Alternative pre-mRNA splicing is a ubiquitous mechanism of gene regulation that allows for precise control of the expression of functionally distinct protein isoforms encoded by a single gene. In particular, signal-induced alternative splicing is a powerful mechanism by which a cell can fine-tune precise protein function in response to changes in cellular environment. Several studies in the past year have demonstrated widespread coordinate regulation of alternative splicing in response to cellular activation as a means to alter cellular function in response to changing environmental conditions. However, the proteins and pathways that coordinate the signal-induced splicing of multiple genes has not yet been investigated in a comprehensive manner. Recently we identified a broad set of genes that undergo changes in splicing in response to T cell activation, and we have begun to group these genes based on similar patterns of regulation, sequence homology, and sensitivity to specific RNA-binding proteins. In this proposal we will extend these studies to gain a more complete understanding of the sequences, proteins and mechanisms that coordinately regulate the program of alternative splicing events that are induced upon T cell activation. First, we will continue our collaborative use of splicing microarrays to analyze how depletion of specific splicing regulatory proteins alters the signal-responsive alternative splicing profile in a T cell line. We will also utilize a bioinformatics approach to identify sequence motifs that correlate with specific patterns of splicing regulation among the genes we identify. Secondly, we will perform systematic mutagenesis of splicing minigenes to characterize how sequence context influences the function of the one sequence element that we have already correlated with activation-induced alternative splicing. A similar approach will also be used to identify and/or validate additional regulatory sequences that control distinct subsets of signal-induced splicing. Finally, we will use in vitro assays to identify and characterize the proteins that function via these identified regulatory sequences. An ultimate goal of these research aims is to define sufficient mechanistic information regarding genes that undergo signal-induced alternative splicing, that we may accurately predict genes which are regulated at the level of alternative splicing in response to a particular stimuli. These studies will also have a broad impact on our understanding of combinatorial splicing regulation, as well as provide detailed insight into essential, but poorly characterized, molecular mechanisms that control proper function of the immune system and are critical to the prevention of human disease.
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