVID-19 pandemic is caused by the positive-sense RNA virus SARS-CoV-2 (1). The genome of this virus is known to hold functional RNAs, a type of biologically essential RNA characterized by conserved sequence, conserved structure, and presence of a pseudoknot (2). Functional RNAs are also good therapeutic targets for antisense oligonucleotides (ASOs) or small molecules making the discovery of novel functional RNAs in SARS-CoV-2 a route for developing therapeutics to counter the disease (3). We built a computational pipeline that functions in two parts. The first part agnostically locates structured RNAs in the genome and determines which of those structures contain pseudoknots; those that contain pseudoknots continue through the pipeline. This part also eliminates terminators and known riboswitches and ribozymes. The second part of the pipeline uses thousands of viral genomic isolates to determine conservation of sequence, structure, and pseudoknots. A known functional RNA within the SARS-CoV-2 genome is a programmed (–1) ribosomal frameshifting element (FSE), which was also analyzed using the pipeline to establish a benchmark for a level of mutation that was acceptable (4). Pseudoknots that showed higher levels of conservation of sequence, secondary structure, and pseudoknot structure than the FSE were considered putatively functional. A final level of analysis was added to the pipeline that eliminated mutations that cause nonsense or missense non-conservative amino acid mutations. The rationale being that it is ambiguous whether the selective pressure acting on those mutations is due to RNA structure or the protein produced. Using this pipeline, seven putatively functional RNAs were identified in the SARS-CoV-2 genome that contain highly conserved sequence, secondary structure, and a pseudoknot. These RNAs are potential novel therapeutic targets.

References:
(1) Manfredonia, I.; Nithin, C.; Ponce-Salvatierra, A.; Ghosh, P.; Wirecki, T. K.; Marinus, T.; Ogando, N. S.; Snijder, E. J.; Hemert, M. J. van; Bujnicki, J. M.; Incarnato, D. Genome-Wide Mapping of Therapeutically-Relevant SARS-CoV-2 RNA Structures. bioRxiv 2020.
(2) Peselis, A.; Serganov, A. Structure and Function of Pseudoknots Involved in Gene Expression Control. Wiley interdisciplinary reviews. RNA. NIH Public Access November 1, 2014, pp 803–822.
(3) Crooke, S. T.; Witztum, J. L.; Bennett, C. F.; Baker, B. F. RNA-Targeted Therapeutics. Cell Metabolism. Cell Press April 3, 2018, pp 714–739.
(4) Sun, Y.; Abriola, L.; Surovtseva, Y. V; Lindenbach, B. D.; Guo, J. U. Restriction of SARS-CoV-2 Replication by Targeting Programmed −1 Ribosomal Frameshifting In Vitro. bioRxiv 2020.

Keywords: SARS-CoV-2, RNA, Pseudoknots

35. 5-methyluridine modifications in mRNA codons impact protein synthesis by the ribosome

Monika K Franco (University of Michigan Program in Chemical Biology), Joshua D Jones (University of Michigan, Department of Chemistry), Mehmet Tardu (University of Michigan, Department of Chemistry), Laura R Snyder (University of Michigan, Department of Chemistry), Robert T Kennedy (University of Michigan, Department of Chemistry), Kristin S Koutmou (University of Michigan Program in Chemical Biology, University of Michigan, Department of Chemistry)

Abstract not available online - please check the booklet.

36. RNA Pseudoknot Structure in 16S Helix 18 Destabilizes the Binding and Enzymatic Activity of Ribosomal RNA Modification Enzyme RsuA

Sanjaya Abeysirigunawardena (Kent State University), Kumudie Jayalath (Kent State University), Sean Frisbie (Kent State University )

Abstract:
Ribosomes are ribonucleoprotein complexes that are involved in protein
biosynthesis in cells. Ribosomes are generated from the assembly of
three different ribosomal RNAs (rRNAs) and more than 50 ribosomal
proteins (r-proteins). Though the ribosome assembly is well studied, the
impact of post-transcriptional rRNA modification enzymes and their
respective modifications on ribosomal assembly is not completely
understood. In response to the lack of understanding of preferred
substrates and the structural prerequisites for the stable binding and
function of the rRNA modification enzymes, we study thermodynamics
of protein RsuA to rRNA. My study focused on understanding the impact
of pseudoknot structure of 16S helix 18 on the binding of RsuA. Several
rRNA mutants that are incapable of forming 16S helix 18 pseudoknot
was generated by using site-directed mutagenesis. FRET-based binding
assay are used to measure the thermodynamic stability of RsuA-rRNA
complexes. These findings will enable us to elucidate thermodynamically
preferred mechanism of RsuA binding to rRNA.

References:
https://www.mdpi.com/2218-273X/10/6/841

Keywords: Helix 18, 16s RNA, Enzymatic Activity

38. Probing the impact of HOPS on RNAs with single molecule tracking

Guoming Gao (Biophysics Graduate Program and Department of Biophysics, University of Michigan, Ann Arbor, MI 48109, USA), Nils G Walter (Department of Chemistry, Biophysics, and Center for RNA Biomedicine, University of Michigan, Ann Arbor, MI 48109, USA)

Abstract:
Proteins and RNAs can form functional biological condensates, also known as membraneless organelles, via liquid-liquid phase separation (LLPS). The partitioning of different proteins and RNAs between the dilute phase and the condensed phase provides delicate regulation over their functions, from promoting biochemical reactions and specific intermolecular interactions, to sequestering key molecules from downstream processing or signaling. Hyperosmotic phase separation (HOPS) is a recently discovered LLPS triggered by the osmotic compression of cell volume. A majority of homo-multimeric proteins are shown to undergo HOPS in several cell types. Moreover, HOPS is much faster than most cellular LLPS processes (within ~10 s versus over minutes to hours), and thus HOPS condensates could be first responders sensing cell volume change and priming other stress responses. However, it was unclear whether RNAs contribute to HOPS and how HOPS impacts the diffusion behaviors and functions of different RNAs. Here, we used live-cell single molecule RNA tracking to explore the dwelling of mRNAs, lncRNAs, and miRNAs in HOPS condensates, and measure the change in their diffusion behaviors in the presence of HOPS. A theoretical model for RNA’s role in HOPS is proposed based on RNA interactions with enigmatic RNA binding sites (EnigmRBS) on protein surfaces.

Keywords: LLPS, Single Molecule

39. The p38 MAPK Hog1 remodels the translatome of the fungal pathogen Cryptococcus neoformans.

David Goich (Microbiology and Immunology, University at Buffalo), Amanda L. M. Bloom (Microbiology and Immunology, University at Buffalo), John C. Panepinto (Microbiology and Immunology, University at Buffalo)

Abstract:
Cryptococcus neoformans is an environmental fungus that causes severe opportunistic infections in immunocompromised individuals, particularly people living with HIV/AIDS. Upon entry into the host, C. neoformans rapidly alters its proteome to accommodate a variety of stressors, including elevated temperature, reactive oxygen species (ROS) in phagocytes, nutrient deprivation, and more. Our preliminary data indicate that these processes are dependent in part on the p38 MAPK, Hog1, which couples the translational response to stress-sensing signal transduction. Using polysome profiling, translational output assays, and mRNA repression kinetics, we investigated whether two putative kinases downstream of Hog1, Hrk1 and Cmk2, are similarly defective in translatome reprogramming. We also investigated the role of these proteins in the translational response to fludioxonil, an antifungal with Hog1-dependent activity. We found that while hrk1∆ demonstated delayed recovery from translation repression, deletion of Cmk2 caused dysregulation of eIF2α. Our data suggests that both of these putative effectors of Hog1 play a role in translational adaptation to stress. Ongoing investigations will determine if these proteins play stress-specific roles, as well as the role of each in the translational response to fludioxonil.

