Views of the dynamic human spliceosome

The spliceosome is the cellular machinery responsible for removing introns from gene transcripts and ligating together exons to form mature messenger RNAs. It therefore serves as a critical player in regulating human gene expression and proteome complexity. We are working to provide a structural framework to understand the molecular mechanisms of the spliceosome. This task is challenged by size and dynamics of the complex as it forms from over 100 components into several assembly intermediates and functional states. Using an in vitro splicing system in human cell extracts in which we modify the pre‐mRNA substrate upon which the complex is assembled, we have new made advances in capturing the spliceosome at distinct intermediate states relative to spliceosome chemistry. Mass spectrometry analyses of the proteins in the different spliceosome intermediate complexes indicate changes in composition, which we then relate to differences in the structure of the complexes that we observe by electron microscopy. Using a combination of crosslinking, chemical modification and mass spectrometry strategies, we have also identified proteins that change in their association with the RNA substrate as the splicing reaction is catalyzed. Taken together, these observations are building a picture of the molecular rearrangements that occur in the spliceosome during the transition between first and second step chemistry.