| dcterms.abstract | High-throughput studies in recent years unveiled unexpected complexity of the transcriptome in yeast and humans. One particular type of change in transcriptome architecture, transcription initiated within an open reading frame, reduces the length of the 5'-UTR and has the potential to generate altered protein products. Budding yeast gametogenesis, also called sporulation, is a developmental process where diploid cells undergo meiosis to produce highly differentiated, stress-resistant gametes in response to nutrient deprivation. Accompanied by massive changes in gene expression, sporulation provides a great system to study transcriptional and post-transcriptional gene regulation. Among the meiotic transcriptome of budding yeast, internally initiated transcripts have been observed previously. Nonetheless, no analyses on next-generation sequencing datasets have been carried out to determine internal transcriptional initiations during sporulation. To systematically identify novel internal transcription start sites unique to a certain period in sporulation, I have developed a customized bioinformatical pipeline that performs unbiased transcription architecture comparison. By applying this approach to an RNA-Seq dataset of a highly synchronized meiotic time-course, dramatically different sets of genes with novel internal initiations are detected in early and mid-sporulation, suggesting that internal initiations are highly dynamic during sporulation. One key regulator in the transcription cascade of sporulation, the transcription factor Ndt80, is a master inducer of ~300 mid-sporulation genes that are required for meiotic divisions and spore formation. Motif analysis on the upstream sequences of internally initiated transcripts reveals a significant enrichment for the binding site of Ndt80 in the 43 genes that display mid-sporulation specific internal initiation. To further study the detailed regulation, my research focused on one candidate MRK1, which is one of the budding yeast homologs of GSK-3 kinases. Mutation analysis reveals direct regulation of MRK1 internal initiation by Ndt80, which results in the expression of an N-terminally truncated protein isoform. Additionally, functional analyses show that MRK1 promotes sporulation in redundancy to its paralog, RIM11, through the internally initiated isoform. Taken together, my findings using budding yeast sporulation as a model system suggest internal transcriptional initiation to be a dynamic, regulated process with potential functional impacts on development. | |
