Protein RNA Rearrangements in the Spliceosome

ABSTRACT Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events — which are not understood mechanistically — will elucidate interactions and rearrangements among core components and may serve as a paradigm for rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which spliceosomal dynamics impact splicing fidelity. The Aims of this project focus on two newly-discovered aspects of the spliceosome and snRNAs: (1) We will investigate the role of inositol hexakisphosphate (IP6) in spliceosome function. This small molecule was recently identified unexpectedly by cryo-EM as part of splicing complexes. No biochemistry or genetics has so far explored its importance. We use mutants in the IP6 biosynthetic pathway to explore its contribution to splicing and whether inositol polyphosphate (IPP) isoform levels may influence splice events. (2) One hole in our current understanding of RNA biology is the role of snRNA mutations in disease. Two mutations were recently described in the 5'-end of U1 snRNA, associated with glioblastoma (GBM) and other cancers. We will use our in silico snRNA genome to interrogate TCGA whole genome datasets (WGS) and investigate, in particular, U2 snRNA mutations that we find highly enriched in bladder cancer and bladder cancer organoid lines.