Date of Award
Doctor of Philosophy (PhD)
Biochemistry & Molecular Biophysics
Kristen W. Lynch
PSF is a ubiquitously expressed and essential nuclear protein that influences many aspects of the genome maintenance and gene expression pathways. Although previous studies have identified numerous protein cofactors and nucleic acid targets of PSF, insufficient work has been done to understand how it is regulated to accomplish its various functions in a coordinated manner. Previous research in the Lynch laboratory demonstrated that, in T cells, PSF is a downstream target of the serine/threonine kinase GSK3. Phosphorylation of PSF T687 by GSK3 promotes interaction of PSF with another multifunctional nuclear factor, TRAP150. This interaction prevents PSF from binding RNA and regulating alternative splicing of CD45 exon 4, though the mechanism for TRAP150’s effect on PSF is unknown. In this dissertation, I probe this regulatory mechanism using several biochemical and biophysical tools. First, I use a combination of mammalian co-immunoprecipitation reactions and GST pull-downs to isolate the minimal domains of PSF and TRAP150 responsible for their interaction. Interestingly, TRAP150 binds the highly conserved DBHS core of PSF using a previously unannotated 70-residue region. Using electrophoretic mobility shift assays and UV-induced protein/RNA crosslinking, I next localize PSF’s mRNA-binding ability to noncanonical RRM2, and I show that TRAP150’s interaction with the PSF RRMs is sufficient to ablate PSF/mRNA interaction. Further, I provide evidence from RASL-Seq showing that PSF regulates ~40 alternative splicing events in T cells. Critically, TRAP150 has an antagonistic effect on PSF’s regulation of these events. Finally, I use limited proteolysis assays to provide evidence that PSF undergoes a phosphorylation-dependent conformational change in unstimulated versus stimulated T cells. Together, these data suggest that phosphorylation of PSF T687 by GSK3 induces a conformational shift in PSF that allows TRAP150 to bind its RRMs and, in turn, decrease its ability to bind RNA and regulate alternative splicing. Together, the results described here point to a generalizable model of PSF regulation predicated on selective posttranslational modification and induced conformational changes that influence its interactions with different cofactors and targets.
Yarosh, Christopher, "The Regulation of Psf Activity in T Cells by Trap150 and Gsk3" (2016). Publicly Accessible Penn Dissertations. 2115.