The precise removal of intervening sequences from primary transcripts by splicing is an essential step in the production of functional messenger RNA in every eukaryotic cell. Our goal is to understand the mechanisms of pre-mRNA splicing and the molecular interactions involved. Our studies employ the powerful techniques available in the yeast S. cerevisiae. The immediate objectives are: (1) to identify and characterize splicing factors and (2) to elucidate critical protein/RNA and protein/protein interactions within the splicing complex (spliceosome). We are particularly interested in understanding how RNA-dependent ATPases and RNA helicases, such as Prp16 and Prp22, affect conformational rearrangements in the spliceosome, essential to drive the splicing reaction forward and to ensure fidelity. Our studies focus on the second step of splicing; this step encompasses the recognition of the 3' splice site and the joining of the exons. We have developed assay systems to halt the reaction at various distinct stages during step 2; activity can be restored by the addition of purified components. Our studies demonstrated that in response to ATP hydrolysis by Prp16, several protein factors (i.e. Slu7, Prp22, Ssf1) enter the spliceosome. They play roles in the selection of the 3' splice site and are essential for the formation of mature mRNA. The biochemical characterization of second step factors is complemented by genetic approaches. These take advantage of our collection of mutants in individual components; genetic interactions identify likely physical and functional connections not only between splicing factors, but also between splicing factors and components of other pre-mRNA processing machineries.