Title

Investigating the molecular mechanisms regulating Dscam alternative splicing in Drosophila

Date of Completion

January 2002

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

The Drosophila Dscam gene encodes an axon guidance receptor that plays an important role in determining the specificity of neuronal wiring. The Dscam gene contains 115 exons, 95 of which are alternatively spliced and organized into 4 clusters. The exon 4, 6, 9, and 17 clusters contain 12, 48, 33, and 2 mutually exclusive alternative exons respectively. Thus, it is possible that 38,016 different Dscam mRNAs can be produced. I have shown that Drosophila Dscam pre-mRNA splicing is regulated both temporally and spatially and have examined how the alternative splicing of the Dscam exon 4 cluster is regulated. To identify proteins that may regulate exon 4 alternative splicing, I performed an RNAi screen in S2 cells. Individual depletion of 32 different pre-mRNA processing factors from S2 cells has identified 16 proteins involved in Dscam alternative splicing. Depletion of the SR protein B52 and either subunit of the general splicing factor U2AF (dU2AF50/dU2AF38) had the most striking effect. These proteins seem to act antagonistically to regulate the alternative splicing of exon 4.4—whereas B52 acts as a positive regulator, dU2AF acts as a negative regulator. ^ The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible since they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. I have demonstrated that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In particular, this method should allow for the function of individual Dscam isoforms to be determined. In addition, these results have implications regarding the mechanism of RNAi in Drosophila. ^