The ELAV gene (embryonic lethal, abnormal visual system) is essential for nervous system development. It has RNA binding motifs, and is specific for nervous tissue. The protein acts to alternatively splice a target to a tissue specific isoform. Conserved sequences between diverged Drosophila species may give a hint as to where the regulatory sequences reside for alternative splicing. A reporter GFP will be used to detect the spliced RNA and quantitative the expression. The candidate will investigate the neural specific splicing of an ELAV dependent intron target, neuroglian. She will define cis sequences of the regulated intron that are identified by the ELAV protein using a biochemical approach and then use a genetic approach to identify other proteins by mutagenesis and screening. The biochemical approach will involve straight-forward gel mobility shift assays combined with a deletion analysis of the intron sequences required for alternative splicing in an artificial splicing assay, which mimics nrg. In this assay putative proteins will be identified. The method of identification is in vivo splicing using the LacZ reporter construct. The second part of the proposal is a screening method which should derive mutations in alternative splicing. One aspect uses a reporter for in vivo selection, the other uses a rather complicated screen after EMS induced mutations.
