Defense Date

6-11-2024

Graduation Date

Summer 8-9-2024

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

School

School of Science and Engineering

Committee Chair

Mihaela Rita mihailescu

Committee Member

Michael Cascio

Committee Member

Jeffrey Evanseck

Committee Member

Nathan Baird

Keywords

s2m, SARS-CoV-2, kissing dimer, miR-1307-3p, microRNA, virus, nucleocapsid, NEAT1, triple helix, G quadruplex

Abstract

The studies within this dissertation highlight the importance of RNA structure and function, highlighting potential mechanisms in the pathogenesis of the SARS-CoV-2 virus and hallmarks of ALS. Specifically, the s2m, a 41-nt structure found within the hyper variable region of the 3’ UTR of the SARS-CoV-2 virus, was investigated as this element has no definitive role in viruses but its high conservation in several viral families warrants investigation. We used various biophysical techniques to determine if a palindromic sequence in the terminal loop could participate in dimerization activities, which may have implications in recombination and the longevity of the virus. Further, we noted the existence of two potential binding sites for miR-1307-3p on the s2m and probed the ability of this motif to interact with this host element. Additionally, we tracked the s2m for acquired mutations throughout the duration of the pandemic and found the existence of a G-to-U point mutation at the 15th position correlated with the rise in prevalence of the Delta variant, and we thus examined the ability of the variant motif to participate in dimerization and host binding. Our results found that the nucleocapsid protein of the virus facilitates conversion to an extended duplex structure of two s2m elements and further showcases a stepwise decrease in affinity for miR-1307-3p as new viral variants emerge.

The second project in this work encompasses a molecular mechanism that may alter the structural integrity of paraspeckles. Paraspeckles are subnuclear bodies found in hyperabundance upon ALS onset in the neurons of patients. The molecular bodies are built on scaffolds of the non-coding RNA NEAT1. We and others identified five regions with the potential to form G-quadruplex structures in this RNA, with the 5th G-repeat residing near the 3’ end of the molecule and within 20 nts of the well-characterized triple helix. Previous work in our group shows that the fold of the triple helix is dependent on the proper folding of the G-quadruplex. Thus, this work used a 3’ exonuclease to determine the effects these tertiary structures have on regulating the degradation of NEAT1. Our results show that the triple helix and G-quadruplex work in tandem to slow the effects of degradation, and the G-quadruplex adds enhanced stability to the molecule as this region is preserved.

Overall, these studies highlight the importance of RNA structure in functionality as well as identify possible mechanistic targets for viral and neurodegenerative therapies.

Language

English

Additional Citations

Imperatore, J. A.; Cunningham, C. L.; Pellegrene, K. A.; Brinson, R. G.; Marino, J. P.; Evanseck, J. D.; Mihailescu, M. R. Highly Conserved S2m Element of SARS-CoV-2 Dimerizes via a Kissing Complex and Interacts with Host miRNA-1307-3p. Nucleic Acids Research 2022, 50 (2), 1017–1032. https://doi.org/10.1093/nar/gkab1226.

Cunningham, C. L.; Frye, C. J.; Makowski, J. A.; Kensinger, A. H.; Shine, M.; Milback, E. J.; Lackey, P. E.; Evanseck, J. D.; Mihailescu, M.-R. Effect of the SARS-CoV-2 Delta-Associated G15U Mutation on the S2m Element Dimerization and Its Interactions with miR-1307-3p. RNA 2023, 29 (11), 1754–1771. https://doi.org/10.1261/rna.079627.123.

Frye, C. J.; Shine, M.; Makowski, J. A.; Kensinger, A. H.; Cunningham, C. L.; Milback, E. J.; Evanseck, J. D.; Lackey, P. E.; Mihailescu, M. R. Bioinformatics Analysis of the S2m Mutations within the SARS‐CoV‐2 Omicron Lineages. J Med Virol 2022, 10.1002/jmv.28141. https://doi.org/10.1002/jmv.28141.

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