Keywords: Translation

40. The novel role of m6A-mRNA binding protein YTHDF1 in the regulation of heart function

Volha A Golubeva (Medical Scientist Training Program and Biomedical Sciences Graduate Program, Center for RNA Biology, OSU, Columbus OH), Lisa Dorn (Medical Scientist Training Program, Center for RNA Biology, OSU, Columbus OH), Federica Accornero (Center for RNA Biology, Davis Heart and Lung Research Institute and Department of Physiology and Cell Biology, OSU, Columbus OH)

Abstract not available online - please check the booklet.

41. Alternative polyadenylation of transcripts encoding trans-acting siRNAs in red clover

Caleb Gooden (Department of Plant and Soil Sciences, University of Kentucky), Randy D. Dinkins (USDA-ARS-FAPRU), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky)

Abstract:
Root nodulation is a process in which host roots are infected with a soil bacterium and form nodules that better fix nitrogen from the atmosphere. In agriculture, nodules help to reduce the need for fertilizer and are thus part of sustainable practice. Studies in Medicago truncatula [1] have shown the process of root nodulation to be suppressed by the expression of a regulatory long non-coding RNA (TAS3). Over-expression of TAS3 and the miR390 pathway with which it interacts resulted in prioritization of lateral root formation over nodulation. Thus, TAS3 is an attractive target for manipulation to improve nitrogen fixation in legumes and enhance their agronomic value.

In Arabidopsis, TAS3a transcripts may end at one of two alternative polyadenylation sites, the choice of which determines whether functional or non-functional TAS3a RNAs are made [2]. Specifically, RNAs ending at the promoter-proximal site will lack the characteristic miR390 target site and thus be non-functional. TAS3a transcript ending at the distal site will possess the miR390 target site, and thus can serve as substrates for miR390-directed cleavage and subsequent production of siRNAs that regulate the responses to auxin.

We hypothesized that if two polyadenylation (poly(A)) sites are present in the TAS3 region of Trifolium pratense (red clover), then differential use of these sites would be seen in lateral roots and nodules. To test whether red clover TAS3 transcripts end at different poly(A) sites, we utilized 3’-RACE to identify and sequence TAS3 RNA isoforms. The results confirm that transcripts encoding the red clover TAS3 may end at one of two poly(A) sites. Further bioinformatics analysis showed that a miR390 target sequence is situated between the two poly(A) sites. These results confirm that red clover TAS3 transcripts undergo alternative polyadenylation much as is seen in Arabidopsis and M. truncatula, and that alternative polyadenylation may regulate TAS3 function in red clover. Current research focuses on measuring the differential use of these sites in roots and nodules using quantitative PCR (qPCR) and 3’-RACE assays. Using these results as a guide, we plan on developing transgenic approaches to manipulate the relative usages of these sites to further establish their importance on nodulation.

References:
1. Hobecker KV, Reynoso MA, Bustos-Sanmamed P, Wen J, Mysore KS, Crespi M, et al. The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth. Plant Physiol. 2017;174(4):2469-86.
2. Hunt AG. RNA regulatory elements and polyadenylation in plants. Front Plant Sci. 2011;2:109.

Keywords: alternative polyadenylation, siRNA, nodulation

42. Gene-specific Pulldown for targeted in vivo RNA structure probing

Samantha 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:
When Escherichia coli experiences environmental stressors it initiates regulatory responses to maintain homeostasis. In response to a specific stress, small non-coding RNAs (sRNAs) change the expression of target mRNAs by altering mRNA stability and translation efficiency. The sRNA RyhB is expressed under iron-limiting conditions and regulates the iron sparing response.¹ 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 bind and that the interaction may induce a structural rearrangement to alter gene expression. To analyze the role of RNA structure in RyhB regulation four putative RyhB targets (cirA, fliA, sufAB, frdA) were selected. Then gene expression profiling was performed, and a gene-specific pulldown was developed for targeted in vivo structure probing. This research will provide a deeper understanding of the regulatory systems 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.

Keywords: sRNA, RyhB, RNA structure probing

43. Translation reinitiation regulates oocyte maturation

Lydia Grmai (Department of Cell Biology, University of Pittsburgh), Hyung Don Ryoo (Department of Cell Biology, New York University School of Medicine), Deepika Vasudevan (Department of Cell Biology, University of Pittsburgh)

Abstract not available online - please check the booklet.

44. Characterization of putative SAM-I riboswitches of L. monocytogenes

Ian Hall (Department of Chemistry, University of Michigan), Sarah Keane (Department of Chemistry and Program in Biophysics, University of Michigan)

Abstract:
Listeria monocytogenes is a saprophytic bacterium which can infect humans causing listeriosis, a life-threatening foodborne illness. In the U.S. about 2,500 cases of listeriosis and 500 related deaths are reported each year. Understanding the bacteria’s virulence and metabolism are essential for improving strategies to prevent and treat listeriosis. Notably regulation of the bacteria’s virulence gene expression is primarily controlled by positive regulatory factor A (PrfA) and an RNA thermosensor (RNAT) that resides in the 5′ untranslated region (UTR) of the prfA messenger (m)RNA. We previously described the mechanism of the prfA RNAT’s temperature dependent translation regulation.¹ PrfA translation research revealed a novel trans interaction between the prfA RNAT and S-adenosyl methionine (SAM) response element A (SreA)², a putative SAM-I riboswitch identified in tiling arrays of the L. monocytogenes genome.³
The present work biophysically characterizes SreA and six other putative SAM-I riboswitches of L. monocytogenes (SreA-G). These elements reside in front of genes for SAM biosynthesis pathways and/or SAM related transport pathways. A key hypothesis of this work is whether the genomic location of a riboswitch is related to its regulatory strength. To answer this question and characterize these presumed SAM-I riboswitches, we initiated studies on the ligand binding, transcription termination, and structure of SreA-G. The ligand affinity and specificity of SreA-G are being determined by isothermal titration calorimetry (ITC) using cognate (SAM) and near-cognate (SAH) ligands. The transcription termination of the riboswitches with SAM or SAH are being evaluated by a ³²P GTP body labeled assay. The aptamer domains of the riboswitches were studied by small angle X-ray scattering (SAXS) with and without SAM, and in all cases, we observe compaction of the structure with SAM. Additional structural studies of aptamer domains via X-ray crystallography are underway.

References:
1. Zhang, H.; Hall, I.; Nissley, A. J.; Abdallah, K.; Keane, S. C., A Tale of Two Transitions: The Unfolding Mechanism of the prfA RNA Thermosensor. Biochemistry 2020, 59 (48), 4533-4545.
2. Loh, E.; Dussurget, O.; Gripenland, J.; Vaitkevicius, K.; Tiensuu, T.; Mandin, P.; Repoila, F.; Buchrieser, C.; Cossart, P.; Johansson, J., A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell 2009, 139 (4), 770-9.
3. Toledo-Arana, A.; Dussurget, O.; Nikitas, G.; Sesto, N.; Guet-Revillet, H.; Balestrino, D.; Loh, E.; Gripenland, J.; Tiensuu, T.; Vaitkevicius, K.; Barthelemy, M.; Vergassola, M.; Nahori, M.-A.; Soubigou, G.; Régnault, B.; Coppée, J.-Y.; Lecuit, M.; Johansson, J.; Cossart, P., The Listeria transcriptional landscape from saprophytism to virulence. Nature 2009, 459 (7249), 950-956.

Keywords: SAM-I riboswitch, Listeria monocytogenes

45. Establishing the presence of hidden breaks in the chloroplast rRNA of various plants

Cooper Halliday (University of Pittsburgh at Johnstown), Kaylin Gaudette (University of Pittsburgh at Johnstown), Laura Ritchey (University of Pittsburgh at Johnstown)

Abstract:
In plant cells, light is captured in specialized organelles known as chloroplasts. These structures turn light energy into chemical energy in the process of photosynthesis. Like mitochondria, chloroplasts contain their own DNA as well as their rRNA. It was discovered that there are two hidden breaks in the rRNA of the 23S large ribosomal subunit. (1-3) The existence of hidden breaks has been detected in the rRNAs of plants, insects (in the 28S rRNA), and in rodents. (1) These breaks, in plants, are believed to be important for translation within the chloroplast and are the result of specific nuclease-mediated (1,2) covalent breaks in the backbone of the rRNA. We have hypothesized that stress conditions such as cold and hot temperatures affect the expression of these breaks. We extracted RNA from Oryza sativa and Spinacia oleracea subjected to temperature stress conditions and analyzed the RNA using qualitative assays to identify the presence of the hidden breaks. We also aim to use sequencing gels and RT-qPCR to look at the nucleotide-specific location in the sequence that the hidden break occurs in the rRNA for each of the plants. Once grown, Coffea arabica and Arabidopsis thaliana will be run through the same procedures to have a wider organism pool for comparison.

