Divergence of Transcription Regulation and the Function of a Conserved Elongation Factor in Budding and Fission Yeast

Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet mechanistic details of this evolution remain elusive. By comparing the transcriptional landscape of distantly related budding and fission yeast, we catch a glimpse of divergence in gene regulation. Using an adapted Precision Run-On sequencing (PRO-seq) approach, we have mapped the positions of RNA polymerase (RNA Pol II) active sites genome-wide in S. pombe and S. cerevisiae at base-pair resolution. We further mapped preferred sites of transcription initiation in each organism, exposing the origins of nascent transcription. Performing PRO-seq in strains lacking Spt4, a highly conserved elongation factor subunit, results in globally elevated levels of transcribing Pol II within genes in both species. Messenger RNA abundance, however, does not reflect the increases in RNA Pol II density, indicating a possible global elongation rate defect. Unexpectedly, we identify a novel pause in early elongation, specific to S. pombe, which requires Spt4, suggesting regulatory potential similar to promoter-proximal pausing in mammals. Overall design: Examination transcriptional landscapes in S. cerevisiae and S. pombe and the changes that result from the deletion of Spt4.

哺乳动物基因表达的复杂调控体系源自更为简单的真核生物系统，但其演化的分子机制细节仍未阐明。通过比对亲缘关系较远的出芽酵母与裂殖酵母的转录组景观，我们得以窥见基因调控的演化分化路径。我们采用改良的精准延伸测序（Precision Run-On sequencing, PRO-seq）技术，在单碱基分辨率下完成了粟酒裂殖酵母（S. pombe）与酿酒酵母（S. cerevisiae）全基因组范围内RNA聚合酶II（RNA Pol II）活性位点的定位。此外，我们还分别定位了两种酵母的转录起始偏好位点，进而揭示了新生转录的起源。对缺失高度保守的延伸因子亚基Spt4的菌株开展PRO-seq实验后，我们发现两种酵母的基因内部活跃转录的Pol II水平均出现全局性升高。然而，信使RNA（messenger RNA, mRNA）的丰度并未与RNA聚合酶II的密度升高保持一致，这提示存在全局性的延伸速率缺陷。出乎意料的是，我们发现了粟酒裂殖酵母特有的、依赖Spt4的早期延伸阶段新型暂停现象，这提示其具备与哺乳动物启动子近端暂停类似的调控潜能。实验整体设计：对酿酒酵母（S. cerevisiae）与粟酒裂殖酵母（S. pombe）的转录景观进行检测，并分析Spt4基因缺失所引发的转录景观变化。