Genomic Approaches to Understanding Pre-mRNA Splicing Regulation in Schizosaccharomyces Pombe

The protein-coding regions of most eukaryotic genes are interrupted by non-coding introns, which must be excised from the pre-messenger RNA (pre-mRNA) prior to translation. Pre-mRNA splicing is performed by the spliceosome, a macromolecular machine that assembles anew on every intron, catalyzing both the removal of the intron and ligation of exons. The central role of pre-mRNA splicing in regulating gene expression is seen in the large number of human diseases which are associated with mutations in this pathway. Nevertheless, the mechanisms by which the complex splicing pathway are regulated remain poorly understood. For example, while it has been established that the spliceosome is co-transcriptionally recruited to introns via interactions with the C-terminal domain of RNA polymerase II, the mechanism by which this enables spliceosome assembly is unknown. To better understand the mechanisms of splicing regulation, our work harnesses the power of the unicellular fission yeast, Schizosaccharomyces pombe, which shares many features of mammalian splicing systems. The splice site sequences found within S. pombe introns are marked by a degeneracy similar to that seen in human introns. Additionally, nearly 50% of S. pombe genes contain an intron, and almost half of those contain multiple introns, a prerequisite for exon-skipping. Indeed, our lab recently demonstrated the first examples in S. pombe of environmentally-regulated exon skipping of deeply evolutionarily conserved exons. Here I describe our work developing a sequencing-based screen that uses a custom-designed barcoding scheme to simultaneously measure changes in in vivo levels of pre-mRNAs in the background of thousands of arrayed mutants. This approach was originally used to assay a library of non-essential genes in S. pombe, enabling identification of scores of strains that displayed defects in splicing of two endogenous pre-mRNAs. This work identified splicing phenotypes associated with factors with described roles in other RNA-processing pathways, such as heterochromatic silencing and 3’end processing, highlighting the interconnected nature of RNA processing. More recently, this approach has been used to interrogate a collection of conditional mutants generated by random mutagenesis to identify canonical splicing mutants. Identification of the causative mutations by whole genome sequencing has led to the identification of novel mutant alleles in the known splicing factors Sap61, Prp22, Cdc28, and Prp1. Our ongoing characterizations of these alleles coupled with recently published structures of several spliceosome complexes suggests that they will provide fascinating insights into the mechanisms by which the spliceosome can function to control regulation. Given the high level of homology between the splicing apparatus in S. pombe and humans, we expect that the results of these studies may provide important insights not only into S. pombe biology, but into mechanisms of mammalian regulation and disease