References:
1. Nishimura, K., et al. Plant J 63 (5), 766-77 (2010).
2. Liu, J., et al. Plant Physiol 168 (1), 205-221, (2015).
3. Ritchey, L.E., et al. Nucleic Acids Res 45 (14), el35 (2017).

Keywords:

46. Modulating RNA Splicing of DNA Topoisomerase IIα in Human Leukemia K562 Cells: Use of CRISPR/Cas9 Gene Editing to Impact Sensitivity/Resistance to the Anticancer Agent Etoposide

Victor A. Hernandez (Pharmaceutics and Pharmacology, The Ohio State University ), Jessika Carvajal (Pharmaceutics and Pharmacology, The Ohio State University ), Jonathan L. Papa, Nicholas Shkolnikov (Pharmaceutics and Pharmacology, The Ohio State University ), Hatice Gulcin Ozer (Biomedical Informatics, The Ohio State University ), Jack C. Yalowich, Terry S. Elton (Pharmaceutics and Pharmacology, The Ohio State University ), Junan Li (Pharmaceutics and Pharmacology, The Ohio State University )

Abstract:
The human DNA topoisomerase IIα (170 kDa, TOP2α/170) is an essential enzyme that resolves DNA topological entanglements that form during chromosome condensation and segregation, making it indispensable for the survival of highly proliferating cells including cancer cells. Hence, TOP2α is a prominent target for anticancer therapies. Some of the most widely used TOP2 targeting drugs such as etoposide, stabilize a TOP2α/170-DNA cleavage complex preventing the religation of broken DNA strands and exerting cytotoxic effects by the accumulation of DNA damage.
Acquired chemoresistance to TOP2 targeting drugs continues to be a major obstacle in cancer treatment in the clinic. Our lab developed etoposide resistant human leukemia K562 cells, designated K/VP.5 in which levels of TOP2α/170 were decreased along with identification of a novel C-terminal truncated isoform of TOP2α, TOP2α/90. TOP2α/90 is the translation product of novel alternatively spliced mRNA via intron 19 (I19) retention and processing. TOP2α/90 is a determinant of acquired resistance to etoposide through a dominant-negative effect related to heterodimerization with TOP2α/170.
It was hypothesized that the E19/I19 splice site (SS) is inefficiently recognized by the spliceosome and results in decreased levels of TOP2α/170 and drug resistance. Therefore, CRISPR/Cas9 was utilized for gene editing of TOP2α in K/VP.5 cells to enhance I19 removal and circumvent acquired TOP2α-mediated drug resistance. Gene edited clones were identified by qPCR and verified by sequencing. Characterization of a K/VP.5 clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 protein, and increased TOP2α/170 protein. In addition, gene editing to optimize the TOP2α E19/I19 SS in K/VP.5 cells circumvented resistance to TOP2α-targeted agents.
Conversely, experiments were performed whereby the E19/I19 SS in etoposide sensitive K562 cells was gene edited to decrease/eliminate splicing at this boundary. Inhibiting splicing of the E19/I19 SS in K562 cells resulted in a decrease in full-length TOP2α/170 expression and induction of resistance to etoposide. Together, results support the role of alternative splicing at the E19/I19 boundary as a determinant of resistance to TOP2α-targeting agents.

Keywords: CRISPRCas9, Alternative RNA Splicing , Drug Resistance

47. Strand Invasion during the Fluoride Riboswitch Cotranscriptional Folding Pathway

Laura M Hertz (Department of Chemical and Biological Engineering at Northwestern University), Angela M Yu (Electrical and Computer Engineering Department at University of Washington), Julius B Lucks (Department of Chemical and Biological Engineering at Northwestern University)

Abstract not available online - please check the booklet.

49. Stress-Induced Increase in Small ncRNAs Generated from Regulatory Regions of the Mitochondrial Genome

Kathryn E. Holzmacher (Department of Biochemistry at University at Buffalo), Jonathan E. Bard (Center of Excellence in Bioinformatics and Life Sciences, and Department of Biochemistry at University at Buffalo), Theodore L. Mathuram (Department of Biochemistry at University at Buffalo), Natalie A. Lamb (Center of Excellence in Bioinformatics and Life Sciences at the University at Buffalo), Norma J. Nowak (Center of Excellence in Bioinformatics and Life Sciences, and Department of Biochemistry at University at Buffalo), A. Blumental-Perry (Department of Biochemistry at University at Buffalo)

Abstract:
Rationale: Alveolar Epithelial Type (AET) 2 cells serve as progenitors for the repair of destructed alveoli. We identified AET2-specific mitochondria-associated retrograde signaling via mtDNA-encoded non-coding (nc) RNA, mito-ncR-805. mito-ncR-805 levels are increased in the mitochondria and nucleus during stress, and positively regulate mitochondrial metabolism and respiration. We propose that mito-ncR-805 represents a subtype of mitochondrial small RNAs that protect cells under stress. To identify transcripts with similar functions we compared mitochondrial transcriptomes of unstressed and stressed cells.
Methods: CS-exposures of MLE12 cells were used to study adaptational transcriptomes. Stress signaling was evaluated by Western blot. Mitochondrial small RNA transcriptome was evaluated by constructing and analyzing strand-specific RNAseq libraries of transcripts 100 bp and shorter. A bioinformatic pipeline was specifically developed for the analysis of mitochondrial transcripts. Identified transcripts were validated by Northern blot and qPCR analyses.
Results: CS exposure increased the generation of 3 small RNAs. The transcripts had a specific processing pattern and originated from the control regions of the mitochondrial genome (D-loop and Rnr genes). The presence of the transcripts in cells and their elevation during smoke exposure was confirmed by Northern. Mitochondrial origin was validated in either mitochondrial-depleted cells (grown in EtBr), or in cells where mitochondrial transcription was inhibited by 6G-rhodamine. Cellular localization of Rnr-derived transcripts is currently being evaluated.
Conclusion: We demonstrate the stress-dependent generation of 4 distinct transcripts that originate from control regions of the mitochondrial genome. This suggests a possible regulatory function in coordinating the activities in both mitochondrial and nuclear genomes. We propose that those small RNAs aid in signaling which aim to restore mitochondrial homeostasis.

References:
Blumental-Perry A, Jobava R, Bederman I, Degar AJ, Kenche H, Guan BJ, Pandit K, Perry NA, Molyneaux ND, Wu J, Prendergas E, Ye ZW, Zhang J, Nelson CE, Ahangari F, Krokowski D, Guttentag SH, Linden PA, Townsend DM, Miron A, Kang MJ, Kaminski N, Perry Y, Hatzoglou M. Retrograde signaling by a mtDNA-encoded non-coding RNA preserves mitochondrial bioenergetics. Commun Biol. 2020 Oct 30;3(1):626. doi: 10.1038/s42003-020-01322-4. PMID: 33127975; PMCID: PMC7603330.
Lee C, Zeng J, Drew BG, Sallam T, Martin-Montalvo A, Wan J, Kim SJ, Mehta H, Hevener AL, de Cabo R, Cohen P. The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metab. 2015 Mar 3;21(3):443-54. doi: 10.1016/j.cmet.2015.02.009. PMID: 25738459; PMCID: PMC4350682.

