Mechanisms Underlying Regulation of Non-Coding RNA Transcription

描述（由申请人提供）：基因组内RNA聚合酶II （Pol II）转录的调控对细胞存活和分化至关重要。在真核生物中参与这种调节的一种蛋白质复合体是Paf1复合体（Paf1C），它被研究得最好的是促进转录延伸和组蛋白修饰。Paf1C的一个不太为人所知的功能是它对非编码RNA （ncRNA）转录物终止的贡献，而ncRNA具有无数重要的细胞功能。与Paf1C一样，ncrna与包括癌症在内的一系列人类疾病有关。这项研究计划的长期目标是阐明酿酒葡萄球菌控制ncrna转录的潜在机制。在Specific Aims 1和2中，Paf1C在整个基因组中ncRNAs转录中的作用将通过高密度平铺阵列定义，并通过RNA分析和使用已建立的遗传报告结构直接证实。此外，有针对性的生化实验将描述包括Paf1C亚基在内的蛋白质网络如何在分子水平上共同调节ncRNA的转录和功能。在Specific Aim 3中描述的基因筛选将识别损害ncRNA转录终止的新型paf1突变。表征这些突变将揭示Paf1C的ncRNA功能是否与Paf1C的其他既定功能重叠，例如促进组蛋白修饰。总的来说，这些研究ncRNA调控和保守的Paf1C功能的实验将定义参与中心调控机制的蛋白质网络的分子相互作用，这反过来将揭示Paf1C和ncRNA的扰动如何导致疾病进展。

DESCRIPTION (provided by applicant): Regulation of RNA polymerase II (Pol II) transcription within a genome is essential for cell survival and differentiation. One protein complex involved in this regulation in eukaryotes is the Paf1 complex (Paf1C), which is best studied for promoting transcription elongation and histone modifications. A less well understood function of the Paf1C is its contribution to termination of noncoding RNA (ncRNA) transcripts, which have a myriad of important cellular functions. Like Paf1C, ncRNAs have been implicated in a range of human diseases including cancer. The long-term goal of this research proposal is to elucidate the underlying mechanisms via which transcription of ncRNAs is controlled in S. cerevisiae. In Specific Aims 1 and 2, the role of the Paf1C in the transcription of ncRNAs throughout the genome will be defined with high-density tiling arrays and confirmed directly with RNA analysis and the use of established genetic reporter constructs. Additionally, targeted biochemical experiments will characterize how a network of proteins, including the Paf1C subunits, work together on a molecular level to regulate ncRNA transcription and function. A genetic screen, described in Specific Aim 3, will identify novel paf1 mutations that impair ncRNA transcription termination. Characterizing these mutations will reveal whether the ncRNA functions of Paf1C overlap with the other established roles of Paf1C, such as promoting histone modifications. Overall, these experiments addressing ncRNA regulation and conserved Paf1C functions will define the molecular interplay of a network of proteins involved in central regulatory mechanisms, which in turn will shed light on how perturbations of Paf1C and ncRNAs can lead to disease progression. PUBLIC HEALTH RELEVANCE: Non-coding RNAs (ncRNAs) perform a myriad of functions important for cell growth. Experiments in this proposal will determine the molecular mechanisms and coordinated action of a network of proteins that are responsible for the regulation of ncRNA synthesis throughout the genome. Shedding light on these key cellular processes is important because perturbations of these regulatory proteins and ncRNAs can lead to human diseases such as cancer.