Aging is a complex, incompletely understood phenomenon that substantially impacts human health on numerous levels. Major hallmarks of the aging process at the molecular level include changes in genomic stability and gene expression that stem from the decreased efficiency or reprogramming of core nuclear processes such as chromatin maintenance, DNA damage repair and RNA processing. Ultimately, these deleterious changes lead to the development of aging-related disorders such as cancer, immune dysfunction and neurodegeneration. To avert these harmful states, cells must tightly regulate the multifunctional nuclear proteins responsible for controlling critical nuclear operations. Despite their importance in promoting cell health, our comprehension of how these proteins are regulated in a signal-responsive manner remains largely incomplete. The goal of this proposal is help remedy this situation by understanding how one such nuclear factor, PSF, is regulated to control its functional capabilities. PSF (SFPQ) is a 707 amino acid protein that plays a role in, among other things, the DNA damage response, transcription, and several steps of the RNA biogenesis pathway. Recent work in the Lynch lab has identified PSF as a downstream target of the serine/threonine kinase GSK3. Phosphorylation of PSF T687 by GSK3 promotes interaction of PSF with TRAP150, and this interaction abrogates PSF's ability to bind at least one known RNA target. However, it is so far unclear how phosphorylation regulates TRAP150 association or how mechanistically TRAP150 alters PSF's function. To fully understand this regulatory regime, I will first determine the molecular features that underpin PSF-TRAP150 interaction by using co-immunoprecipitation and pulldown assays to define the minimal complex interface. I will next employ pulldowns, electrophoretic mobility shift assays and UV crosslinking experiments to biochemically analyze the effects of GSK3 phosphorylation and TRAP150 association on PSF's ability to bind other biologically important protein partners and DNA and RNA targets. Finally, I will use limited proteolysis, mass spectrometry, and hydrogen/deuterium exchange mass spectrometry to investigate how phosphorylation, TRAP150 association, and nucleic acid binding affect PSF conformation. Together, these experiments will allow me to develop testable models of regulation that will guide us in our long term efforts to study the role of PSF and other nuclear factors in processes such DNA double strand break repair and mRNA alternative splicing that have implications for aging and aging-related disorders.
NIH ExPorter
NIH ExPorter