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

哺乳动物的基因表达复杂调控网络源自更为简单的真核生物系统，然而该演化过程的分子机制细节仍未得到清晰解析。通过比较亲缘关系较远的出芽酵母（即酿酒酵母Saccharomyces cerevisiae, S. cerevisiae）与裂殖酵母（即粟酒裂殖酵母Schizosaccharomyces pombe, S. pombe）的转录全景，我们得以一窥基因调控的演化分化模式。借助优化后的精准运行测序（Precision Run-On sequencing, PRO-seq）技术方案，我们在单碱基分辨率下完成了全基因组范围的RNA聚合酶II（RNA polymerase II, RNA Pol II）活性位点定位。我们进一步绘制了两种生物各自的转录起始偏好位点，阐明了新生转录的起源机制。在缺失高度保守的延伸因子亚基Spt4的菌株中开展PRO-seq实验，结果显示两个物种的基因内部活跃转录的RNA聚合酶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密度的升高呈现对应关系，这提示可能存在全局性的转录延伸速率缺陷。出乎意料的是，我们在裂殖酵母中发现了一种全新的早期延伸暂停现象，该现象的发生依赖于Spt4，这提示其具备与哺乳动物中启动子近端暂停类似的调控潜能。整体实验设计：对酿酒酵母与裂殖酵母的转录全景进行检测，并分析Spt4基因缺失所引发的转录变化。