Keywords: Alveolar Epithelial Type 2 Cells, Mitochondrial Stress Signaling , Small non-coding RNA

50. RNA Thermometer Thermal Stability Modulated by pH-Sensitive Molecular Switching

Md Ismail Hossain (Department and institution: Department of Chemistry & Biochemistry, Ohio University, Athens, OH), Erin R. Murphy (Department and institution: Department of Biomedical Sciences, Ohio University Athens, OH), Jennifer V. Hines (Department and institution: Department of Chemistry & Biochemistry, Ohio University, Athens, OH)

Abstract:
Bacterial resistance to current medicines continues to be a significant global health threat. Consequently, there is a need to develop new therapeutic approaches that prevent or overcome bacterial infections. One emerging area of investigation is targeting regulatory RNA, which mediates gene expression in bacteria. In order for drug discovery studies to be successful, a detailed understanding of the molecular mechanism of the RNA-mediated regulation is needed. In this study, we investigated the effect of pH on the thermal stability of a set of RNA thermometers (RNATs) found in the untranslated leader region of Shigella shuA, shuT, and ompA mRNA. ^1,2 Shu (Shigella heme utilization) proteins have essential roles in utilizing heme as a sole source of iron. Outer membrane protein A (ompA) is a virulence factor required for the cell adhesion, biofilm formation, invasion, and spread of the bacteria. UV monitored thermal denaturation was used to determine the melting temperature (T^m) and thermodynamic parameters for model RNAs of each RNAT. At low ionic strength, the T^m of the shuT and ompA RNA thermometers were pH dependent. However, at high ionic strength, only the T^m of shuT was pH dependent. The T^m of shuA was not pH-dependent at either low and high ionic strength, but it was significantly higher than that of shuT and ompA. We also investigated the effect of pH on RNAT binding of a 16S rRNA mimic using a fluorophore-quencher labeled model RNA. For ompA, the EC₅₀ for binding the 16S rRNA mimic was pH dependent. These results indicate that in some RNA thermometers, pH-sensitive molecular switching may play a role in modulating gene expression regulation.

References:
1. Kouse, Andrew B., Francesco Righetti, Jens Kortmann, Franz Narberhaus, and Erin R. Murphy. "RNA-mediated thermoregulation of iron-acquisition genes in Shigella dysenteriae and pathogenic Escherichia coli." PloS one 8, no. 5 (2013): e63781.
2. Murphy, Erin R., Johanna Roßmanith, Jacob Sieg, Megan E. Fris, Hebaallaha Hussein, Andrew B. Kouse, Kevin Gross et al. "Regulation of OmpA translation and Shigella dysenteriae virulence by an RNA thermometer." Infection and immunity 88, no. 3 (2020): e00871-19.

Keywords: RNA Thermometer, shuT, shuA, OmpA, Molecular switching

51. Determining the role of the 3’-5’ reverse polymerase in Myxococcus xanthus

Brandon Iwaniec (Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Chemistry and Biochemistry, The Ohio State University)

Abstract:
tRNA^His guanylyltransferase-like proteins (TLPs) are able to catalyze nucleotide addition to RNA substrates in the opposite direction (3'-5') of all known canonical RNA polymerases. This biochemical activity is seen in vitro with bacterial TLPs, which can add nucleotides to the 5'-ends of tRNA substrates. However, no bona fide biological substrates have been identified to date in any bacterial species that has a TLP. Myxococcus xanthus, a bacterium, exhibits defects in starvation-induced fruiting body formation and sporulation when its MXTLP gene is deleted, suggesting that this enzyme plays a role in maturation or maintenance of some biologically relevant RNA species. Here, we used a variety of in vitro biochemical assays to gain a more in-depth picture of the biological role of MxTLP in M. xanthus. Addition of an essential G_-1 nucleotide to the 5'-end of tRNA^His is an important function of members of the tRNA^His guanylyltransferase (Thg1) enzyme family. This activity has also been associated with one TLP, and therefore we sought to test whether MxTLP could exhibit a similar function. However, we demonstrated that MxTLP is not apparently needed for addition of the G_-1 nucleotide in vivo in M. xanthus, based on primer extension assays of RNA isolated from MXTLP deletion strains. This observation is also consistent with the RNase P-dependent pathway for obtaining G_-1 in bacteria, and our in vitro histidylation assays confirmed that the form of G_-1-containing tRNA^His preferred by the M. xanthus histidyl tRNA synthetase is the form that would most likely result from post-transcriptional RNase P cleavage, rather than from addition by MxTLP. We also demonstrated that MxTLP can incorporate labeled nucleotides into RNA isolated from vegetative and developing cells, but with different patterns of incorporation, indicating that MxTLP may play distinct roles in each life cycle. Efforts to identify the substrates that are acted on during each life cycle stage using pull-down approaches are underway, and these results will be used to provide the first look at the potential biological function of a TLP in any bacterial system.

Keywords: Reverse polymerization , tRNA, Myxococcus xanthus

52. Reverse (3'-5') RNA polymerases: Applications beyond tRNA(His) maturation

Malithi I. Jayasinghe (Department of Chemistry and Biochemistry, The Ohio State University), Krishna J. Patel (Department of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the booklet.

53. Identification of human homologue of mtDNA encoded retrograde signaling molecule, mmu-mito-ncR805

T. L. Mathuram (Department of Biochemistry, University at Buffalo, NY, USA), Y. Perry (Department of Surgery, University at Buffalo, NY, USA), A. Blumental-Perry (Department of Biochemistry, University at Buffalo, NY, USA)

Abstract:
We previously discovered mtDNA-encoded small ncRNA, mmu-mito-ncR805 (1), which increases in Alveolar Epithelial Type II (AETII) during the stress of smoking (SS). Recovery from SS characterizes by distribution of mmu-mito-ncR805 to the nucleus. Redistribution helps to preserve mitochondrial bioenergetics. Mmu-mito-ncR-805 maps to the D-loop of the mtDNA at the beginning of light strand promoter (LSP). Its sequence is not conserved between mouse and human. Nevertheless, identification of human homologue is important because mitochondrial stress and malfunction drives multiple human pathologies. The goal of this study was to identify human homologue of mmu-mito-ncR805 based on genomic location, kinetics and function during stress. Existing databases that contained transcripts originating from the D-loop of human mitochondrial genome were re-analyzed and 4 potential transcripts were identified. Their presence in the relevant human cell lines was confirmed via Northern blot analysis. RNA probes were designed to exclude cross-reactivity to Nuclear Mitochondrial DNA (NUMTS). Mitochondrial origin of the transcripts was further confirmed by their absence in cells with inhibited mitochondrial transcription (2). Mitochondria specific ~260 bp transcript (hsa-mito-ncR-D-loop-805) that originate from the transcription initiation site of human D-loop was further analyzed, and found to increase in its levels during SS. hsa-mito-ncR-D-loop-805 demonstrated dotted pattern throughout the cell, with dots juxtaposed, but not overlapping with mitochondrial protein Tom20. The dots became smaller, and some nuclear localization was detectable during recovery from SS. We therefore identified hsa-mito-ncR-D-loop-805 as a candidate to be a human homologue of mmu-mito-ncR805 retrograde signaling molecule. Our current research is validating hsa-mito-ncR-D-loop-805 levels, kinetics and retrograde signaling function in primary human AETII cells.

References:
1. Blumental-Perry A, Jobava R, Bederman I, Degar AJ, Kenche H, Guan BJ, Pandit K, Perry NA, Molyneaux ND, Wu J, Prendergas E, Ye ZW, Zhang J, Nelson CE, Ahangari F, Krokowski D, Guttentag SH, Linden PA, Townsend DM, Miron A, Kang MJ, Kaminski N, Perry Y, Hatzoglou M. Retrograde signaling by a mtDNA-encoded non-coding RNA preserves mitochondrial bioenergetics. Commun Biol. 2020 Oct 30;3(1):626. doi: 10.1038/s42003-020-01322-4. PMID: 33127975; PMCID: PMC7603330.
2. Gear AR. Rhodamine 6G. A potent inhibitor of mitochondrial oxidative phosphorylation. J Biol Chem. 1974 Jun 10;249(11):3628-37. PMID: 4275428.

