GENOME-WIDE ANALYSIS OF NASCENT TRANSCRIPTION IN SACCHAROMYCES CEREVISIAE

该子项目是利用资源的许多研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其他NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 虽然大多数mRNA丰度的研究分析的是稳态水平，但为了了解基因调控，直接测量RNA合成至关重要。我们结合了核运行（NRO）测定，它使用RNA标记和捕获新生转录本，与高通量DNA测序，以检查指数增长的酿酒酵母的转录。NRO样品中测序读段的积累不仅在RNA聚合酶启动子募集水平上，而且在多个募集后步骤，特别是启动子近端暂停，与转录调控一致。例如，除了一个酵母核心组蛋白基因外，其他所有基因都显示出强的启动子近端暂停，这表明在S期快速诱导组蛋白合成需要过渡到生产性延伸。我们检查了NRO样本的反义转录本，并观察到这些310个基因，但我们没有发现广泛的双向转录从一个单一的趋异启动子的证据。相反，反义数据似乎反映了在相反方向上驱动转录的不同启动子的活性。通过计算每个基因的NRO转录与总RNA的比率，我们可以估计RNA的稳定性，我们发现最稳定和最不稳定的转录本编码的蛋白质的功能作用与这些稳定性一致。在对酵母进行短暂的热休克处理后，还分析了转录，这表明大多数热休克诱导基因通过增加其RNA合成来增加其RNA丰度。总之，NRO测定和高通量测序的组合允许评估酵母中RNA聚合酶活性的全基因组，鉴定RNA合成和RNA稳定性的调节步骤。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Although most studies of mRNA abundance analyze steady-state levels, it is critical to directly measure RNA synthesis in order to understand gene regulation. We combined the nuclear run-on (NRO) assay, which uses RNA labeling and capture of nascent transcripts, with high throughput DNA sequencing to examine transcription of exponentially growing Saccharomyces cerevisiae. The accumulation of sequencing reads in the NRO sample is consistent with transcriptional regulation not only at the level of RNA polymerase promoter recruitment but also at multiple post-recruitment steps, particularly promoter-proximal pausing. For example, all but one yeast core histone gene showed strong promoter-proximal pausing, suggesting that transition to productive elongation is necessary for rapid induction of histone synthesis in S phase. We examined the NRO sample for antisense transcripts and observed these for 310 genes, but we found no evidence for widespread bidirectional transcription from a single divergent promoter. Instead, the antisense data appear to reflect the activity of distinct promoters driving transcription in opposite directions. By calculating the ratio of NRO transcription to total RNA for each gene, we could estimate RNA stability, and we found that the most stable and unstable transcripts encode proteins whose functional roles are consistent with these stabilities. Transcription was also analyzed after a brief heat shock treatment of yeast, which revealed that most heat shock-inducible genes increase their RNA abundance by increasing their RNA synthesis. In sum, the combination of the NRO assay and high throughput sequencing allows an assessment genome-wide of RNA polymerase activity in yeast, identifying regulatory steps of RNA synthesis and RNA stability.