Advisor(s)
Jun Zhang
Committee Member(s)
Aaron Lucius
David Schneider
Rui Lu
Todd Green
School
College of Arts and Sciences
Document Type
Dissertation
Department (new version)
Chemistry
Date of Award
1-6-2025
Abstract
RNA-binding proteins (RBPs) play critical roles in RNA processing and regulation. Among these RBPs, U1-70K is a key protein involved in RNA processing, nuclear speckle formation, and has implications in neurodegenerative diseases such as Alzheimer's. Despite its significance in transcription, RNA processing, nuclear speckle formation, and nonsense-mediated decay, studies on U1-70K have been limited primarily to its well-structured N-terminal domain due to challenges in solubilizing and expressing the full-length protein. In this dissertation, we address these challenges by optimizing cell strain and co expressing DnaK and DnaJ, enabling the successful expression, solubilization, and purification of full-length U1-70K. Using fluorescence polarization binding assays, we demonstrate that the RNA Recognition Motif (RRM) and Basic Acidic Dipeptide repeat domain 1 (BAD1) of U1-70K are necessary and sufficient for binding to the SR family protein SRSF1, interacting specifically with the RRM1 domain and the RS tail, respectively. We further show that the kinase SRPK1 phosphorylates the BAD1 domain iv at nine sites, inhibiting its binding to SRSF1. NMR chemical shift perturbation (CSP) analysis was employed to pinpoint the residues on SRSF1 RRM1 that mediate binding to U1-70K RRM. Additionally, western blot calibration curves were used to determine the concentration thresholds for phase separation and revealed that both the BAD domains and the RRM contribute to U1-70Ks’s liquid-liquid phase separation (LLPS). Upon phosphorylation with SRPK1, U1-70K’s solubility was increased. In vitro phase separation results correlate with U1-70K’s localization to nuclear speckles in HeLa cells. Notably, we found that Aβ42 induces U1-70K aggregation in vitro under conditions where it would typically phase separate, shedding light on the co-aggregation observed in Alzheimer’s pathology. In conclusion, this dissertation elucidates the role of U1-70K in binding SRSF1 through interactions between its structured RRM domains and the flexible BAD1 and RS tail domains, with phosphorylation playing a regulatory role. The findings also highlight the contribution of U1-70K’s RRM and BAD domains to phase separation and co?aggregation with Aβ42, offering insights into its involvement in nuclear speckle localizations and Alzheimer’s disease.
ProQuest ID
Recommended Citation
Paul, Trenton Myers, "Characterization Of The Roles Of U1-70K In Alternative Splicing And Nuclear Speckle Formation" (2025). All ETDs from UAB. 7251.
https://digitalcommons.library.uab.edu/etd-collection/7251
