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CFIm68 Regulates PolyA Site Usage Through a Novel UGUA Dimeric Motif

Herron, Richard (2022) CFIm68 Regulates PolyA Site Usage Through a Novel UGUA Dimeric Motif. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Alternative polyadenylation (APA) has in recent studies been linked to the mechanistic target of rapamycin (mTOR) signaling pathway. We previously used the cTag-PAPERCLIP technique to discover an APA shift in Trim9, an E3 ubiquitin ligase with a role in neurodevelopment, in Tsc1-knockout neurons in mouse brain in vivo. In this study, we characterized the molecular mechanisms of Trim9 APA shift and established human relevance. In both mouse Neuro2a and human BE2C cells, mTORC1 signaling pathway activities regulated the balance between Trim9/TRIM9 isoforms. We further identified that CFIm68 promotes Trim9-S/TRIM9-S expression, and we found an evolutionarily conserved UGUA dimeric motif (UGUAYUGUA) that was critical for TRIM9-S polyadenylation site (PAS) usage. Through PAPERCLIP profiling, we identified additional CFIm68-regulated PASs with a UGUA dimeric motif in BE2C cells, and we experimentally validated the contribution of the UGUA dimeric motif to PAS usage in BMPR1B, MOB4, and BRD4 through reporter assays. We also showed that loss of CFIm68 alters the balance between BRD4-L and BRD4-S at both mRNA and protein levels. Lastly, we demonstrated that the UGUA dimeric motif was a transferable cis-element for CFIm68 regulation using a JUNB reporter. Importantly, a single UGUA motif was insufficient to confer CFIm68 regulation in JUNB. Our results demonstrate a role of CFIm68 in Trim9/TRIM9 and BRD4 APA regulation and identify a previously unknown functional role of the UGUA dimeric motif in selected CFIm68-regulated PASs. Furthermore, our results suggest a potential connection between neurological disorders with mTOR pathway dysregulation (“mTORopathies”) and neurodevelopment through Trim9/TRIM9. Lastly, our results also suggest that BRD4 isoform imbalance could be a possible outcome from CFIm dysregulation in cancer.

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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Herron, Richardrsh35@pitt.edursh350000-0003-0212-9070
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorHwang, Hun-Wayhunway.hwang@pitt.eduhunway.hwang
Committee ChairChu, Charleenctc4@pitt.eductc4
Committee MemberDonnelly, Chrischrisdonnelly@pitt.educhrisdonnelly
Committee MemberArndt, Karenarndt@pitt.eduarndt
Committee MemberClemens, Paulapclemens@pitt.edupclemens
Date: 22 December 2022
Date Type: Publication
Defense Date: 22 June 2022
Approval Date: 22 December 2022
Submission Date: 1 August 2022
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 146
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cellular and Molecular Pathology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Alternative Polyadenylation, CPSF
Date Deposited: 22 Dec 2022 14:24
Last Modified: 22 Dec 2022 14:24
URI: http://d-scholarship.pitt.edu/id/eprint/43437

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