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 signaling induced alternative splicing. Alternative splicing is a major determinant of diversity within the human proteome and is a critical mechanism for modulating protein expression. In particular, an increasing number of genes have been shown to undergo alternative splicing in response to extracellular stimuli. Moreover, mistakes in alternative splicing have been linked to numerous human diseases. Despite the significance of alternative splicing to human health, relatively little is understood about the 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 signaling-induced regulation of alternative splicing.
The Specific Aims of this proposal are 1) To identify the cis-regulatory sequences that determine the pattern of CD45 splicing, 2) To identify and characterize the RNA-binding proteins that regulate CD45 splicing, and 3) To identify components of the pathway(s) by which T cell activation induces changes in CD45 splicing. The sequence requirements for CD45 regulation will be determined by assaying the effects of mutations within CD45 minigenes on the splicing of CD45. Protein that bind to the functional elements within CD45 RNA will be identified by biochemical purification, and both in vitro and cell-based functional assays will be used to determine the mechanisms by which these proteins regulate CD45 splicing. Finally, a cell-based screen will be used to identify and characterize mutant cells that are deficient in activation-induced alternative splicing, in order to determine the pathway(s) that lead from the initial signaling event to the regulation of splicing. Together these studies will lead to greater insight as to the mechanisms by which activation of signaling pathways leads to regulation of alternative splicing, and in so doing, are likely to suggest potential targets for therapeutic modification of pre-mRNA splicing.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM067719-01
Application #
6596585
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
1
Fiscal Year
2003
Total Cost
$277,089
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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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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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
Martinez, Nicole M; Lynch, Kristen W (2013) Control of alternative splicing in immune responses: many regulators, many predictions, much still to learn. Immunol Rev 253:216-36
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

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