The long-term objective of this project is to understand how extracellular stimuli can induce a single gene to encode functionally distinct protein isoforms through the process of signal-induced alternative splicing. Alternative splicing is a major determinant of diversity within the human proteome and is a critical mechanism for modulating protein expression. An increasing number of genes have been shown to undergo alternative splicing in response to extracellular stimuli, particularly in the nervous and immune systems where cells must react robustly to changing environmental conditions. Moreover, mistakes in alternative splicing, including inhibition of signal-induced splicing regulation, have been linked to numerous human diseases. However, relatively little is understood about the proteins and mechanisms that regulate this process. This proposal seeks to understand the regulated splicing of the CD45 gene that occurs in response to T cell activation, as a model for characterizing mechanisms of signal-induced alternative splicing. Previous studies have demonstrated that the RNA-binding protein PSF represses inclusion the CD45 variable exons upon activation, by associating with a splicing silencer complex on the regulated exons. To determine how extracellular stimuli and sequence context influence the assembly and function of the CD45 signal-responsive silencer complex, the Specific Aims of this proposal are as follows: (1) To identify the proteins that associate within the silencer complex on the divergent CD45 variable exons 4 and 5 by RNA-affinity purification, (2) To identify proteins that regulate PSF recruitment to the CD45 silencer complexes, by purification of functionally distinct PSF-associated complexes from resting and activated cells and use of in vitro and cell-based assays to confirm the functional relevance of PSF associated proteins, and (3) To determine how signaling proteins influence the differential activity of the silencer complex by validation of candidate regulatory signaling molecules and analysis of post-translational modifications of PSF and associated proteins. Together these studies will provide a comprehensive understanding of how extracellular stimuli alters the function of PSF with exonic silencer complexes to achieve induced repression of the CD45 variable exons. As such, these studies will provide a new paradigm for mechanisms of signal-induced splicing regulation. In addition, the identification of a minimal signal-responsive silencer complex, and an understanding of how such a complex forms on distinct exons, will ultimately inform efforts to predict exons that may be regulated by similar mechanisms. Many human cell types must be able to alter their protein expression rapidly and precisely in response to a given stimuli, in order to function appropriately and prevent disease states. Current studies demonstrate that an abundant mechanism for regulating protein expression and cellular function in response to extracellular stimulation is that of signal-induced alternative splicing. The studies in this proposal seek to elucidate one mechanism by which cellular activation regulates alternative splicing, as a model for understanding the broader phenomena of signal-responsive splicing regulation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Bender, Michael T
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University of Pennsylvania
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Vaquero-Garcia, Jorge; Barrera, Alejandro; Gazzara, Matthew R et al. (2016) A new view of transcriptome complexity and regulation through the lens of local splicing variations. Elife 5:e11752
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Shankarling, Ganesh; Lynch, Kristen W (2013) Minimal functional domains of paralogues hnRNP L and hnRNP LL exhibit mechanistic differences in exonic splicing repression. Biochem J 453:271-9

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