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

描述（由申请人提供）：RNA衰变是人类细胞中基因调控的关键机制，控制RNA丰度和对应激的反应。本提案的目标是确定由普遍存在的人蛋白激酶/核糖核酸内切酶RNase L介导的应激激活的RNA衰变的分子基础（Aim 1和Aim 2），并建立工具以克服该途径研究中的相当大的实验限制（Aim 3）。 RNase L激活干扰素和细胞因子的产生，并抑制广谱的人类病毒，因此大量的研究集中在其抗病毒作用上。RNase L是前列腺癌中的肿瘤抑制基因，并且是脂肪细胞分化和凋亡的重要介质，使其成为用于诊断和治疗大量人类疾病的有吸引力的靶标。然而，这些应用的一部分挑战，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有限知识。我们的建议旨在通过获得负责RNA切割的关键蛋白质和蛋白质复合物的详细结构和分子理解来解决这些限制。 该建议的重点是RNase L的结构和生物化学的研究和两个非传统的人类RNA聚合酶OAS 1和OAS 3所需的RNase L的激活。在我们最近的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识别领域的良好记录，使我们的实验室成为RNase L介导的RNA衰变的结构和功能分析的理想场所。我们提出了三个独立但互补的具体目标。 在本提案的目的1中，我们将阐明OAS 1和OAS 3合成2-5A的分子机制，2-5A是RNA酶L激活RNA衰变所需的特异性辅因子。这项工作是重要的，因为它将奠定基础， 研究2-5A的合成机制和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。这项工作将提供一个详细的分子理解独特的蛋白激酶/RNase驱动应力激活的RNA衰变在人类细胞。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地检测2-5A的合成。这项工作将消除该领域中由于缺乏特异性和非侵入性地监测RNaseL途径激活的工具而造成的重大障碍。我们工作的长期目标将是通过理解该途径中分子组分的结构、功能和调节来理解RNase L介导的RNA衰变的机制。

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.