Interplay between signaling and splicing during T cell activation : a study on MKK7 / Nicole M. Martinez.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Martinez, Nicole M., author.

Contributor:

Lynch, Kristen W., degree supervisor.

Raj, Arjun, degree committee member.

Shorter, James, degree committee member.

Kohli, Rahul, 1985- degree committee member.

Pear, Warren S., degree committee member.

Wilusz, Jeremy E., degree committee member.

Nagengast, Alexis A., degree committee member.

University of Pennsylvania. Department of Biochemistry and Molecular Biophysics.

Language:

English

Subjects (All):

Penn dissertations--Biochemistry and molecular biophysics.

Biochemistry and molecular biophysics--Penn dissertations.

Local Subjects:

Penn dissertations--Biochemistry and molecular biophysics.

Biochemistry and molecular biophysics--Penn dissertations.

Physical Description:

xi, 216 leaves : illustrations (some color) ; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2015.

Summary:

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.

Notes:

Ph. D. University of Pennsylvania 2015.

Department: Biochemistry and Molecular Biophysics.

Supervisor: Kristen W. Lynch.

Includes bibliographical references.

OCLC:

945583663