A combined genetic and biochemical approach will be taken to study the functions of two yeast splicing factors, Spp2p and Prp31p. Spp2p functions together with Prp2, one of 5 DEAD(H) box proteins thought to function as RNA-dependent ATPases or RNA helicases at different steps of spliceosome assembly or splicing. It is not known how each of these DEAD(H) box proteins is specifically targeted to the splicing complex, nor what protein targets or RNA ligands are recognized by them. Spp2p is the first protein identified that may interact with a DEAD(H) box splicing factor. Studies of Spp2p are designed to help understand how Prp2p and Spp2p assemble into the spliceosome and function in splicing. Prp2p is thought to function after most of the spliceosome is assembled, when it might promote rearrangements of the spliceosome necessary to initiate the first catalytic reaction of splicing. Genetic and biochemical experiments suggest that Spp2p interacts with Prp2p and is required for association of Prp2 with the spliceosome. Using biochemical assays, it will be tested whether Spp2p directly interacts with Prp2p, and if so, which regions of the two proteins are necessary for this interaction. To identify the niche within the spliceosome where Spp2p and Prp2 function, genetic screens will be used to search for protein or RNA molecules that interact with Spp2p. UV-cross-linking assays also will be used to determine if Spp2p specifically binds to pre-mRNA or U2 snRNA, suspected ligands of Prp2p. Prp31p is a U4/U6.U5 tri-snRNP protein necessary for assembly of tri-snRNPs with the splicing complex. It is not known how U4/U6 and U5 snRNPs assemble into U4/U6.U5 tri-snRNPs, or how tri-snRNPs associate with prespliceosomes to form spliceosomes. Prp31 is associated with the U4/U6.U5 tri-snRNP and with the prespliceosome. In prp31 mutants, levels of tri-snRNPs are partly diminished and formation of spliceosomes from pre-spliceosomes is completely blocked. These results suggest that Prp31p may be necessary for assembly of tri-snRNPs from U4/U6 and U5 snRNPs and may tether the tri-snRNP to the splicing complex. UV-cross-linking assays and genetic screens will be employed to investigate whether Prp31p is associated with the pre-spliceosome via contacts with pre-mRNA or snRNAs or by protein-protein interactions. The applicant will examine with which snRNPs Prp31p is associated and with which snRNP molecules Prp31p interacts by co-immunoprecipitation and UV-cross-linking assays as well as genetic screens. Effects of additional prp31 mutations on snRNP biogenesis and spliceosome assembly will also be assayed.
