The broad long-term objectives of this application are to understand in detail pre-mRNA splicing. The regulation of splicing is a key feature of a number of medically relevant agents, such as DNA tumor viruses. It is a fundamental aspect of gene expression of all eukaryotes. In the yeast, Saccharomyces cerevisiae, splicing is relatively simple and predictable. Also, this organism is amenable to genetic analyses, and splicing can be easily analyzed both in vivo and in vitro. Several snRNA genes are known to play important roles in splicing. These genes will be manipulated to establish structure-function relationships for in vitro and in vivo activity. Splicing requires the assembly of a complicated and stable splicing complex, within which the cleavage and ligation reactions take place. In vitro and in vivo approaches will be used to study the pre-mRNA commitment process, i.e., what factors interact with the pre-mRNA substrate early in the assembly process to form a stable complex and "commit" the pre-mRNA to the spliceosome assembly pathway? Splice site cleavage and splice site selection are performed with a high degree of specificity. This specificity will be examined by in vitro and in vivo methods designed to perturb this process and there by identify some of the important, relevant factors. Spliceosome characterization has been performed to date almost exclusively on in vitro assembled complexes. Consequently, in vivo spliceosomes will be identified and characterized.