The ultimate goal of this proposal is to understand how alternative splicing controls cellular development. Alternative splicing involves the differential production of distinct protein-coding messages from a given gene. As such alternative splicing plays an essential and ubiquitous role in generating protein diversity and regulating protein expression. In particular, changes in splicing patterns that occur during development or in response to environmental cues help determine the functional response or differentiation pathway of a cell. The development of T cells provides an especiall rich system for studying the mechanisms and functional consequences of induced alternative splicing, as immature T cells (thymocytes) progress through development via a series of check-points triggered by ligand-dependent signaling pathways. Importantly, defects in these check-points result in immunodeficiencies or autoimmune diseases. Ligand-induced signaling in thymocytes is known to induce changes in transcription profiles~ however, strikingly, the regulation of alternative splicing in thymocytes has been largely unexplored. As a first step toward correcting this dearth of knowledge, recent data has demonstrated that increased expression of the splicing regulatory protein CELF2 during thymic development regulates alternative splicing of the gene encoding the transcription factor LEF1. The regulation of LEF1 splicing, in turn, contributes to the stimulated expression of the T cell receptor (TCR), a necessary step in T cell maturation and function. This proposal seeks to build on this initial data to determine the mechanism(s) by which CELF2 is regulated in response to developmental check-points in the thymus and how this in turn regulates specific alternative splicing events during T cell maturation. Specifically, the aims of this proposal are to (1) determine the precise molecular changes in CELF2 that lead to signal-dependent increases in expression and activity by examining mRNA stability, transcription changes and post-translational modifications induced by cell stimulation, (2) utilize biochemical assays to uncover the molecular mechanisms by which CELF2 regulates splicing and how this changes in a signal-dependent manner and (3) exploit emerging high- throughput technologies to gain a global view of CELF2 RNA binding and function in thymocytes. Together these studies will provide unprecedented insight into alternative splicing programs in developing thymocytes, an essential step toward determining the broad role of splicing regulation in the shaping of a functional immune system. Furthermore, as mis-regulation of CELF2 has been linked to developmental defects and disease of heart, muscle and thymus these studies have direct implications for human health. In addition, these studies will increase our general understanding of mechanisms of developmentally regulated splicing and the connection between signaling pathways and splicing regulatory proteins.

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 progress through developmental check points, function appropriately and prevent disease states. In particular, development of an active immune system involves a complex program of changes in protein expression. The studies in this proposal investigate a newly identified mechanism for controlling protein expression during the development of the immune system, thereby providing unprecedented insight into mechanisms of protein expression through which the activity of the immune system can be controlled.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-GGG-C (02))
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Marino, Pamela
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University of Pennsylvania
Schools of Medicine
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Martinez, Nicole M; Agosto, Laura; Qiu, Jinsong et al. (2015) Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation. Genes Dev 29:2054-66
Mallory, Michael J; Allon, Samuel J; Qiu, Jinsong et al. (2015) Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell signaling. Proc Natl Acad Sci U S A 112:E2139-48
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