Keywords: mmu-mito-ncR805, mtDNA-encoded small ncRNA, retrograde signaling molecule

54. Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout

Lauren M. Aufdembrink (Genetics, Cell Biology and Development, University of Minnesota), Pavana Khan (Genetics, Cell Biology and Development, University of Minnesota), Nathaniel J Gaut (Genetics, Cell Biology and Development, University of Minnesota), Katarzyna P Adamala (Genetics, Cell Biology and Development, University of Minnesota), Aaron E Engelhart (Genetics, Cell Biology and Development, University of Minnesota)

Abstract:
The SARS-CoV-2 pandemic has highlighted the importance of early, rapid and widespread pathogen detection tests that are easily accessible and available to all. We have developed Apta-NASBA, an isothermal, cell-free, synthetic biology-based detection system that combines Nucleic Acid Sequence Based Amplification (NASBA) with transcription of fluorescent aptamers. Despite using the highly active and promiscuous T7 polymerase in the system, we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags. With a limit of detection of 1 pM for the E. coli AggR pathogenic template, Apta-NASBA is highly sensitive and can yield multiplexed, multicolor, real-time fluorescent readout. Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module. This method is compatible with field detection and has potential for use in point-of-care SARS-CoV-2 diagnostics. We will discuss our work with Apta-NASBA and other isothermal amplification technologies.

References:
Aufdembrink LM, Khan P, Gaut NJ, Adamala KP, Engelhart AE. Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout. RNA. 2020 Sep;26(9):1283-1290. doi: 10.1261/rna.075192.120.

Keywords: NASBA, T7 RNA polymerase, Isothermal amplification

55. The democratization of RNA therapeutics

John P. Cooke (Center for RNA Therapeutics, Houston Methodist Research Institute, Houston TX), Daniel L. Kiss (Center for RNA Therapeutics, Houston Methodist Research Institute, Houston TX), Ruli Gao (Center for RNA Therapeutics, Houston Methodist Research Institute, Houston TX), Tulsi Damase (Center for RNA Therapeutics, Houston Methodist Research Institute, Houston TX), Yi Liang, Hongye Li (Center for RNA Therapeutics, Houston Methodist Research Institute, Houston TX), Roman Sukhovershin (Center for RNA Therapeutics, Houston Methodist Research Institute, Houston TX)

Abstract:
The development of mRNA vaccines against COVID-19 brought worldwide attention to the transformative potential of RNA-based therapeutics. Integral to this potential, mRNA therapeutics are not solely the province of big biopharma. RNA therapies are a disruptive technology precisely because small biotech startups and academic researchers can rapidly develop innovative and personalized mRNA constructs. However, most of these small groups lack the key competencies to translate their transformational mRNA therapeutics into the clinic. To facilitate the democratization of mRNA therapeutics, we have built the crucial infrastructure to assist smaller groups as they bring their novel ideas to the clinic. In the Texas Medical Center, our fully integrated hospital-based RNA therapeutics program provides a single-entry point with consultation to ensure the seamless development of RNA therapy candidates into transformative drugs. Our RNA Biology and Bioinformatics faculty assist with construct design; our cGMP-trained personnel and clean rooms support the synthesis, purification, and validation of RNA drugs; plus our Nanomedicine colleagues formulate suitable lipid nanoparticles for local or systemic delivery. We have a Comparative Medicine Program with expertise in GLP preclinical studies; a first-in-human clinical trials unit for Phase 1 and 2a studies; and a large hospital system with a clinical research infrastructure that supports Phase 2 and 3 clinical trials. Furthermore, our industry partner manufactures large batches needed for Phase 2, 3 and/or commercialization. To our knowledge, we are the sole academic center with a fully integrated and operational infrastructure to support both academic groups and startups. We intend to support the translational efforts of small groups of scientists as they attain the near limitless potential of RNA Therapeutics.

Keywords: RNA therapeutics, RNA biology, RNA medicine

56. Study of Trm7 interactions with binding partners Trm732 and Trm734 for 2’-O-methylation of the tRNA anticodon loop in yeast

Kellyn Dolezal (Chemistry & Biochemistry; Northern Kentucky University), Thao Le (Chemistry & Biochemistry; Northern Kentucky University), Holly M. Funk (Chemistry & Biochemistry; Northern Kentucky University), Adrian R. Guy (Chemistry & Biochemistry; Northern Kentucky University), Michael P. Guy (Chemistry & Biochemistry; Northern Kentucky University)

Abstract:
Transfer RNAs undergo post-transcriptional modifications, including many which occur at or near the anticodon loop and are necessary for protein synthesis. In Saccharomyces cerevisiae, Trm7 is required for 2’-O-methylation of tRNA and requires interaction with Trm732 and Trm734 for modifications at positions 32 and 34, respectively. These proteins are widely conserved in eukaryotes, and mutations in the human ortholog of TRM7, FTSJ1, can cause non-syndromic X-linked intellectual disability by decreasing or eliminating methylation activity. Through a genetic assay, we have found Trm7 residues required for interaction with Trm732 or Trm734 and the subsequent methylation of tRNA. To verify that this genetic result is due to impaired interaction of Trm7 variants with Trm732 or Trm734, we are performing immunoprecipitation assays. In addition, we have begun to study interactions of the individual proteins and complexes with tRNA through biochemical methods such as electrophoretic mobility shift assays, to determine the role of each protein in tRNA binding. Through this study of Trm7 variants, we can better understand the interaction of Trm7 with Trm734 and Trm732, which will facilitate the study of conserved homologs in humans.

Keywords: tRNA, modifications, Trm7

57. Utilization of high-pressure NMR for the study of the folded and disordered domains of hnRNP A1

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

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

hnRNP A1 is composed of three domains, two structurally identical RRMs that form the N-terminal protein UP1, and a disordered, glycine rich C-terminal domain (G-CTD). The UP1 domain is responsible for binding the nucleic acid substrates while the G-CTD assembles the multi-protein complexes the protein utilizes to carry out its various functions. It is also a low-complexity domain (LCD) capable of mediating liquid-liquid phase separation.

High-pressure NMR spectroscopy is a powerful technique for examining the biophysical nature of proteins. Application of pressure to a protein increases solvent density and pushes the protein towards a lower molar volume, causing folded proteins to unfold. For disordered domains, pressure has been shown to have just minor effects on NMR parameters.

The utilization of high-pressure NMR spectroscopy for the study of full length hnRNP A1 revealed unique properties for both the structured and disordered domains. It revealed the LCD, at standard conditions, adopts a disordered, but compact conformation that is stabilized by polar and electrostatic interactions. The application of pressure results in a disruption of the intra-protein interactions and the compact nature of the domain.

While pressure normally unfolds proteins, the structured UP1 domain failed to unfold at high pressure. To study this phenomenon, UP1 variants were examined to determine what properties of the protein are responsible for its stability at high pressure. Results have revealed that communication between the two RRM domains is vital for maintenance of the protein’s stability during the application of high-pressure.

References:
Levengood JD, Peterson J, Tolbert BS, Roche J. Thermodynamic stability of hnRNP A1 low complexity domain revealed by high-pressure NMR. Proteins. 2021 Jul;89(7):781-791.

Keywords: RRM, IDP, high-pressure NMR

58. Quantitative modeling of ligand-binding thermodynamics in the cyclic RNA-binding oligomeric protein TRAP

Weicheng Li, Andrew Norris (Department of Chemistry and Biochemistry, The Ohio State University), Katie Lichtenthal, Skyler Kelly (Department of Biological Sciences, SUNY Baffulo), Melody L Holmquist, Vicki Wysocki, (Department of Chemistry and Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, SUNY Baffulo), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the booklet.

