Discovering Alternative Splicing Of Deeply Conserved Exons And Characterizing The Intronome In The Unicellular Yeast S. Pombe Using Lariat Sequencing
Alternative splicing is a potent regulator of gene expression that vastly increases proteomic diversity in multi-cellular eukaryotes. Although it is widespread in vertebrates, little is known about the evolutionary origins of this process owing in part to the absence of phylogenetically conserved events that cross major eukaryotic clades. The unicellular fission yeast, Schizosaccharomyces pombe, is an organism evolutionarily distant from mammals that nonetheless shares many of the hallmarks of the major mammalian alternative splicing form, exon skipping. Further, S. pombe is a highly genetically tractable organism that has been considered as an attractive potential model system in which to study exon skipping. However, evidence of exon skipping from RNA-seq studies in S. pombe has remained elusive. To better search for such evidence I have developed a novel lariat sequencing approach that offers high sensitivity for detecting splicing events, and applied it to study splicing in S. pombe. Using this approach, I discovered multiple examples of exon skipping, several of which involve exons that are conserved with dozens of animals, plants, other fungi and even protists. Strikingly, some of the specific alternative splicing patterns found in S. pombe are identical in mammals. In addition, I discovered hundreds of novel splicing events, many of which occur in genes that were thought to be intronless. Finally, I present only the second large-scale map of the intronic branch point sequences in any organism, allowing me to test a prediction about differences in splice site recognition in introns from non-coding regions versus those that separate protein-coding exons.
Alternative Splicing; Sequencing; pombe
Pleiss, Jeffrey A.
Lis, John T; Alani, Eric
Ph.D. of Genetics
Doctor of Philosophy
dissertation or thesis