The ultimate goal of this project is to understand the mechanisms by which extracellular stimuli regulate protein expression in a cell through the process of signal-induced alternative splicing. Alternative splicing involves the differential joining of sequences within a nascent pre-mRNA transcript, to form distinct mature mRNAs in different cell types or growth conditions. Importantly, these differential splicing patterns typically encode distinct proteins or alter the presence of cis-regulatory elements thereby altering the level of protein expression. As the vast majority of human genes undergo some form of alternative splicing, this process is a major determinant of diversity and expression within the human proteome. Signal-responsive alternative splicing is used to control protein expression in response to cellular stimulation, particularly in the nervous and immune systems in which cells must react robustly to changing environmental conditions. The human CD45 gene, one of the first known examples of signal-induced alternative splicing, contains three variable exons that are preferentially skipped (repressed) in response to immune challenge. This regulated splicing of CD45 is essential for optimal function of T cells in an immune response as indicated by the correlation between mis-regulation of CD45 splicing and human autoimmune disease. Moreover, as one of the best-characterized examples of signal-regulated splicing, CD45 is a distinctively valuable model to dissect the mechanisms by which alternative splicing is controlled. CD45 exon skipping is primarily accomplished in resting cells through the RNA-binding protein hnRNP L, which is further joined by the related proteins PSF and hnRNP LL following cellular stimulation. Remarkably, PSF is inhibited from binding to the CD45 substrate in resting cells due to a phosphorylation-dependent interaction with a poorly characterized protein called TRAP150, providing a new paradigm for signal-dependent regulation of splicing factors. The current proposal will exploit this knowledge regarding CD45 splicing to elucidate the molecular mechanisms by which cellular activation regulates alternative splicing at an unprecedented level of detail. Specifically, this proposal seeks to determine (1) how appropriate formation of the splicing enzymatic complex (spliceosome) is altered by hnRNP L/PSF/hnRNP LL to block exon inclusion, (2) the molecular interactions through which hnRNP L and PSF influence the spliceosome and (3) how phosphorylation of PSF controls it's interaction with TRAP150 to regulate the function of PSF. Each of these questions will be addressed through biochemical methods to identify protein-protein, protein-RNA and RNA-RNA interactions that correlate with regulatory activity, followed by functional assays to determine the mechanistic significance of these interactions. Answers to these questions will yield a complete understanding of the regulation of a physiologically significant example of signal-induced alternative splicing, and will provide novel insight to broadly inform our understanding of the mechanisms by which such regulation can occur.

Public Health Relevance

All human cells 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 stimulaton is that of signal-induced alternative splicing. The studies in this proposal seek to elucidate he molecular mechanisms by which cellular activation regulates alternative splicing at an unprecedented level of detail as required to predict the effect of genetic mutations or manipulate splicing for therapeutic purposes.

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
Schools of Medicine
United States
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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
Cole, Brian S; Tapescu, Iulia; Allon, Samuel J et al. (2015) Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons. RNA 21:2053-66
Yarosh, Christopher A; Tapescu, Iulia; Thompson, Matthew G et al. (2015) TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells. Nucleic Acids Res 43:9006-16
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