High Resolution Global Analyses of the Molecular Mechanisms of pre-mRNA Splicing Regulation

Abstract

Pre-mRNA splicing is an essential component of eukaryotic gene expression, and complex patterns of alternative splicing in higher eukaryotes significantly enhance proteome diversity. Mutations in the splicing pathway have been increasingly identified as drivers of human disease, yet the mechanistic consequences of these mutations remain poorly understood, inhibiting our understanding of these diseases. The fission yeast Schizosaccharomyces pombe offers an outstanding model for understanding complex splicing patterns seen in humans: its genome is rich with introns with highly degenerate splice site sequences, closely resembling those seen in higher eukaryotes, while its facile genetics enable interrogations of the highly conserved splicing machinery. A broad focus of my work has been understanding the mechanisms by which bona fide examples of regulated alternative splicing of specific S. pombe transcripts occurs. While Next Generation Sequencing technologies have had a transformative impact on studies of pre-mRNA splicing, currently a major obstacle in the field which remains poorly appreciated to this day, regards the quantitative limitations associated with these approaches, particularly in detecting and monitoring rare RNA species. To alleviate this problem, we developed a targeted RNA sequencing method termed Multiplexed Primer Extension sequencing (MPE-seq) which enriches for splicing informative reads, allowing for improved quantitative assessments of splicing isoforms, including the intermediates generated during the splicing reaction. I have leveraged this approach to interrogate different aspects of splice site recognition, with a particular focus on alternative splicing. Here I describe the results of a high-throughput forward genetic screen of thousands of temperature-sensitive S. pombe strains designed to identify those with defects in canonical and/or alternative pre-mRNA splicing. We identified scores of alleles, some causing global defects of canonical splicing, while others lead to specific changes in alternative splicing of select transcripts. Among others, whole genome sequencing of candidate strains revealed a pair of mutations in prp10, the S. pombe ortholog to the human protein SF3B1, one of the most commonly mutated genes in myelodysplastic syndromes. Remarkably, while these two variants lie close in three-dimensional space to one another, using MPE-seq I demonstrate the markedly different impacts of these mutations on pre-mRNA splicing patterns genome-wide. These studies provide important insights into how the spliceosome activates its cognate targets, and how disease-related mutations may mis-regulate this process.