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The Roles of RNA Structural Dynamics and Molecular Interaction in Viral Gene Expression

Chiu, Liang-Yuan
http://orcid.org/0000-0002-0584-1023
http://rave.ohiolink.edu/etdc/view?acc_num=case1625243374378648

Abstract Details

2021, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Current HIV antiretroviral therapies target multiple steps of the viral lifecycle. Expanding potential targets can bene t HIV therapy by increasing the types of drugs used in combination. Alternative splicing is a potential step to target because it allows 9 kb HIV viral genome to produce more than 40 different mRNA transcripts critical to viral replication. Alternative splicing is regulated through various interactions between cellular host proteins and specific structural elements present on the viral ribonucleic acid (RNA) that can act together either to enhance or repress splicing at specific sites. In order to better understand the splicing mechanisms, it is important to characterize the structures of the molecules responsible and understand how these molecules interact to perform their functions. For this reason, this research aims to shed light on RNA elements and proteins that play a role in HIV splicing, namely splice site acceptor 3 (ssA3). The A3 3'-splice site of HIV-1 is required for Tat mRNA production. The inefficient utilization of this splice site has been attributed to the presence of a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The cellular protein HnRNP H binds to ESS2P element to inhibit the splicing process. However, there is no structural and mechanistic information about how hnRNP H recognizes ESS2P to inhibit the splicing. In our studies, we aim to characterize the three-dimensional structures of hnRNP H and ESS2P. The domain organization of hnRNP H/F proteins is modular consisting of N-terminal tandem quasi-RNA Recognition Motifs 1 and 2 (HqRRM1,2) and a third C-terminal qRRM3 embedded within glycine-rich repeats. The tandem qRRMs are connected through a 10-residue linker with most of the amino acids strictly conserved between hnRNP H and F. We probed the structural dynamics of its HqRRM1,2 domain with X-ray crystallography, NMR spectroscopy, and small angle X-ray scattering (SAXS). We observed that HqRRM1,2 contains multiple conformations in solution by SAXS. These exchangeable conformations are located on the linker region and RNA recognition sites. Moreover, we also identified that the alternative structure exist in ssA3 by NMR. The three-dimensional structure of the major conformation of ESS2P has been determined using NMR spectroscopy and SAXS. The hairpin structure reveals that apical loop of ESS2P is the binding site for hnRNP A1 to inhibit splicing. The minor conformation of ESS2p potentially provides the binding site for hnRNP H with an exposed G-tract. Collectively, this work provides evidence that RNA itself can form different unique scaffolds to trap different RNA binding proteins to regulate splicing.
Blanton Tolbert (Advisor)
Fu-Sen Liang (Committee Chair)
Thomas Gerken (Committee Member)
Gregory Tochtrop (Committee Member)
Shane Parker (Committee Member)
Biochemistry; Biophysics
HIV; ESS2p; Splicing; NMR; ssA3

Recommended Citations

Citations

  • Chiu, L.-Y. (2021). The Roles of RNA Structural Dynamics and Molecular Interaction in Viral Gene Expression [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1625243374378648

    APA Style (7th edition)

  • Chiu, Liang-Yuan. The Roles of RNA Structural Dynamics and Molecular Interaction in Viral Gene Expression. 2021. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1625243374378648.

    MLA Style (8th edition)

  • Chiu, Liang-Yuan. "The Roles of RNA Structural Dynamics and Molecular Interaction in Viral Gene Expression." Doctoral dissertation, Case Western Reserve University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1625243374378648

    Chicago Manual of Style (17th edition)

case1625243374378648
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.