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(Unpublished)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Unpublished |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