This proposal aims to use genetic, in vivo and in vitro approaches to investigate the role of individual SR type """"""""basal"""""""" splicing factors in regulated splicing events. The proposal focuses largely on the activities of two proteins, B52 and U1 70K. Both proteins contain RRM regions and Ser/Arg rich regions. B52 is a member of the group of SR proteins that can be both necessary for basal splice site function and which can alter splice site choice in a concentration dependent manner. U1 70K is a protein which binds to the first loop of the U1snRNA. Both proteins have functional homologs in vertebrates and loss of function mutations exist for both in Drosophila. In addition, biochemical work in other systems has shown that different SR proteins are differentially effective in activating particular splice sites. The B52 protein is chosen for study as a representative of the SR proteins, and also because a specific dominant allele seems to alter the splicing of a number of loci (eg w, dsx, Ubx) in which alternative splicing can lead to phenotypically notable consequences. Studies on B52 first catalogue the tissue and temporal expression of two alternative splice variants of the RNA which encode proteins differing by 5 aa in the spacer region between the two RRMs. In vitro experiments are then envisioned to determine the preferred RNA bindng specificity for the wild type and mutant forms of the protein using either SELEX techniques or characterization of binding sites on possible in vivo targets suchs as w, dsx or Ubx. This is particularly important since the cause of the dominant mutant phenotypes is not clearly understood and since only the dominant allele seems to interact in two (w and dsx) of the genetic tests described in the proposal. Additional tests of the dominant B52 allele will involve testing its ability to function as a basal splicing factor in complementation of S100 extracts from Drosophila or Hela cells and tests of its ability to alter 5' splice site choice in appropriate globin constructs (the canonical SR protein as splice site regulator assay). To attempt to assay differences between specific and non-specific activities of the dominant allele, mutations will be made in the B52 binding sites of some or all of the potential target genes and then assayed for changes in binding and for changes in assembly of splicing complexes. These experiments will (I think) be carried out in vitro without corresponding in vivo functional tests. The possibility of interactions between variants of B52 and other splicing proteins will be analyzed by far-western analysis. These include tests of interaction with the U1 70K protein. Various in vivo studies will be attempted using loss of function or variant dominant alleles of B52. Some will involve looking at mutant transgenes in clones of cells which have lost wild type function. Tests will be made of wild type and dominant sequences altered in their RRM or SR domiains. A related set of experiments will be performed with the U1 70K gene involving production of total loss of function alleles and tests of the relative importance of the RRM and SR domains for function in vitro and in vivo. In addition, since preliminary work suggests that U1 70K, like another U1 associated protein, is necessary to establish appropriate female function of Sxl, tests will be done to determine the effects of 70K mutations on Sxl splicing and to see if 70K is regulated in the female germline. Finally, it is proposed to do a screen of existing lethal P- element insertions to identify genes which fail to complement the B52 dominant/+. The rationale is that the B52 dominant mutation interferes with at least some aspects of normal splicing and sensitizes the system such that loss of half the dose of any other near limiting splicing component will be revealed in a detectable phenotype. Previous results show that such an interaction can be seen in animals the are also tra-2/+; tra/+ in the dominant background. Additionally, at least one potential spontaineous mutation which enhances the dominant B52 phenotype has been found, although it remains unmapped.
| Nagengast, Alexis A; Stitzinger, Shane M; Tseng, Chin-Hsiu et al. (2003) Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping. Development 130:463-71 |
