Title

Mechanisms governing alternative splicing

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternative splicing is regulated through a complex network of both cis-acting elements, present in the mRNA, and trans-acting factors, which interact with the cis-acting elements either directly or indirectly. In order to gain more insight into the mechanism of alternative splicing, we sought to study both trans-acting effectors and cis-acting sequence elements that influence and identify alternative splicing events. We began with an in depth study of the discrete functions of the RS domains of SR proteins. I have demonstrated that minimal RS domains, both endogenously and synthetically derived, can function to activate enhancer dependent splicing in vitro, independent of other SR protein domains. I have also shown that the subcellular localization of RS domain containing proteins is intimately linked to the arginine and serine content of the protein and that appropriate subcellular localization is essential for splicing activation. ^ Next, we asked if we could identify novel alternative exons using bioinformatics. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Using comparative genomics, I identified candidate exons expected to undergo alternative splicing in Drosophila melanogaster and experimentally verified their usage as alternatively spliced exons in vivo. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data. Identification of conserved alternative splicing events improves our understanding of how an exon is specified as a modulated exon. Taken together, our studies of cis-regulatory elements and trans-acting factors are a fascinating glimpse into the complex regulatory networks that govern alternative splicing. ^