59. Deploy RNA G1-G4 dendrimers self-assembled from motifs for drug sheltering with controllable layer-by-layer release

Xin Li (Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, Dorothy M. Davis Heart and Lung Research Institute, James Comprehensive Cancer Center, The Ohio State University), Peixuan Guo (Center for RNA Nanobiotechnology and Nanomedicine, College of Pharmacy, Dorothy M. Davis Heart and Lung Research Institute, James Comprehensive Cancer Center, The Ohio State University)

Abstract:
Dendrimers are polymeric molecules with defined and monodisperse branched structures, which have been used as drug delivery platforms due to their unique properties. Taking advantage of the self-assembly and programmability property of RNA, we used three-way junction RNA nanoparticles as the building block for dendrimer construction to achieve simple synthesis procedure and favorable biodistribution profile. Oligo ribonucleotides produced by solid phase chemical synthesis allow the large-scale manufacture of homologous RNA dendrimers. Employing concepts from RNA nanotechnology enabled the controllable production of four generations of RNA dendrimers from G₁, G₂, G₃, to G₄ with temperature-triggered layer-by-layer release capability. The conjugation of various functional groups into individual RNA strands and the incorporation of functionalized RNA strands into the dendrimers at different sites have been reported. RNA dendrimers conjugated with multiple copies of anticancer drug, paclitaxel, showed comparable cancer cell inhibition effect to free drugs. Furthermore, the encapsulation of cell binding ligands and hydrophobic drugs within the dendrimer significantly reduced the efficiency of cell binding and protein binding respectively, demonstrating the shielding effect of RNA dendrimers. The results imply a potential application of RNA dendrimer for delivery, shielding and controlled release of hydrophobic drugs in vivo.

References:
1. X. Li, M. Vieweger and P. Guo, Nanoscale, 2020, 16514–16525.
2. S. Guo, M. Vieweger, K. Zhang, H. Yin, H. Wang, X. Li, S. Li, S. Hu, A. Sparreboom, B. M. Evers, Y. Dong, W. Chiu and P. Guo, Nat. Commun., 2020, 11, 972.
3. A. Sharma, F. Haque, F. Pi, L. S. Shlyakhtenko, B. M. Evers and P. Guo, Nanomedicine Nanotechnology, Biol. Med., 2016, 12, 835–844.

Keywords: RNA nanotechnology, RNA dendrimer, Drug delivery and shielding

60. Androgen Receptor messenger RNA Metabolism

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

Abstract not available online - please check the booklet.

61. Pum3 as a molecular player in transcriptional termination and R-Loop-Associated DNA Damage

chenyu Lin (The James Comprehensive Cancer Center, The Ohio State University), Wayne Miles (The James Comprehensive Cancer Center, The Ohio State University)

Abstract:
Pum3 as a molecular player in transcriptional termination and R-Loop-Associated DNA Damage

Pum3 was reported to correlate with resistance to genotoxic exposure ssDNA break via preventing PARP1 degradation by caspase3. Given it is vital for the maintenance of homeostasis that DSBs within transcriptionally active regions undergo accurate repair. However, it remains unknown how Pum3 modulates DSBs response and DNA repair. Our research identified Pum3 as RNA/DNA hybrid interactome that promotes R-loop and transcriptional termination. Pum3 is recruited to the DSB site in a DNA-RNA-hybrid-dependent manner, as playing a critical role in promoting Pol III-mediated R-loop processing and imitating subsequent homologous recombination-mediated repair. Pum3-deficient induces defective HR, accumulation of unrepaired DSBs. Pum3 is overexpressed in PRAD and LIHC cancers, and higher expression of Pum3 correlates with lower survival probability in these cancers. Depletion of Pum3 increases the sensitivity of cancer cells to DNA damage-based therapy-cisplatin. Thus, our data indicated that the function of Pum3 in the presence of R-loops around DSBs within transcriptionally active regions promotes accurate repair of DSBs, which may have implications for cancer therapy.

References:
hPuf-A/KIAA0020 modulates PARP-1 cleavage upon genotoxic stress. (PMID: 21266351)

Keywords: Pum3, R-loop , DNA damage

62. Coacervate rescue of nucleic acid enzyme activity damaged by UV

Haiyun Liu (Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA), Philip C Bevilacqua (Department of Chemistry; Center for RNA Molecular Biology; Department of Biochemistry, Microbiology, and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA)

Abstract:
According to the RNA world hypothesis, RNA had the roles of catalyst and genetic information carrier in the emergence of life. However, lack of proofreading in RNA replication and the unstable chemical nature of RNA compared to DNA make it more challenging for RNA to retain its catalytic activity. Additionally, ribozymes were under strong UV radiation of the early earth which lacked an ozone layer. On the other hand, membraneless compartments such as coacervates have been hypothesized as plausible prebiotic compartments due to their ability to enrich cations, nucleotides and RNA. Moreover, enhancements of activity of various ribozymes by coacervates have been described in previous studies. We hypothesize that coacervates can rescue the activity of intact ribozymes, as well as damaged, mutated, and fragmented ones. To test this hypothesis, we are using UV light to damage a DNAzyme, and attempting rescuing it with PDAC/D10 coacervate. Our latest design and results will be presented.

References:
Poudyal, R.R., Guth-Metzler, R.M., Veenis, A.J. et al. Template-directed RNA polymerization and enhanced ribozyme catalysis inside membraneless compartments formed by coacervates. Nat Commun 10, 490 (2019). https://doi.org/10.1038/s41467-019-08353-4

Keywords: Astrobiology, Nucleic acid enzyme, Coacervate

63. Investigation of structural switch that regulates pre-miR-92a processing by NMR

Yaping Liu (University of Michigan), Sarah Keane (University of Michigan)

Abstract:
MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumorigenesis. miRNAs are initially expressed as a long primary transcripts (pri-miRNA) which are processed to yield pre-miRNAs and ultimately mature miRNAs. The miR-17-92a cluster, also known as ‘oncomiR-1’ is a polycistronic pri-miRNA that plays a pivotal regulatory role in cellular processes, including the cell cycle, proliferation and apoptosis. OncomiR-1 includes six constitute miRNAs, each processed with different efficiencies as a function of both developmental time and tissue type. However, the structural mechanism that regulate the differential processing still remain unclear.
NMR is a key technique that has significantly advanced our understanding of RNA structure and dynamics. In vitro processing assays indicate that pre-miR-92a processing is inhibited relative to other miRs within oncomiR-1. We are interested in uncovering the structural basis for the differential processing by NMR. NPSL2 has been identified as an inhibitor of pre-miR-92a processing. Therefore, in this work we determined the solution structure of NPSL2. Our data suggest that NPSL2 is dynamic, and we hypothesize that the dynamics are linked to regulation of processing. Our ongoing efforts will investigate the mechanism of the structural switch that promotes premiR-92a processing in the context of the full-length oncomiR-1 RNA.

References:
1. Kim, V. N., Han, J. & Siomi, M. C. Nat Rev Mol Cell Biol (2009) 10, 126-139. doi:10.1038/nrm2632.
2. Mogilyansky, E. & Rigoutsos, I. Cell Death Differ (2013) 20, 1603-1614. doi:10.1038/cdd.2013.125. PMC3824591.
3. He, L. et al. Nature (2005) 435, 828-833. doi:10.1038/nature03552. PMC4599349.
4. O'Donnell, K. A., Wentzel, E. A., Zeller, K. I., Dang, C. V. & Mendell, J. T. Nature (2005) 435, 839-843. doi:10.1038/nature03677.
5. Chakraborty, S. & Krishnan, Y. Nucleic Acids Res (2017) 45, 9694-9705. doi:10.1093/nar/gkx613. PMC5766152.
6. Chaulk, S. G., Xu, Z., Glover, M. J. & Fahlman, R. P.. Nucleic Acids Res (2014) 42, 5234-5244. doi:10.1093/nar/gku133. PMC4005684.

