Dissecting spliceosome function with small-molecule inhibitors

In eukaryotes, a crucial step in gene expression is pre-mRNA splicing by the spliceosome. The spliceosome is a macromolecular machine that removes intervening intron sequences from over 95% of human transcripts to create a functional template for protein synthesis. Precise splicing is essential for correct gene expression, and mutations in the spliceosome are associated with diseases including cancer. Mass spectrometry has identified over 100 spliceosome proteins, but deciphering the function for the vast majority of them has been challenging due to the highly dynamic and complex nature of the spliceosome. My dissertation focuses on small-molecule inhibitors as tools to expand the currently limited mechanistic understanding of the spliceosome: First, I developed a high-throughput assay to rapidly and efficiently screen large compound libraries for small molecules that inhibit splicing. I found three new splicing inhibitors and determined their effect on spliceosome assembly and splicing chemistry. Second, I used synthetic inhibitor analogs for structure-activity studies to identify chemical groups that are responsible for compound activity. I found that the same structural features are required for in vitro and in vivo splicing inhibition, and that the cellular response was mainly due to inhibition of the spliceosome. Third, I used biochemical assays to show that three structurally distinct inhibitors bind to the same site of the spliceosome core protein SF3B1, and that SF3B1 has a functional role throughout the multi-step splicing process. SF3B1 is of particular interest because it is often mutated in cancer, which makes it a promising target for the development of novel chemotherapeutics. My work provides a fast, reliable assay to identify small-molecule splicing inhibitors, a series of in vitro and in vivo assays to characterize their effect on complex assembly, splicing chemistry, and cellular phenotype, and an excellent example of how inhibitors can be utilized to decipher the function of spliceosome proteins. In addition to expanding the mechanistic model of one of the most complex macromolecular machines in the cell, identifying the function of individual spliceosome parts in healthy situations is the necessary first step to determine how changes of these functions in aberrant situations can lead to cancer.