Mechanistic analyses of a novel RNA polymerase I transcription checkpoint

摘要 细胞的生长和增殖速率与核糖体生物合成速率成正比。这 这种关系在癌症中得到强调，其中对核糖体合成的持续需求是 普遍存在。核糖体生物合成的第一步是核糖体（r）DNA被RNA转录 聚合酶I（Pol I）;在癌症中，该过程被解除管制。尽管有这些既定的联系 核糖体生物合成和癌细胞增殖之间的调节网络， rDNA基因座上的转录延伸仍然不清楚。 我们已经发现了一个新的调控检查点，可以监控rDNA的转录扰动， 并通过Pol I催化亚基RPA 194的降解来解决。此检查点已激活 我们新鉴定的Pol I化学抑制剂（BMH-21）。此应用程序的目标是 通过定义因子和激活该检查点的分子机制来表征该检查点。 此外，这项工作将确定酶降解的分子基础， 在rDNA基因座上Pol I和II的相互调节。 该建议将追求三个主要目标：（1）表征监测Pol I延伸的因素; (2)定义Pol I延长检查点如何被激活和实施，以及它如何影响Pol II;以及 (3)确定介导RPA 194降解的泛素-蛋白酶体系统组分。到 为达致这些目标，我们会致力达致三个具体目标。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鉴定，并说明它们如何调节Pol I 转录。目的2实现生物化学定义的转录测定和基因组分析， 确定Pol I转录扰动如何激活检查点及其对聚合酶的影响 选择性目的3明确了泛素-蛋白酶体系统因子在控制稳定性中的作用 以及Pol I的转录。总之，这些研究为Pol I的调节提供了前所未有的见解。 转录和Pol I失活后的核转录重组。一个深入， 对Pol I转录调控机制的理解将支持寻求治疗的策略。 控制人类疾病中rRNA合成失调。

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.