Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Kristen Lynch

Abstract

Alternative splicing is the process by which an exon is preferentially included or excluded from an mRNA transcript. Recent global sequencing studies have shown that >95% of the transcriptome undergoes some form of alternative splicing. Such regulation often alters protein isoform expression, as is especially apparent in T cells of the immune system that change their expression of RNA and protein according to signaling cues. The focus of this thesis is on one alternative exon in the pre-mRNA of transcription factor LEF1 and its regulation by the splicing factor CELF2. LEF1 is crucial for T cell function as it upregulates the expression of TCRα. Upon signal induction in T-cells, CELF2 promotes the inclusion of exon 6 in LEF1 (LEF1-E6) in the final mRNA transcript. This increase in LEF-E6 inclusion generates an isoform of LEF1 that is preferentially active in promoting transcription of TCRα. CELF2 regulates LEF1-E6 inclusion upon stimulation by increasing its binding to two conserved elements (USE60 and DSE120) in the upstream and downstream introns flanking exon 6. My goal is to understand how the increase of binding of CELF2 to the USE60 and DSE120 upon stimulation results in an increase in LEF1-E6 inclusion. Using a combination of in vivo minigene assays, in vitro splicing assays and UV-crosslinking assays I correlate the binding of CELF2 to the function of the USE60 and DSE120. I show that the USE60 and DSE120 do not work synergistically to enhance inclusion but function antagonistic to each other. The USE60 is a repressor of splicing while the DSE120 is an enhancer. In order to achieve an increase in exon 6 inclusion only upon stimulation, CELF2 binding is highly regulated between the USE60 and DSE120. In unstimulated T cells, binding is biased towards the repressive USE60 and upon stimulation the increase in CELF2 binding happens purely on the activating DSE120. This bolus of CELF2 binding on the DSE120 upon stimulation leads to an increase in exon 6 inclusion. These studies reveal a model where binding of CELF2 to the DSE120 is inhibited in unstimulated cells and this inhibition is relieved upon stimulation.

Available for download on Saturday, September 15, 2018

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Included in

Biochemistry Commons

Share

COinS