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.
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.
|Chiou, Ni-Ting; Shankarling, Ganesh; Lynch, Kristen W (2013) hnRNP L and hnRNP A1 induce extended U1 snRNA interactions with an exon to repress spliceosome assembly. Mol Cell 49:972-82|
|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|
|Vu, Ngoc T; Park, Margaret A; Shultz, Jacqueline C et al. (2013) hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L. J Biol Chem 288:8575-84|
|Motta-Mena, Laura B; Smith, Sarah A; Mallory, Michael J et al. (2011) A disease-associated polymorphism alters splicing of the human CD45 phosphatase gene by disrupting combinatorial repression by heterogeneous nuclear ribonucleoproteins (hnRNPs). J Biol Chem 286:20043-53|
|Heyd, Florian; Lynch, Kristen W (2011) Degrade, move, regroup: signaling control of splicing proteins. Trends Biochem Sci 36:397-404|
|Heyd, Florian; Lynch, Kristen W (2011) PSF controls expression of histone variants and cellular viability in thymocytes. Biochem Biophys Res Commun 414:743-9|
|Motta-Mena, Laura B; Heyd, Florian; Lynch, Kristen W (2010) Context-dependent regulatory mechanism of the splicing factor hnRNP L. Mol Cell 37:223-34|
|Heyd, Florian; Lynch, Kristen W (2010) Phosphorylation-dependent regulation of PSF by GSK3 controls CD45 alternative splicing. Mol Cell 40:126-37|
|Shankarling, Ganesh; Lynch, Kristen W (2010) Living or dying by RNA processing: caspase expression in NSCLC. J Clin Invest 120:3798-801|
|Topp, Justin D; Jackson, Jason; Melton, Alexis A et al. (2008) A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4. RNA 14:2038-49|
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