The removal of introns from nuclear pre-mRNAs requires the assembly of a large macromolecular machine termed the spliceosome. In humans, it has been estimated that the spliceosome must be able to recognize and precisely excise as many as 106 different introns of varying lengths and sequences. The precise identification of intron boundaries and their excision by the spliceosome is accomplished through the highly ordered and dynamic assembly of five small nuclear ribonucleoprotein particles and more than sixty proteins onto a pre-mRNA. During many of the assembly steps, RNA helices are formed and/or broken in reactions requiring enzymes termed ATP- dependent RNA helicases. Intriguingly, genetic analyses in yeast have indicated that ATP-hydrolysis by RNA helicases can function in proofreading steps that insure the fidelity of splice site choice. Prp16p is an RNA helicase that catalyzes a conformational change in the spliceosome acts to insure the fidelity of intron branch site selection. Recent results have suggested that ATP hydrolysis by Prp16 may lead to the creation of a binding site for the 3' splice site. The first major goal of the proposed research is to identify the RNA substrates for the RNA helicase activity of Prp16p within the spliceosome, and to characterize the rearrangements of RNA/RNA and RNA/protein interactions that occur as a consequence of ATP hydrolysis by Prp16p. The nature of the interactions that may serve to connect ATP hydrolysis by Prp16p to the fidelity of branch site recognition will also be determined. Second, we will determine if ATP hydrolysis by Prp16p leads to changes in the interactions of U2/U6 helix I, a helix that has a critical function in the second step. Third, because the rearrangements facilitated by Prp16p may culminate in the formation of a binding site for the 3' splice site, we will determine if changes in the interactions involving the 3' splice site are directly tied to ATP hydrolysis by Prp16p. A detailed picture of the role of a specific member of the RNA helicase family in splicing should help elucidate the principles by which they function. Because these enzymes have important roles in all aspects of cellular RNA metabolism, the results of these studies will be relevant to the study of their functions in other biological contexts.