The goal of this proposal is to understand the mechanisms involved in the function of the conserved arginine-serine (R/S) domain containing pre-mRNA splicing factor U2 snRNP auxiliary factor (U2AF) in the fruit fly, Drosophila melanogaster. This proposal capitalizes on the strengths of Drosophila as an experimental organism and allows a multidisciplinary approach to biological problems. Using a combination of genetics, molecular biology, and biochemistry, we will elucidate the mechanisms by which R/S domains function in pre-mRNA splicing. R/S domains have been found in many mammalian and Drosophila general and regulatory splicing factors but in vivo experiments analyzing their function at physiological levels have not been performed. We have isolated the genes encoding both Drosophila U2AF homologs, have shown that recombinant dU2AF50 has all of the RNA binding and splicing activities of hU2AF65 and have expressed the dU2AF38 in protein in E. coli. Cytogenetic and germline transformation experiments carried out in our lab have shown that previously identified lethal mutations at position 14C1-2 on the X chromosome in an uncharacterized complementation group called 9-21 correspond to the large U2AF subunit, dU2AF50. We have also shown that the dU2AF38 small subunit gene is located on the second chromosome and have identified lethal dU2AF38 mutations. Cytogenetic experiments have localized the dU2AF38 gene to position 21B-C on the second chromosome and we have now isolated lethal mutations in the dU2AF38 small subunit gene demonstrating the gene is essential. These observations provide the basis assaying the function of mutant forms of an essential pre-mRNA splicing factor in a transgenic metazoan organism. There already exist a number of biochemical assays for U2AF function and thus any in vivo phenotype of mutant U2AF derivatives can be directly correlated with altered biochemical activities in vitro. During this funding period in order to accomplish the goals of this proposal, we will 1) Carry out genetic analysis of wild type and mutant U2AF large and small subunit derivatives in vivo; 2) Define the U2AF heterodimer interaction domains in vivo and in vitro; 3) Use Schizosaccharomyces pombe and sequence information for mutant prp-2 alleles to create temperature-sensitive dU2AF50 mutants and test their function in Drosophila; 4) Assay wild type and mutant U2AF heterodimers for splicing activity and splicing complex assembly in vitro; and 5) Structure studies on the RNA binding domain of Sex-lethal and dU2AF50 and the interaction domain of the dU2AF heterodimer. It is clear that many human oncogenes are expressed differentially by alternative pre-mRNA splicing. These oncogene protein isoforms may have different functions in different tissues. Because many human diseases, such as beta-thalassemia, results from defects in pre-mRNA splicing, it is likely that alternative splicing defects may lead to oncogenic transformation of certain somatic cell types.
| Crowder, S M; Kanaar, R; Rio, D C et al. (1999) Absence of interdomain contacts in the crystal structure of the RNA recognition motifs of Sex-lethal. Proc Natl Acad Sci U S A 96:4892-7 |
