Formation of an Exon-Defined A Complex Spliceosome Intermediate Results in CD45 Exon Repression




House, Amy Elizabeth

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Alternative splicing is a common means for genetic regulation in higher eukaryotes, and variability in splicing patterns results in a significant increase in protein diversity. Of particular interest is how the splicing machinery is regulated to allow for alternative splicing while maintaining accurate splicing fidelity. CD45 is a trans-membrane tyrosine phosphatase that is expressed on the surface of hematopoetic cells. The CD45 gene contains three variable exons that are partially repressed in na?T cells and are preferentially skipped upon T cell activation. Importantly, it has been shown that the alternative splicing of CD45 pre-mRNA affects the resultant protein's function within the cell emphasizing the importance of this RNA processing event. As such, CD45 provides an excellent model system in which to study signal-responsive alternative splicing. Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles de novo on a pre-mRNA template in a stepwise and highly dynamic process. The inherent dynamic nature of the spliceosome suggests the potential for regulation at numerous points along the assembly pathway. Previously, a 60-nucleotide exonic silencer (ESS1) was identified within variable exon 4 of the CD45 gene. ESS1 is bound by at least three hnRNP proteins (hnRNP L, PTB, hnRNP E2) of which hnRNP L is the main functional component. Subsequent work showed that ESS1 does not regulate CD45 exon 4 splicing by the typical mechanism of preventing exon definition. Instead, spliceosome assembly is stalled after the exon has been recognized by the spliceosome such that both U1 and U2 are bound to the splice sites suggesting that the stalled complex represents an A-like exon-defined complex (AEC). Additionally, hnRNP L is sufficient to block spliceosome assembly at the AEC. Further biochemical analysis of the AEC suggests that the stall in assembly may be due to altered interactions between the spliceosomal snRNAs and/or proteins and the pre-mRNA substrate. Overall, this inhibition represents a new mechanism for splicing regulation, and suggests that the formation of an AEC intermediate is an important transition in the spliceosome assembly pathway.

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