Alternative splicing provides cells with the ability to generate distinct proteins from the same gene, thus increasing protein diversity and controlling protein expression. Almost all multi-exon mammalian genes undergo alternative splicing in a tissue specific or signal-induced manner, however the regulatory mechanisms and functional consequences of such splicing changes have only been well-characterized for a small fraction of genes. Cells of the immune system are particularly expected to utilize alternative splicing to control cellular activity, as these cells must respond dynamically to their environment receiving danger signals and converting these into effector functions. Specifically, T-cells are stimulated through their T-cell receptors to trigger signaling cascades that lead to proliferation and cytokines secretion. However, the understanding of gene regulation upon T-cell activation has focused on transcriptional changes, with much less investigation of the contribution of alternative splicing to T-cell effector functions. Studies in our laboratory seek t fill this critical gap in our understanding of both alternative splicing and T-cell biolgy. Next-generation sequencing has recently led to the identification of 168 genes that undergo robust signal-induced alternative splicing in response to T-cell signaling. Detailed investigation of prototypical examples from among these 168 genes will lead to a deeper understanding of how splicing of these genes is regulated, and how such alternative splicing impacts the function of the cell. This proposal is focused on one gene, MKK7, based on the robustness of splicing change and ontology relevant to T-cell biology. MKK7 is a kinase that activates the JNK-signaling pathway that is essential for T-cell activity. Moreover, preliminary data suggests that splicing of MKK7 is regulated by a mechanism distinct from those previously implicated in signal-induced alternative splicing. To investigate the regulatory mechanisms governing alternative splicing of MKK7, standard minigene and mutagenesis approaches will first be used to define the cis-regulatory elements that are sufficient to confer signal-induced changes in MKK7 splicing. RNA-affinity purification and UV crosslinking will then be used to identify RNA-binding proteins that associate with these cis-regulatory elements, and the functional relevance of such proteins to MKK7 splicing will be determined by knock-down in cells. Finally, the functional distinction between the alternate protein isoforms of MKK7 that result from activation-induced alternative splicing will be investigated using standard signaling and protein association assays that are in common use in the laboratory. Taken together, the studies outlined in this proposal will reveal unique insight into a novel pathway of signal- induced splicing regulation in T-cells and will provide further understanding as to how alternative splicing is utilized to shape the functional response of the human immune system.

Public Health Relevance

Signal-induced alternative splicing allows cells of the immune system to rapidly change protein expression patterns to achieve effector functions in response to pathogen threat. This proposal aims to understand how alternative splicing is regulated in response to T-cell activation signals and the consequences of this regulation on T-cell activation and effector functions. Understanding the molecular mechanisms and consequences of alternative splicing in the context of a proper immune response will inform on how misregulation of the process can lead to the pathogenesis of human diseases involving the immune system and even pave the way for the development of therapeutics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Gaillard, Shawn R
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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
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
Martinez, Nicole M; Pan, Qun; Cole, Brian S et al. (2012) Alternative splicing networks regulated by signaling in human T cells. RNA 18:1029-40