Title

Musashi regulates alternative splicing

Date of Completion

January 2008

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

The Drosophila Down syndrome cell adhesion molecule (Dscam) gene potentially generates up to 37,224 isoforms via the alternative splicing of 94 exons that are arranged into four clusters (Schmucker et al., 2000; Wojtowicz et al., 2007). Dscam encodes an axon guidance receptor required for proper neural wiring (Schmucker et al., 2000; Wang et al., 2002; Hummel et al., 2003; Zhan et al., 2004) and is expressed in the hemocytes where it functions as an immune receptor to defend the organism against pathogens. The expression of a distinct set of Dscam isoforms in both individual neurons and hemocytes is thought to be critical for the function of both the nervous and immune system (Neves et al., 2004; Watson et al., 2005; Zipursky et al., 2006). Our lab has previously shown that the alternative splicing of Dscam exon 4.2 is regulated both spatially and temporally and that the regulation is evolutionally conserved (Celotto et al., 2001). Thus, we attempted to identify proteins that regulate Dscam exon 4.2 splicing. We found that depletion of musashi (msi; CG5099) by RNAi significantly affected Dscam exon 4.2 splicing. The change in the inclusion of exon 4.2 was confirmed in vivo by quantitative RT-PCR of total RNA from msi mutant pupae. We also find that MSI binds directly to the sequence AUUUUAAUCU located in the intron upstream of exon 4.2. Moreover, using a high-throughput microarray system we identified two additional MSI splicing regulatory targets, the CG18076 (shot) and CG2674 (M(2)21AB) pre-mRNAs. Finally, consistent with our observation that MSI is a splicing regulator, we show that MSI localizes to the nucleus. Together, these results demonstrate that MSI is an important splicing regulator in Drosophila that simultaneously controls the alternative splicing of many pre-mRNAs. ^