GENOME-WIDE ANALYSIS OF NASCENT TRANSCRIPTION IN SACCHAROMYCES CEREVISIAE

酿酒酵母新生转录的全基因组分析

• 批准号: 8365819
• 负责人: STANLEY FIELDS
• 金额: $ 2.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365819
• 关键词: Automobile Driving; Biology; DNA Sequence; DNA-Directed RNA Polymerase; Data; Electroconvulsive Therapy; Funding; Fungal Genome; Gene Expression Regulation; Genes; Genetic Transcription; Grant; Heat-Shock Response; Histones; Label; Measures; Messenger RNA; National Center for Research Resources; Nuclear; Principal Investigator; Proteins; RNA; RNA Stability; RNA chemical synthesis; Reading; Research; Research Infrastructure; Resources; Role; Run-On Assays; Running; S Phase; Saccharomyces cerevisiae; Sampling; Source; Sum; Transcript; Transcriptional Regulation; United States National Institutes of Health; Yeasts; cost; genome-wide; genome-wide analysis; promoter

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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 尽管大多数对mRNA丰度的研究都分析了稳态水平，但至关重要的是直接测量RNA合成以了解基因调节。我们将使用RNA标记和捕获新生转录本的核跑步（NRO）测定与较高的吞吐量DNA测序相结合，以检查酿酒酵母的指数生长的转录。 NRO样品中测序读数的积累与转录调节不仅在RNA聚合酶启动子募集水平上，而且在多个效率后步骤，尤其是启动子抗速度暂停下。例如，除一个酵母核心组蛋白基因以外的所有均显示出强启动子抗性的暂停，这表明过渡到生产性伸长对于在S期快速诱导组蛋白合成是必不可少的。我们检查了NRO样品的反义转录本，并观察到了310个基因，但是我们没有发现单个发散启动子的广泛双向转录的证据。取而代之的是，反义数据似乎反映了朝相反方向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稳定性，我们发现最稳定，最不稳定的转录本编码功能作用的蛋白质与这些稳定性一致。在对酵母进行短暂的热休克处理后，还分析了转录，这表明大多数可热休克诱导的基因通过增加其RNA合成而增加了其RNA丰度。总而言之，NRO分析和高通量测序的组合允许在酵母中评估RNA聚合酶活性的全基因组，从而确定RNA合成和RNA稳定性的调节步骤。