Sex-lethal (Sxl) is a splicing regulator and controls sexual development in Drosophila by altering the splicing mode. Sxl protein has the ability to act from a distance and interacts with other splicing factors to cause this switch in splicing mode.
The aim of the proposed research is to define the interactions that Sxl protein makes in switching the splicing mode. This will require 1) defining the minimal sequences needed for regulation. The minimal sequences will give us the site of the interactions and should eventually allow us to separate what Sxl protein recognizes on the RNA from the sequence requirements of the cofactors. We will also 2). Analyze and clone an existing mutant that affects Sxl autoregulation. Complementation analysis with known X-linked mutations that affect Sxl splicing suggest that this mutant is a new locus involved in Sxl splicing regulation. 3). Perform two different screens to search for additional gene members involved in the regulatory process. a) Screen for proteins that interact with Sxl protein with the yeast two-hybrid-interaction system. It is anticipated that this approach will identify proteins that directly interact with Sxl protein. b). A mutagenesis screen on fruit flies carrying a white reporter gene that splices sex-specifically. This screen scores for genes that affect the efficiency of Sxl autoregulation within the fly by looking for changes in eye color.
The first aim will involve making constructs by site directed mutagenesis and deletion and alteration of Sxl sequences. These will be transformed into the germline to assay the effects of the changes in vivo. Genetic crosses and recombination, ethyl methansulfonate mutagenesis, P-element tagging, cloning and the screening of cDNA libraries will be used in the second and third aims. Monoclonal antibodies will be generated against the proteins of the new genes that are cloned. RNA splicing is a fundamental biological event. Understanding how splicing occurs and how it can be altered is important to understanding how genes are regulated.The insulin receptor, heart smooth muscle myosin heavy chain isoforms, regulators of blood complement activators, HIV and c-src are only a few of a long list of genes that undergo alternate splicing. Some of the thalessemias result from aberrant splicing and both the tau protein and beta-amyloid peptides that are the major constituent of the neurofibrillary tangles in patients with Alzheimer's disease are alternatively spliced.
