Structural Analysis of Catalytic and Post-catalytic Spliceosome Complexes
Pre-mRNA splicing is an essential step in eukaryotic gene expression. Splicing is catalyzed by the spliceosome, a large and highly dynamic macromolecular machine in the cell. Errors in splicing have been linked to many diseases, including cancer. The spliceosome is responsible for precisely joining the protein coding sequences that will be translated to protein. It is composed of five small nuclear RNA (U1, U2, U4, U5, and U6) along with over 100 unique proteins. Understanding the RNA/RNA and RNA/protein interactions that position the pre-mRNA substrate in the spliceosome for the two catalytic steps of splicing is key to determining the molecular mechanisms of this dynamic complex.
In this thesis, we compared two states of the spliceosome complexes, catalytic and post-catalytic. We investigated the role of pre-mRNA sequences in the spliceosome complex stalled prior to the second step of splicing. We next developed a new strategy that traps the spliceosome after the second step of splicing, but before the mRNA is released. We show that structural rearrangements of the RNA substrate exist as the spliceosome transitions from the first to second step of splicing. Using mass spectrometry assays, we identify proteins that stably associate with affinity-purified catalytic and post-catalytic complexes. We show enrichment of SF3B components and loss of an ATPase, DHX35, in the post-catalytic complex. In conjunction with biochemical studies, we use electron microscopy to determine structures of both complexes to further gain insights into the spliceosome's role in splicing. This study serves as a foundation for understanding the mechanisms underlying the later stages of spliceosomal progression.