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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 尽管大多数 mRNA 丰度研究都分析稳态水平，但直接测量 RNA 合成对于了解基因调控至关重要。我们将核连载 (NRO) 测定（使用 RNA 标记和捕获新生转录本）与高通量 DNA 测序相结合，以检查指数生长的酿酒酵母的转录。 NRO 样本中测序读数的积累不仅在 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）上都与转录调控一致。例如，除了一个酵母核心组蛋白基因外，所有酵母核心组蛋白基因都显示出强烈的启动子近端暂停，这表明向生产性延伸的转变对于在S期快速诱导组蛋白合成是必要的。我们检查了 NRO 样本中的反义转录本，并观察了 310 个基因，但我们没有发现来自单个不同启动子的广泛双向转录的证据。相反，反义数据似乎反映了驱动相反方向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，并且我们发现最稳定和不稳定的转录物编码的蛋白质的功能作用与这些稳定性一致。在对酵母进行短暂热激处理后，我们还对转录进行了分析，结果表明，大多数热激诱导基因通过增加 RNA 合成来增加其 RNA 丰度。总之，NRO 测定和高通量测序的结合可以评估酵母中全基因组的 RNA 聚合酶活性，确定 RNA 合成和 RNA 稳定性的调控步骤。