Alternative splicing of mRNA is one of the major ways in which higher organisms control expression and functional diversity of their gene products. Aberrations in control of alternative splicing decisions are implicated in various genetic disorders that affect human development and physiology, as well as in tumor progression and metastasis. The ability to manipulate alternative splicing decisions could offer therapies for such conditions and also for genetic disorders caused by mutations in non- essential exons. Nevertheless, current knowledge of the molecular mechanisms that control alternative splicing is rudimentary. The goal of this project is to gain a deeper understanding of these mechanisms, using the Ubx gene of Drosophila as a model system amenable to combined genetic, molecular and biochemical approaches. The proposed investigations will test the hypothesis that the choice between alternative 5, splice sites in Ubx RNAs is controlled by the assembly of opposing regulatory complexes that recruit U1 snRNP to the upstream site in modes that either promote or prevent subsequent spliceosome assembly. The role of identified trans-acting factors will be investigated through biochemical studies of protein-RNA and protein-protein interactions, detailed characterization of the effects of loss- and gain-of function mutations in vivo, and analysis of mutations in target cis-acting regulatory elements using established transgenic and cell culture systems. Trans-acting factors already identified that play important roles in regulation of Ubx splicing include the Snf protein (associated with U1 snRNP) and hrp48, a member of the hnRNP A/B protein family; Rbp1, a member of the SR protein family, is implicated through the analysis of cis-acting elements. Proposed experiments will test the hypothesis that Snf provides an interaction site for recruitment of U1 snRNP in a negative regulatory mode, and that hrp48 mediates this recruitment to the regulated 5' splice site by interaction with a specific sequence on the pre-mRNA, whereas Rbp1 opposes the action of hrp48 by helping to recruit U1 snRNP in a productive mode to the same 5' splice site. These factors are all homologous to human proteins that are either components of U1 snRNP (Snf=U1A) or have been implicated as potential splicing regulators. Hence, these investigations will be highly relevant to the regulation of splicing in humans.
