Genome-wide Transcriptional Starting Sites Analysis In Fission Yeast

Transcriptional starting sites plays an essential role in identification ofcore promoter regions and further revelation of transcriptional regulationin eukaryotes.Fission yeast Schizosaccharomyces pombe serves as an importantmodel organism with a plethora of transcriptome studies. However, theposition and structure of its core promoter regions which play a key role ingene expression regulation still remains as the least-explored field. Sincecore promoters are often100base pairs surrounding transcriptional startingsites (TSS), it is of importance to perform a genome-wide analysis oftranscriptional starting sites analysis in fission yeast. To solve this problemwe applied CAGE (Cap Analysis of Gene Expression) technology, incombination with next-generation sequencing strategy, in yeast to globallycapture the TSSs. We also analysed these sites through bioinformaticsapproach. This thesis included the techniqal part of development of CAGEand a preliminary analysis of TSS sites obtained from high-throughputsequencing. In this thesis we discussed different methods of total RNAisolation, optimization of RNase I digestion condition as well as a way toconstruct CAGE tags library. Through the serial of optimizations, weobtained CAGE library with a ribosomal RNA contamination below10%,indicating the high efficiency in TSS selection. This thesis also covered thepreliminary bioinformatics analysis of CAGE data derived fromhigh-throughput sequencing, and found that77%of CAGE tags aremapped to the5'end of annotated genes, another indication of a highefficiency in TSS capture. Other interesting findings include: S.pombeshares a similar complexity in their promoter shapes with higher eukaryotes; Most of the yeast genes belong to the broad-promoter class;More than50%of yeast genes have alternative promoters; Antisensetranscripts in yeast also have broad-typed and sharp-typed core promoters.Our research will provide important information for the further study intranscriptional regulation in yeast and other eukaryotes.