Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Kristen W. Lynch

Abstract

Pre-mRNA alternative splicing (AS) has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. In this thesis, I describe the use of high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to provide evidence for the translation of AS mRNAs. To increase proteome coverage and improve protein quantitation, I optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, I generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. With my analyses, we improved identification of splicing-derived proteoforms by 28% and provided peptide evidence for 554 alternate proteoforms arising from 274 genes. Our increased detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T cell stimulation, as well as fluctuations in sub-cellular localization of proteins.

In this thesis, I also identify 18 histone modifying enzyme (HME) genes whose transcripts are AS upon T cell PMA stimulation. Additionally, I report that RNA binding proteins CELF2 and PSF regulate the splicing of 14/18 HME genes. For several reasons, I focus on the class IIa histone deacetylase HDAC7 as a model system to understand the connections between T cell stimulation, AS and epigenetics. Specifically, I find that in response to T cell activation, HDAC7 is alternatively spliced to favor an isoform that includes exon 9. This isoform, termed iE9, is more highly expressed and stable than shorter isoform lacking E9, termed deltaE9. Expression of the iE9 isoform alone resulted in altered gene and protein expression of key T cell surface receptors that receive and propagate T cell activation signals, such as CD3, CD28 and CD69. Thus, we have uncovered a positive feedback loop that helps sustain T cell activation signals. Moreover, I observed that expression of either HDAC7 protein isoform leads to dysregulated histone PTMs. Overall, the work presented in this thesis underscores the contribution of alternative splicing to the diversity and regulation of the proteome, as well as the regulation of chromatin marks.

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