Interplay Between Signaling and Splicing During T Cell Activation: A Study on Mkk7

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Doctor of Philosophy (PhD)
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Biochemistry & Molecular Biophysics
alternative splicing
positive feedback loop
T cell activation
Allergy and Immunology
Immunology and Infectious Disease
Medical Immunology
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Alternative splicing is a mechanism of gene regulation that expands the genome's coding capacity. The ability to regulate alternative splicing in response to external signals is critical for immune responses. I profiled alternative splicing during T cell activation using next-generation sequencing and identify hundreds of genes that are regulated by alternative splicing during activation of a T cell line and primary human T cells. Alternative splicing in response to T cell receptor signaling is enriched for genes encoding signaling proteins; suggesting interplay between signaling and splicing regulation. In this thesis I describe the selection of the JNK kinase MKK7, as a model system to understand the connections between signaling and splicing in T cells. Specifically, I find that that in response to T cell activation, MKK7 is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a MAPK docking site within MKK7 that is disrupted in the larger isoform. Consistently, skipping of exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and upregulation of the c-Jun target gene TNF-alpha. Notably, signaling through JNK itself is necessary and sufficient to promote activation-induced repression of exon 2 of the MKK7 gene. Thus, JNK-induced MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. Through minigene based analysis I identify sequences within the introns flanking MKK7 exon 2 that are necessary and sufficient to promote activation-induced skipping. Analysis of RNA-protein interactions and knockdown experiments identified CELF2 as a protein that binds to MKK7 introns in an activation-dependent manner and represses exon 2 inclusion. The repressive activity of CELF2 on MKK7 exon 2 is mediated through a JNK-dependent increase in CELF2 protein, that results from stabilization of the CELF2 mRNA. Finally, I show that ~25% of T cell receptor-mediated alternative splicing events are dependent on JNK signaling. These JNK-dependent events are also significantly enriched for CELF2-dependence. Together, the data presented in this thesis demonstrates a broad role for the JNK-CELF2 axis in controlling splicing during T cell activation, including a specific role in potentiating JNK signaling.

Kristen W. Lynch
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