The central role of splicing in the eukaryotic gene expression pathway is well established, and yet our understanding of how spliceosomal snRNPs recognize introns and assemble into an active spliceosome remains in shadow. The recent explosion of cryo-EM structure models has exposed the spliceosome?s complex journey from one state to the next, detailing its elaborate changes in composition and conformation. A surprise has been just how big some of the conformational changes are ? so big that we have no understanding of how they might occur. The challenge now is to understand the molecular basis by which these amazing transitions take place. The veil has yet to be lifted on the structural details of how U2 selects the branchpoint, a process of keen interest that is affected by recurrent splicing factor mutations in many cancers. How do the RNA elements and splicing proteins required for branchpoint recognition select the appropriate site? With the recent structures, we can infer a kaleidoscope of changing U2 interactions with itself, with other RNAs and with proteins. Years ago, we showed that a U2 stem IIa to stem IIc RNA rearrangement promotes the first catalytic step of splicing, and the structures now suggest that this rearrangement happens as the 3? half of the U2 snRNP swings 150 and the U2- branchpoint helix moves 50 into the catalytic center. How are these movements triggered and what ensures they are completed properly? Recently Karla Neugebauer developed single molecule methods to determine both the position of RNA polymerase on the template and the splicing status of the pre-mRNA. We will use her method with reporters in which we have engineered changes in the gene that delay RNA polymerase and affect splicing outcomes. How does the timing of polymerase transit affect RNA processing? To address these exciting questions in a comprehensive way, we propose the following specific aims. We will (1) determine how early interactions between the U2 snRNP and the intron lead to the establishment of the extended U2-intron pairing upon ATP hydrolysis, (2) use the recent cryo-EM structures as a guide to characterize factors controlling the dynamics of U2 snRNA during splicing, and (3) determine mechanisms by which the dynamics of transcript elongation impact splicing decisions. The overarching hypothesis of this application is that combined structure and function analysis of the core components of the spliceosome will provide the mechanistic and structural basis for understanding the regulation of this central step in eukaryotic gene expression. The recent structures of the yeast spliceosome, the power of yeast genetics and biochemistry, and the fundamental conservation of the splicing machinery promise to translate directly into new understanding of the mechanisms of gene regulation in eukaryotes, including humans, where defects in splicing are increasingly recognized as contributors to disease, and interventions that address splicing are increasingly recognized as pathways to treatment and cures.
Splicing is a fundamental step in the process of gene expression, which we will explore using the simple yeast Saccharomyces cerevisiae. Recently recurrent mutations in the human gene (SF3B1) homologous to a yeast splicing factor we have studied for 20 years were found to influence tumor progression through their effects on splicing. The fundamental splicing mechanisms altered by these human cancer mutations are not completely understood, and in part of this project we will examine those fundamental mechanisms in yeast, where more rapid and comprehensive analysis can be undertaken.
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