Structural and Functional Bases of Stress-Activated RNA Decay Mediated by RNase L

RNase L 介导的应激激活 RNA 衰变的结构和功能基础

• 批准号: 9267489
• 负责人: Alexei Korennykh
• 金额: $ 30.13万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9267489
• 关键词: 2-5A Synthetase; Active Sites; Address; Ankyrin Repeat; Antiviral Agents; Apoptosis; Automobile Driving; Binding; Biochemical; C-terminal; Cells; Complex; Crystallization; DNA-Directed RNA Polymerase; Detection; Diagnostic; Dimerization; Disease; Double-Stranded RNA; Endoribonucleases; Engineering; Family; Foundations; Gene Expression Regulation; Goals; Hela Cells; Hepatitis C; Human; In Vitro; Inflammation Mediators; Inherited; Interferons; Knowledge; Laboratories; Length; Light; Link; Luciferases; Malignant neoplasm of prostate; Mediating; Mediator of activation protein; Molecular; Molecular Structure; Monitor; N-terminal; Obesity; Pathway interactions; Positioning Attribute; Production; Protein Kinase; Proteins; Publications; RNA; RNA Decay; RNA analysis; Regulation; Reporter; Research; Ribonucleases; Role; Signal Transduction; Signaling Protein; Source; Specificity; Stress; Structure; Technology; Testing; Tumor Suppressor Genes; Viral Cancer; Virus; Virus Diseases; West Nile virus; Work; adipocyte differentiation; base; cofactor; cytokine; dimer; human disease; in vivo; member; oligoadenylate; protein complex; protein structure; prototype; public health relevance; response; sensor; therapeutic target; tool

DESCRIPTION (provided by applicant): RNA decay is a key mechanism of gene regulation in human cells, controlling RNA abundance and responses to stress. The goal of this proposal is to determine the molecular basis for stress- activated RNA decay mediated by the ubiquitous human protein kinase/endoribonuclease RNase L (Aim 1 and Aim 2) and to build tools to overcome the considerable experimental limitations in studies of this pathway (Aim 3). RNase L activates the production of interferons and cytokines, and inhibits a broad spectrum of human viruses, so a large body of research focuses on its antiviral effects. RNase L is a tumor suppressor gene in prostate cancer and is an essential mediator of adipocyte differentiation and apoptosis, making it an attractive target for diagnostics and treatment of a large number of human diseases. However, a part of the challenge with these applications, and a major challenge in understanding the RNase L-mediated RNA decay, is our limited knowledge of the molecular structures and mechanisms controlling this pathway. Our proposal aims to address these limitations by obtaining a detailed structural and molecular understanding of the key proteins and protein complexes responsible for RNA cleavage. This proposal is focused on structural and biochemical studies of RNase L and of two unconventional human RNA polymerases OAS1 and OAS3 required for activation of RNase L. During preliminary work described in our recent publications, we determined several crystal structures of these proteins or their domains. This progress and our established track record in the field of signal transduction and protein/RNA recognition position our laboratory ideally for structural and functional analysis of RNA decay mediated by RNase L. We propose three independent but complementary specific aims. In Aim 1 of this proposal we will elucidate the molecular mechanism by which OAS1 and OAS3 synthesize 2-5A, a specific cofactor of RNase L required for activation of RNA decay. This work is important because it will lay the foundation for rational studies of the machinery for synthesis of 2-5A and for therapeutic targeting of this machinery in diseases. In Aim 2 we will determine the crystal structure of RNase L. This work will provide a detailed molecular understanding of the unique protein kinase/RNase driving stress-activated RNA decay in human cells. In Aim 3, we will engineer light-emitting reporters of 2-5A, which will enable rapid and specific detection of 2-5A synthesis in vitro and in vivo. This work will eliminat a significant obstacle in the field due to the lack of tools to monitor the activation of the RNaseL pathway specifically and non-invasively. The long-term goal of our work will be to understand the mechanism of RNase L-mediated RNA decay by understanding structures, functions, and regulation of the molecular components in this pathway.

描述（由申请人提供）：RNA 衰减是人类细胞中基因调控的关键机制，控制着 RNA 丰度和对应激的反应。该提案的目标是确定由普遍存在的人类蛋白激酶/核糖核酸内切酶 RNase L 介导的应激激活 RNA 衰变的分子基础（目标 1 和目标 2），并构建工具来克服该途径研究中相当大的实验限制（目标 3）。 RNase L 可激活干扰素和细胞因子的产生，并抑制多种人类病毒，因此大量研究都集中在其抗病毒作用上。 RNase L 是前列腺癌中的抑癌基因，是脂肪细胞分化和凋亡的重要介质，使其成为诊断和治疗大量人类疾病的有吸引力的靶点。然而，这些应用面临的部分挑战，以及理解 RNase L 介导的 RNA 衰变的主要挑战，是我们对控制该途径的分子结构和机制的了解有限。我们的建议旨在通过对负责 RNA 切割的关键蛋白质和蛋白质复合物获得详细的结构和分子了解来解决这些限制。 该提案的重点是 RNase L 以及激活 RNase L 所需的两种非常规人类 RNA 聚合酶 OAS1 和 OAS3 的结构和生化研究。在我们最近出版物中描述的初步工作中，我们确定了这些蛋白质或其结构域的几种晶体结构。这一进展以及我们在信号转导和蛋白质/RNA 识别领域的既定记录使我们的实验室成为对 RNase L 介导的 RNA 衰变进行结构和功能分析的理想选择。我们提出了三个独立但互补的具体目标。 在本提案的目标 1 中，我们将阐明 OAS1 和 OAS3 合成 2-5A 的分子机制，2-5A 是激活 RNA 衰变所需的 RNase L 的特定辅因子。这项工作很重要，因为它将为理性奠定基础 研究 2-5A 合成机制以及该机制在疾病中的治疗靶向。在目标 2 中，我们将确定 RNase L 的晶体结构。这项工作将为驱动人类细胞中应激激活的 RNA 衰变的独特蛋白激酶/RNase 提供详细的分子理解。在目标 3 中，我们将设计 2-5A 的发光报告基因，这将能够在体外和体内快速、特异性地检测 2-5A 的合成。这项工作将消除该领域由于缺乏专门且非侵入性监测 RNaseL 通路激活的工具而存在的重大障碍。我们工作的长期目标是通过了解 RNase L 介导的 RNA 衰减的结构、功能和该途径中分子成分的调节来了解该机制。