DESCRIPTION (from the application): Our long term goals are to understand how splice sites for pre-mRNAs are recognized by the spliceosome and to determine how the coupling of pre-mRNA processing with transcription by RNA polymerase II (pol II) contributes to this decision. The general strategy is to characterize pre-mRNA splicing in vitro, as it allows precise control over the reaction conditions. The central hypothesis is that complexes assembled on exonic splicing enhancers (ESE) assist in the recruitment of the spliceosome to adjacent introns, and that the elongation transcription machinery aides this assembly by interacting with splicing factors. Three specific aims are proposed: Understand the mechanisms involved in the positive control of fru pre-mRNA alternative splicing. It is hypothesized that the fru ESE increases U1 snRNP binding to the regulated 5' splice site. We will carry out an in-depth study to analyze the structure and function of a splicing enhancer that controls the activity of the fru female specific 5' splice site. We will examine the role of the fru ESE in spliceosomal assembly, and analyze the positional effects of the dsx/fru ESE complex in activating a 3' or a 5' splice site. Characterize the architecture and assembly of the dsx splicing enhancer complex. It is hypothesized that the assembly of the dsx ESE complex requires highly cooperative interactions. We propose to carry out a detailed structural analysis of the protein complex formed on the dsx ESE. We will determine the function of Tra, Tra2, and RBP1 in the recruitment of the splicing machinery, define the cooperative assembly and stoichiometry of the dsx ESE heterotrimeric complex, map protein-protein interactions within the complex, and characterize mutants in Tra and Tra2. Determine how transcription of pre-mRNAs by polymerase II affects ESE dependent splicing. We have established an in vitro assay to analyze the efficiency of pre-mRNA splicing when coupled to transcription. Using this assay we will test the hypothesis that the level of C-terminal domain (CTD) phosphorylation influences ESE dependent splice site activation and determine at which step during transcription the splicing machinery associates with nascent pre-mRNA.