Mechanistic analyses of a novel RNA polymerase I transcription checkpoint

新型RNA聚合酶I转录检查点的机制分析

• 批准号: 9979913
• 负责人: Marikki Laiho
• 金额: $ 47.95万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979913
• 关键词: Acute; Address; Affect; Anabolism; Animal Model; Biochemical; Biogenesis; Biological Assay; Biology; Catalytic Domain; Cell Proliferation; Cell Survival; Cell model; Cells; Cellular biology; Chemicals; Chromatin Remodeling Factor; Clinical; Complex; DNA; DNA-Directed RNA Polymerase; Data; Development; Developmental Biology; Disease; Ensure; Enzymes; Epigenetic Process; Equilibrium; Eukaryotic Cell; Foundations; Future; Gene Expression; Gene-Modified; Genetic Transcription; Genomics; Goals; Growth; Health; Histone Acetylation; Human; Knowledge; Learning; Malignant Neoplasms; Mammalian Cell; Mammals; Mediating; Molecular; Monitor; Nuclear; Oncogenic; Pharmacology; Polymerase; Predisposition; Process; Property; Proteolysis; RNA; RNA Polymerase I; RNA interference screen; Recombinant DNA; Regulation; Ribosomal DNA; Ribosomal RNA; Ribosomes; Role; Signal Transduction; Specific qualifier value; Stress; System; Testing; Therapeutic; Therapeutic Intervention; Transcription Elongation; Transcription Initiation; Transcriptional Regulation; Treatment Efficacy; Ubiquitin; Ubiquitin-mediated Proteolysis Pathway; Work; Yeasts; base; cancer cell; cancer therapy; cell growth; epigenetic drug; genome wide screen; genome-wide; histone methylation; human disease; inhibitor/antagonist; insight; mammalian genome; multicatalytic endopeptidase complex; new therapeutic target; novel; pre-clinical; response; small molecule; targeted treatment; therapeutic target; tool; ubiquitin-protein ligase; yeast genome

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.

抽象的 细胞的生长和增殖速率与核糖体生物合成速率成正比。这 癌症中强调了这种关系，其中对核糖体合成的持续需求是 流行。核糖体生物合成的第一步是通过 RNA 转录核糖体 (r) DNA 聚合酶 I (Pol I)；在癌症中，这个过程不受控制。尽管有这些已建立的联系 核糖体生物合成和癌细胞增殖之间的调控网络 rDNA 基因座的转录延伸仍然不明确。 我们发现了一个新的监管检查点，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。该检查点已激活 由我们新发现的 Pol I 化学抑制剂 (BMH-21) 产生。该应用程序的目标是 通过定义激活该检查点的因素和分子机制来表征该检查点。 此外，这项工作将确定酶降解的分子基础，以及 rDNA 基因座上 Pols I 和 II 的相互调节。 该提案将追求三个主要目标：（1）描述监测 Pol I 伸长的因素； (2) 定义 Pol I 伸长检查点如何激活和执行以及它如何影响 Pol II；和 (3) 鉴定介导 RPA194 降解的泛素-蛋白酶体系统组件。到 为了实现这些目标，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 药物将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详细说明它们如何调节 Pol I 转录。目标 2 实施生化定义的转录测定和基因组分析，以 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。目标 3 定义了泛素-蛋白酶体系统因素在控制稳定性中的作用 总而言之，这些研究为 Pol I 的调控提供了前所未有的见解 转录和 Pol I 失活后核转录的重组。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。