Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Kristen W. Lynch

Abstract

Alternative splicing is a key step in gene regulation and involves the differential selection of splice sites to generate different pre-mRNA transcripts. It has been shown that 90-95% of pre-mRNAs are alternatively spliced in human cells. Pre-mRNA splicing is catalyzed the spliceosome, which consists mainly of the U1, U2, U4, U5 and U6 snRNP, and about a hundred of non-snRNP proteins. Splicing regulators that bind to enhancer or silencer elements on the pre-mRNA can alter assembly of these spliceosome components. Understanding how splicing regulators control spliceosome assembly will bring insights to the prediction of splice site choices. In our lab, we used CD45 as a model gene for studying alternative splicing and spliceosome assembly. The exonic silencer sequence (ESS1) within CD45 exon 4 is bound by hnRNP L to induce its skipping. HnRNP L represses spliceosome assembly at a step after the binding of U1 and U2 snRNP on either side of the exon. My goal is to understand how hnRNP L perturbs U1 snRNP binding at the 5' splice site (5' ss) to cause the skipping of CD45 exon 4.

Using psoralen- and UV-crosslinking analysis, U1 snRNP and other protein components that associate with the sequences around the 5' ss within the hnRNP L-repressed spliceosome complexes were compared with the control complexes. These studies revealed that hnRNP L recruits hnRNP A1 to the 3' end of exon 4 to induce an extended pairing interaction between the U1 snRNA and the 5' ss. Splicing assays in vitro and in cells further demonstrated that hnRNP A1 and the U1 snRNA binding at the 3'end of exon 4 are required for hnRNP L-mediated skipping of exon 4. Further analysis of other exons repressed by hnRNP L or A1 suggests the potential for the extended U1 pairing interactions with these exons. These data imply that induction of U1 interactions with the exonic region nearby a 5' ss could be a widespread mechanism in inducing the skipping of exon. To further determine how the extended U1 binding affects the subsequent spliceosome assembly steps, hnRNP L-repressed spliceosome complexes were purified. This analysis revealed that association of U6 snRNP with the 5'ss, and recruitment of NTC components, are blocked in the hnRNP L-repressed complexes. Moreover, enhancing binding of U6 to the 5'ss overcomes the effect of the extended U1 interaction, thereby increasing the splicing of the hnRNP L-repressed substrate. These results provide the first example showing that the U1/U6 switch, a structural rearrangement during the catalytic activation of the spliceosome, is a naturally occurring point for regulating alternative splicing. This study also suggests that splicing regulators that alter U1 binding, or spliceosome components that associate with the 5' ss after U1 binding, are important factors for determining the usage of the 5'ss.

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