IDENTIFICATION OF SPLICING PATHWAY MUTATIONS VIA TARGETED SEQUENCING

Abstract

The identification of splice sites and catalytic removal of introns via the spliceosome is an essential and regulated component of eukaryotic gene expression. While ever-increasing numbers of human genetic diseases can be linked to defects in the splicing pathway, our molecular understanding of how these mutations disrupt this complex process remains incomplete. To identify mutations which impact the splicing pathway I have developed a series of targeted-sequencing based quantitative genetic screens in S. pombe, a yeast species which is genetically tractable yet retains features of complex splicing patterns that have been lost in the S. cerevisiae lineage. Through these screens I have isolated conditional alleles of spliceosome components and investigated the mechanism by which some of these mutations disrupt splicing. Furthermore, I have identified genes in other nuclear processes, such as transcription and chromatin remodeling, which contribute to splicing outcomes. These findings suggest splicing is largely co-transcriptional and suggest specific genes by which transcription is functionally linked to splicing. Finally, I describe a novel approach for enriching RNA-sequencing libraries for splicing-informative reads. This technique allows for precise quantitation and discovery of rare splice products, including splice intermediates, which are poorly captured in other RNA-sequencing methods.