Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to many human genetic diseases, and pre-mRNAs containing multiple introns and exons can be alternatively spliced in a cell type, cell cycle, or developmentally regulated manner by joining different pairs of 5'and 3'splice sites. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. The overall goal of this research proposal is to understand the mechanisms involved in splice-site recognition and pairing of pre-mRNAs. During the previous funding period, we have developed quantitative assays to provide new insights into the mechanisms of splice-site pairing. In the next phase of investigation, we propose to determine the ?molecular events that lock splice sites into a pairing position and to analyze how the combinatorial contribution of multiple splicing signals influence exon inclusion. Specifically, we will determine the biochemical steps that lead to splice-site pairing in A complex (Aim 1). We will test the hypothesis that ATP hydrolysis during A complex formation drives the irreversible juxtaposition of alternative splice sites or exons. In Aim 2 we will determine how the spliceosome executes commitment to splice-site pairing. We will use immuno-depletion and RNAi approaches to test the hypothesis that a subset of U2 snRNP components and associated proteins (CUS2/Tat-SF-1, Prp5, SF3a120, and UAP56) is necessary for irreversible splice-site pairing. Aim 3 describes a systematic and quantitative approach to determine how the probability of exon definition and inclusion is influenced by the combinatorial contributions of variable splice sites, enhancers, silencers, and the exon/intron architecture. We will test the hypothesis that measures of exon inclusion can be quantitated and used to improve the predictability of constitutive and alternative splicing within the human genome. These experiments are important because 1) the commitment to splice-site pairing constitutes arguably the most crucial step during the splicing reaction because it determines the splicing patterns of pre- mRNAs, and because 2) a quantitative framework of combinatorial exon recognition will elucidate mechanisms of splicing regulation and allow to predict the intrinsic pattern of splicing from sequence analysis.