Our lab is interested in regulation of alternative splicing and we have begun several projects to study alternative splice site choice using C. elegans as a model system. To this end, we have developed the Intronerator as a computational system for using EST information in C. elegans to identify alternatively spliced genes. We have identified over 800 alternatively spliced genes using this system (1). By comparing genomic sequence of C. briggsae and C. elegans we have identified regions of conservation near some of these spliced genes that are candidates for putative cis -regulatory elements for alternative splicing (2). We are developing splicing reporter assays to test the activity of these conserved elements in regulation of alternative splicing. One alternatively spliced gene we are studying in depth is the
let-2 gene. Alternative splicing involves the use of two mutually exclusive exons, exons 9 and 10. In embryos, exon 9 is primarily used and in adults exon 10 is primarily used, with a gradual switch in the usage occurring during the larval stages (3). We have created a splicing reporter construct in which the alternatively spliced region of
let-2 is fused to green fluorescent protein. The almost exclusive usage of exon 9 in embryos is maintained in this construct. A series of cis -mutations that we have devised in this reporter indicate that the unusual beginning of the intron between exon 10 and exon 11, the intron starts with GC instead of the canonical GT, is important for promoting the splicing of exon 9. Cis mutagenesis of the alternatively spliced regions of this gene to identify splicing regulatory elements is continuing. In addition, we have made a splicing reporter for this gene in which only the adult form of the message is in frame with GFP. Animals containing this reporter do not express detectable GFP until the L2 stage. We are using this reporter in a visual screen to identify genes involved in the regulation of
let-2 alternative splicing. Our progress in this screen will be presented. 1. Kent, W.J. and Zahler, A.M. 2000. Nucleic Acids Research 28: 91-93. 2. Kent, W.J. and Zahler, A.M. 2000. Genome Research 10: 1115-1125. 3. Sibley M.H. et al. 1993. Journal of Cell Biology 123: 255-264