Summary Pre-mRNA splicing is essential for gene expression in all eukaryotes and errors in splicing cause genetic disorders and many other diseases. A thorough understanding of the molecular mechanisms of pre-mRNA splicing has the potential to provide useful approaches for human disease therapy. The splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a large RNA/protein complex composed of five snRNPs (U1, U2, U4, U5, U6) and many non-snRNP related protein factors. The spliceosome undergoes dramatic changes in a splicing cycle, generating the E, A, Pre-B, B, Bact, B*, C, C*, P, and ILS complexes. Genetic, biochemical, and structural studies in the past four decades have generated tremendous information on the mechanism of splicing, but significant knowledge gaps remain. Our goal in the next 5 years is to use a combination of structural, biochemical, and genetic/genomics approaches to understand the mechanism and modulation of alternative 5’ ss recognition, the mechanism of exon definition, and the mechanism and function of transcription and splicing coupling. We envision that these projects will fill several significant knowledge gaps and advance our understanding of the mechanism of splicing.