Keywords: MicroRNNAs, oncomiR-1, NPSL2

64. Selectivity mechanisms of an aminoacyl-tRNA trans-editing factor

Xiao Ma (Department of Chemistry and Biochemistry, the Ohio State University), Marina Bakhtina (Department of Chemistry and Biochemistry, the Ohio State University), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, the Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, the Ohio State University)

Abstract:
Aminoacyl-tRNA synthetases (aaRSs) are responsible for charging their cognate tRNAs with the correct or cognate amino acid. The structural features of aaRS catalytic domains provide a high degree of selectivity for both the amino acid and tRNA; however, given the similar stereochemical properties of many amino acids, errors in tRNA charging can occur, especially for the smaller and isometric amino acids. Organisms in all three domains of life possess editing enzymes that function in trans to deacylate mischarged tRNAs. Caulobacter crescentus ProXp-ala recognizes the terminal base pair, C1:G72, on the acceptor stem of tRNA^Pro to deacylate mischarged Ala-tRNA^Pro but not Ala-tRNA^Ala. We used NMR spectroscopy, binding and activity assays to examine the structural details of this discrimination. We also found that the A76 of tRNA^Pro was essential for its binding to ProXp-ala, thus supporting a previously proposed interaction with a highly conserved Lys. Ongoing work continues to refine a 3D model of the enzyme-product complex of ProXp-ala bound to a tRNA^Pro acceptor stem. These studies will improve our understanding of substrate recognition by a bacterial trans-editing factor with the potential to inform new antibiotic drug design.

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

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

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

Keywords: NMR, Protein-RNA

65. Human spliceosomal snRNA sequence variants generate variant spliceosomes

Justin W. Mabin (Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health), Peter W. Lewis (Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health), David A. Brow (Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health), Heidi Dvinge (Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health)

Abstract:
Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4 and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than one thousand-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1 to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing non-canonical snRNAs may contribute to different tissue- and cell-type specific alternative splicing patterns.

Keywords: snRNA, snRNA biogenesis, snRNA variants, spliceosomes, pre-mRNA splicing

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

Ananya Mahapatra (Genome, Cellular and Developmental Biology, Indiana University), Sarah Deffit (Medical Sciences Program, Indiana University School of Medicine), Pranathi Vadlamani (Medical Sciences Program, Indiana University School of Medicine), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine),

Abstract not available online - please check the booklet.

67. Human patient derived iPSCs as a system for studying cardiac dysfunctions in Myotonic Dystrophy type 1

Sarah N. Matatov (Department of Biochemistry University of Illinois, Urbana-Champaign, Illinois, USA), Chaitali Misra (Department of Biochemistry University of Illinois, Urbana-Champaign, Illinois, USA), Auinash Kalsotra (Department of Biochemistry, Cancer CenterIllinois, Carl R. Woese Institute of Genomic Biology, University of Illinois, Urbana-Champaign, Illinois, USA )

Abstract:
Myotonic Dystrophy type 1 (DM1), is the most prevalent form of adult-onset muscular dystrophy. DM1 is caused by a CTG trinucleotide repeat expansion in the 3’-UTR of the DMPK gene. Over 80% of DM1 patients experience heart dysfunctions making this the second leading cause of death for affected individuals. The CUG expanded repeat in RNA creates a toxic gain-of-function phenotype, which causes RNA aggregation into ribonuclear foci. These RNA foci sequester Muscleblind-like Splicing Regulators (MBNL) and cause over-expression of the CUGBP Elav-Like Family Member 1 (CELF1) family of splicing factors. Our lab recently demonstrated that aberrant expression of a non-muscle splice isoform of RNA-binding protein RBFOX2 triggers the characteristic cardiac conduction delay, atrioventricular heart blocks, and spontaneous arrhythmogenesis in DM1 hearts. To further study the mechanisms by which non-muscle RBFOX2 alters the electrophysiological properties and thereby induces cardiac arrhythmias in DM1, we generated a human cardiomyocyte cell culture model for DM1. We acquired induced pluripotent stem cells (iPSC) from a healthy donor, a patient with a mild case of DM1 (238 repeats), and another patient with severe case of DM1 (1150 repeats). We successfully established a robust protocol for differentiating these iPSC lines into beating cardiomyocytes with reliable morphologic, molecular, and functional features. Importantly, we demonstrate that the differentiated experimental cells faithfully recapitulate the classical characteristics of DM1, including ribonuclear foci formation, MBNL sequestration, misregulation of alternative splicing, cardiac conduction delay, and contractility defects. Using these human patient-derived cardiac culture systems, we are now investigating the mechanistic and functional roles of specific RNA binding proteins in DM1 cardiac pathogenesis.

Keywords: Muscular Dystrophy, Cardiac Dysfunction, iPSCs

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

Chaitali Misra (Departments of Biochemistry, University of Illinois, Urbana-Champaign), Ullas V. Chembazhi (Departments of Biochemistry, University of Illinois, Urbana-Champaign), Sarah N. Matatov (Departments of Biochemistry, University of Illinois, Urbana-Champaign), Auinash Kalsotra (Departments of Biochemistry, Cancer CenterIllinois, Carl R. Woese Institute of Genomic Biology, University of Illinois, Urbana-Champaign)

Abstract not available online - please check the booklet.

69. Calculations of theoretical pKa values for modified nucleobases

Alan J. Mlotkowski (Chemistry Department - Wayne State University ), Evan Jones (Chemistry Department - Wayne State University ), H. Bernhard Schlegel (Chemistry Department - Wayne State University ), Christine S. Chow (Chemistry Department - Wayne State University )

Abstract not available online - please check the booklet.

70. GSK3α plays a vital role in male fertility by regulating m6A levels in testis

Rushdhi Rauff (Department of Chemistry and Biochemistry, Kent State University), Souvik Dey (Department of Biological Sciences, Kent State University), Wesam Nofal (Department of Biological Sciences, Kent State University), Mustfa Kabi (Department of Biological Sciences, Kent State University), Srinivasan Vijayaraghavan (Department of Biological Sciences, Kent State University), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry, Kent State University)

Abstract:
N6-methyladenosine (m6A) is the most abundant nucleotide modification observed in eukaryotic mRNA. N6-methylation levels are dynamic in mRNA, where methylations are added and removed by proteins that are known as writers and erasers, respectively. FTO (Fat mass and obesity-associated protein) is one of the most well-studied methyl eraser proteins found in eukaryotes. It has been known that in stem cells, FTO is a substrate of GSK3 protein. GSK3 enzyme, a serine/threonine protein kinase, is ubiquitous and is expressed as two paralogs, GSK3α and GSK3β, encoded by two genes. The only phenotype of global deletion of Gsk3α is male infertility. Here we hypothesize that GSK3α plays a similar role in controlling m6A levels in testis by interacting with FTO. Our experiments show that FTO is localized to post-meiotic spermatogenic cells within the seminiferous tubules. There is no visible staining for FTO in Sertoli cells, spermatogonia, myoid, and Leydig cells in the interstitial spaces of the seminiferous tubules. Co-immunoprecipitation of FTO with GSK3α, but not with GSK3β was observed when immunoprecipitation was carried out for GSK3α and GSK3β followed by western blots using FTO primary antibodies. A marked increase in FTO expression was seen in day 20 testis up to day 40 adult testis; an expression pattern characteristic of proteins expressed in post-meiotic developing spermatocytes and spermatids. Using HPLC and ELISA-based methods, we observed that m6A levels are significantly lower in GSK3α KO mice compared to WT mice. Hence, our experimental findings illustrate that GSK3α plays a main role in male fertility by regulating m6A levels in testis.

