Nguyen, Elaine
(2022)
Identifying the molecular determinants of the C-terminal LARP1 interaction with 5’ TOP mRNAs.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
TOR (target of rapamycin), a ubiquitous eukaryotic serine/threonine kinase is central to the maintenance of cellular health through its critical role in a number of key biological processes. As a target of TOR, LARP1 connects TOR signaling to protein synthesis through its direct interaction with TOP (5’ Terminal Oligopyrimidine) mRNAs, which in mammalian systems, encode all protein constituents of the ribosome. It is well established that the phosphorylation of LARP1 by TOR regulates TOP translation, but seemingly antithetical functions have been proposed such that LARP1 stimulates, stabilizes, and represses the translation of TOP mRNAs. As a repressor, the C-terminal region of LARP1 including the DM15 is sufficient for mTORC1 control of TOP translation. Despite the ambiguous context surrounding the opposing functions of LARP1, it is evident that the TOR-LARP1-TOP regulatory axis is paramount to cellular homeostasis. Unsurprisingly, LARP1 is associated with a variety of human health disorders including several cancers, and more recently, COVID-19. Understanding the role of LARP1 and how it recognizes its cognate mRNAs is therefore valuable in the pursuit of potential targeted therapies.
Here, we dissected the role of the C-terminal region of LARP1 in TOP mRNA recognition using biochemical and biophysical approaches to probe both components of the interaction. We examined the DM15 region across evolution, RNA characteristics, and regions beyond the DM15. We demonstrated that the DM15 fold is well conserved across evolution with crystal structures of D. melanogaster DM15 (DmDM15) and D. rerio (DrDM15). Both orthologs expectedly retained TOP binding activity with substitutions primarily located distal to the RNA-binding surface that may modulate flexibility in ligand binding. Interestingly, TOP mRNAs encoding the same gene may vary in sequence composition across organisms, but similar secondary structures remain that may contribute to their interaction with the DM15. Expanding beyond the DM15 region, we identified at least one additional amino acid C-terminal to the DM15 that participates in RNA interaction. Finally, we began exploring the potential for an interdomain interaction between the centrally located La-module and the DM15 region of LARP1.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
13 August 2022 |
| Date Type: |
Publication |
| Defense Date: |
28 July 2022 |
| Approval Date: |
18 December 2024 |
| Submission Date: |
25 July 2022 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
161 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
LARP, RNA-binding protein, TOP, DM15, translation regulation |
| Date Deposited: |
18 Dec 2024 20:00 |
| Last Modified: |
19 Dec 2024 13:08 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/43359 |
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