Mechanistic analyses of a novel RNA polymerase I transcription checkpoint

摘要 细胞的生长和增殖速度与核糖体生物合成的速度成正比。这 这种关系在癌症中被强调，其中对核糖体合成的持续需求是 很流行。核糖体生物合成的第一步是由rna转录核糖体dna。 聚合酶I(Pol I)；在癌症中，这一过程是不受调控的。尽管建立了这些联系 在核糖体生物合成和癌细胞增殖之间，控制 RDNA基因座的转录伸长仍然没有明确的定义。 我们已经发现了一种新的调节检查点，它可以监测rDNA的转录扰动 并通过Pol I催化亚基RPA194的降解来解决。此检查点已激活 由我们新发现的Pol I化学抑制剂(BMH-21)。此应用程序的目标是 通过定义激活该检查点的因素和分子机制(S)来描述该检查点。 此外，这项工作将确定该酶降解的分子基础，以及 RDNA基因座POLS I和POLS II的相互调节。 这项提案将追求三个主要目标：(1)确定监测Pol I延长的因素； (2)界定POL一延长检查点如何启动和强制执行，以及它如何影响POL II；以及 (3)鉴定介导RPA194降解的泛素-蛋白酶体系统成分。至 要实现这些目标，我们将追求三个具体目标。在这些目标中，BMH-21和其他表观遗传学 药物将被用作研究聚合酶选择性检查点的工具。目标1描述因素 在酵母和哺乳动物全基因组筛选中识别，并指定它们如何调节Pol I 抄写。AIM 2实施生物化学定义的转录分析和基因组分析 确定Pol I转录扰动如何激活检查点，以及它对聚合酶的影响 选择性。目的3确定泛素-蛋白酶体系统因子在控制稳定性中的作用 和Pol I的转录。总之，这些研究为Pol I的调控提供了前所未有的洞察力 Pol I失活时的转录和核转录重组。 对Pol I转录调控的机械性理解将支持寻求治疗的策略 人类疾病中非调控rRNA合成的控制。

ABSTRACT The growth and proliferation rates of cells are proportional to the rate of ribosome biosynthesis. This relationship is emphasized in cancer, where the continuous demand for ribosome synthesis is prevalent. The first step in ribosome biosynthesis is the transcription of the ribosomal (r) DNA by RNA polymerase I (Pol I); in cancers, this process is deregulated. Despite these established connections between ribosome biosynthesis and cancer cell proliferation, the regulatory networks that control transcription elongation at the rDNA locus remain poorly defined. We have discovered a new regulatory checkpoint that monitors transcription perturbations at the rDNA and is resolved by the degradation of the Pol I catalytic subunit RPA194. This checkpoint is activated by our newly identified chemical inhibitor of Pol I (BMH-21). The goal of this application is to characterize this checkpoint by defining factors and molecular mechanism(s) by which it is activated. Furthermore, this work will identify the molecular basis for the degradation of the enzyme, and the reciprocal regulation of the Pols I and II at the rDNA locus. This proposal will pursue three primary goals: (1) Characterize factors that monitor Pol I elongation; (2) Define how the Pol I elongation checkpoint is activated and enforced and how it impacts Pol II; and (3) Identify ubiquitin-proteasome system components that mediate the degradation of RPA194. To achieve these goals, we will pursue three specific aims. In these aims, BMH-21 and other epigenetic drugs will be used as tools to study the polymerase-selective checkpoint. Aim 1 characterizes factors identified in yeast and mammalian genome-wide screens and specifies how they regulate Pol I transcription. Aim 2 implements biochemically defined transcription assays and genomic analyses to identify how Pol I transcription perturbations activate the checkpoint, and its impact on polymerase selectivity. Aim 3 defines the roles of ubiquitin-proteasome system factors in controlling the stability and transcription of Pol I. In all, these studies provide unprecedented insight into the regulation of Pol I transcription and the reorganization of nuclear transcription upon inactivation of Pol I. An in-depth, mechanistic understanding of Pol I transcription regulation will support strategies seeking therapeutic control of deregulated rRNA synthesis in human disease.