The experiments described in this proposal are designed to gain insight into both the mechanism and regulation of pre-mRNA splicing. Much of the work focuses on a family of conserved proteins known as SR proteins that are both essential for splicing in vitro and also able to modulate the use of alternative splice sites. Studies on the role of snRNAs in splicing catalysis will also be pursued. Four Specific Aims are proposed. 1. ANALYSIS OF SR PROTEIN FUNCTION IN VITRO. Studies will focus on abundant SR proteins including the prototype ASF/SF2, as well as SC35, SRp20 and SRp40. Several outstanding issues will be addressed. One is the question of specificity, both with respect to protein-RNA and protein-protein interactions. A number of assays will be employed to detect differences in the behavior of individual proteins. The effects of phosphorylation of the RS domain on RNA and protein binding will also be examined. 2. ANALYSIS OF SR PROTEIN FUNCTION IN VIVO. Transfection assays involving overexpression of SR proteins in mammalian cells will be continued to provide further insight into the role of SR proteins in modulating splicing, including autoregulation, and in other recently suggested processes such as nuclear-cytoplasmic transport. Cell lines producing different amounts of ASF/SF2 will produced using the chicken B cell line DT40, and effects on cell growth and splicing will be determined. 3. FUNCTION AND REGULATION OF CLK KINASES. Clk/Sty kinase is the prototype of a family of kinases containing N-terminal RS regions and C-terminal catalytic domains. Recent studies showing that Clk/Sty binds to certain SR proteins, phosphorylates serines in the RS domain, and modulates splice site selection in vitro and in vivo will be pursued. The possibility that different Clk family members target distinct SR proteins will be examined. Regulation of the kinases themselves will be studies. 4. FUNCTION OF SNRNAs IN SPLICING CATALYSIS. Genetic studies involving transfection assays will be continued to investigate further the U2-U6-pre-mRNA interactions required to catalyze splicing. The requirements of mutually exclusive base pairing involving U2, U6 and the pre-mRNA will be pursued, as will studies of an intramolecular U6 helix that may be critical for catalysis. Interactions between purified U2, U6 and a model pre-RNA will be studied in vitro.