Keywords: m6A, nucleotide modifications, epitranscriptomics

71. Elucidating the mechanisms of regulation for MDM2 alternative splicing

Matias Montes (Center for Childhood Cancer and Blood diseases Nationwide Childrens Hospital), Dawn Chandler (Center for Childhood Cancer and Blood diseases Nationwide Childrens Hospital)

Abstract:
MDM2, a well-known negative regulator of the tumor suppressor protein p53 undergoes complex alternative splicing. One of its spliced isoforms, MDM2-ALT1 comprised of exons 1 through 3 and 12, is highly expressed in several cancers such as lung carcinoma, liposarcoma and rhabdomyosarcoma (RMS). In RMS, MDM2-ALT1 expression correlates with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress. The way that MDM2 splicing is controlled is not well understood. Here we explore the possibility that a nuclear miRNA, miR29b, interplays with RNA binding proteins to regulate MDM2 splicing.

miR-29b is part of the miR-29 family, and has been reported as one of the miRNAs that localize to the nuclear compartment of cells. However, its function there is still unknown. Additionally, miR-29b has been shown to act as a tumor suppressor. In RMS, we have shown how miR-29b levels are downregulated in both cell lines as well as in patient samples. Here, we show how miR-29b is downregulated after genotoxic stress, which inversely correlates with MDM2-ALT1 expression, similarly to RMS. Furthermore, we show how miR-29b influences the alternative splicing of MDM2, which is not through canonical gene-silencing of splicing factors. We have identified predicted binding sites for the miR-29b in the regulated region of MDM2 pre-mRNA. Moreover, we demonstrate that miR-29b directly binds to the MDM2 pre-mRNA and that the site present in the exon 12 is important for alternative splicing regulation. Finally, we show how Argonaute 1 antagonizes miR-29b influence over the MDM2 alternative splicing, and interaction between AGO1 and miR-29b is increased after genotoxic stress. In summary, in this work we report for the very first time how a nuclear miRNA has direct involvement regulating the alternative splicing of a gene.

Keywords: alternative splicing, miRNAs, MDM2

72. Identifying novel m6A binding proteins using a peptide as a bait which was enriched in a phage display experiment.

David Morgan (Department of Chemistry and Biochemistry - Kent State University ), Rushdhi Rauff (Department of Chemistry and Biochemistry - Kent State University ), Sanjaya Abeysirigunawardena (Department of Chemistry and Biochemistry - Kent State University )

Abstract:
N6-methyladenosine (m6A) is the most abundant nucleotide modification in eukaryotic mRNA. The m6A modification can be added, removed, and recognized by m6A writers, erasers, and readers, respectively. Due to the presence of many m6A modifications in some mRNAs, likely there are more m6A binding proteins that can interact site-specifically. Protein pull-down assays were performed using various methylated and unmethylated RNA targets to identify proteins that are bound to methylated RNA with sequence and structural specificity. Lysates from various cancer cell-lines were used for these pull-down assays. Interestingly, the enriched proteins have sequence similarity to dodecamer peptides selected from phage display experiments using same RNA baits. Our experiments show that m6A readers interacts with RNA using unique strategies.

Keywords: m6A, Nucleotide modifications, Epitranscriptomics

73. Long noncoding RNAs are abundant targets of ADAR3 in glioblastoma

Priyanka Mukherjee (Cell, Molecular and Cancer Biology, Indiana University School of Medicine), Heather A. Hundley (Medical Sciences Program, Indiana University School of Medicine)

Abstract not available online - please check the booklet.

74. Towards structural characterization of RNA-only complexes via small-angle scattering techniques

Aldrex Munsayac (Department of Chemistry, University of Michigan), Ian Hall (Department of Chemistry, University of Michigan), Sarah C. Keane (Department of Chemistry and Program in Biophysics, University of Michigan)

Abstract:
Noncoding RNAs constitute the majority of the transcribed human genome and play a multitude of roles in biological processes; having been shown to regulate gene expression at the levels of transcription, RNA processing, and translation.¹ In the foodborne pathogen Listeria monocytogenes, two riboregulators—the prfA RNA thermosensor (RNAT) and the SreA riboswitch—work in tandem to regulate the expression of PrfA, the major regulator of Listeria virulence gene expression.² However, the precise molecular mechanisms governing the interaction between the prfA RNAT and SreA riboswitch remains undefined. In order to understand how these two cis-regulatory RNA elements are able to function in trans, elucidation of their structure at both the global and local scale is necessary. RNA-only structures account for only 1% of the three-dimensional structures deposited in the protein data bank.³ Furthermore, we expect that both the prfA RNAT and the SreA riboswitch undergo significant remodeling upon interaction, necessitating the development and implementation of new approaches for determining the structures of RNA-RNA complexes. To address this, we propose to employ a hybrid structural biology approach using small-angle X-ray/neutron scattering (SAXS/SANS) techniques complemented with NMR spectroscopy to unambiguously probe RNA-RNA interactions. Here, we lay the groundwork for the development of contrast variation SANS (CV-SANS) for RNA-only complexes. Using the HIV-1 RNA dimerization initiation site (DIS) kissing complex as a model system, we have collected SAXS data and successfully reconstructed the molecular envelope of the complex, which will serve as the foundation for our initial CV-SANS studies. CV-SANS coupled with NMR spectroscopy has the potential to solve very large, dynamic RNA structures.

References:
1. Cech, T.R.; Steitz, J.A. The noncoding RNA revolution—trashing old rules to forge new ones. Cell. 2014, 35, 77-94.

2. Loh, E.; Dussurget, O.; Gripenland, J;, Vaitkevicius, K.; Tiensuu, T.; Mandin, P.; Repoila, F.; Buchrieser, C.; Cossart, P.; Johansson, J. A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes. Cell. 2009, 139(4), 770-779.

3. Das, R. RNA structure: a renaissance begins?. Nature Methods. 2021, 18(5), 439-439.

Keywords: Small-angle scattering, RNA-RNA interactions, RNA structural biology

75. Proximity labeling to monitor in vivo protein dynamics during nonsense-mediated mRNA decay

Sarah L.H. Nock (Department of Genetics and Genome Sciences, Case Western Reserve University), DaJuan L. Whiteside (Department of Genetics and Genome Sciences, Case Western Reserve University), Kristian E. Baker (Department of Genetics and Genome Sciences, Case Western Reserve University)

Abstract not available online - please check the booklet.

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

Elizabeth A. Oser (Department of Chemistry and Biochemistry, The Ohio State University), Tyler McClain (Department of Chemistry and Biochemistry, The Ohio State University), Jane E. Jackman (Department of Chemistry and Biochemistry, The Ohio State University)

Abstract not available online - please check the booklet.

79. A key to understanding RNA’s interactome: photocleavable probes for spatiotemporal control of RNA

Marta Rachwalak (Chemistry, Carnegie Mellon University), Avleen K. Chawla (Chemistry, Carnegie Mellon University), Megan L. Van Horn (Chemistry, Carnegie Mellon University), Anna M. Kietrys (Chemistry, Carnegie Mellon University)

Abstract:
Caging is one of the key methods for imaging and studying interactions and structural aspects of RNA inside the cell. Despite the rapid improvements in caging and cloaking techniques, scientists still struggle to develop universal and effective tools for understanding the complexity and dynamics of the RNA interactome in cellulo. To overcome this absence of appropriate tools, we designed and synthesized a set of photoreversible probes for studying RNA’s function and interactions within cells. An introduction of photocleavable protecting groups (PPGs) ensures control over the biological function of various RNAs (e.g., siRNA, mRNA) and allows for structure and function reactivation, in a spatiotemporal manner, in the living cell. Using the same set of probes in living cells may help to map short- and long-distance interactions in RNA complexes.
Herein, we present a synthesis of photocleavable (INPhRNA) probes designed to block RNA’s biological function through acylation of 2′-OH groups in single stranded regions. Protection of 2’-OH moieties significantly disrupts RNA structure and as such its function. The RNA function may be fully restored via UV-irradiation without any cellular damage. The photosensitive residue of the probes consists of PPG units connected by linkers. By modulating the type of linker and its length, we are able to affect both the solubility and reactivity of the probe. To show the biological applicability of the proposed strategy, we tested the probe’s cytotoxicity and potential for spatiotemporal control over siRNA and mRNA. We believe that the developed caging probes may serve as an efficient method for postsynthetic modification of RNA to protect them from degradation and direct their cellular activity. Additionally, they may be used as tools to explore RNA interactome complexity in vivo.