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

描述(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的丰度和对压力的反应。这项建议的目的是确定由普遍存在的人类蛋白激酶/核糖核酸酶L(目标1和目标2)介导的应激激活核糖核酸衰退的分子基础，并建立工具来克服这一途径研究中相当大的实验限制(目标3)。核糖核酸酶L激活干扰素和细胞因子的产生，并抑制广泛的人类病毒，因此大量研究集中在其抗病毒作用上。核糖核酸酶L是前列腺癌的抑癌基因，是脂肪细胞分化和凋亡的重要调节因子，是人类多种疾病的诊断和治疗靶点。然而，这些应用面临的部分挑战，以及理解核糖核酸酶L介导的核糖核酸衰变的主要挑战，是我们对控制这一途径的分子结构和机制的有限了解。我们的建议旨在通过获得对负责RNA切割的关键蛋白质和蛋白质复合体的详细结构和分子理解来解决这些限制。本研究主要对核糖核酸酶L及其激活所需的两种非常规人类核糖核酸聚合酶OAS1和OAS3进行了结构和生化研究。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分析RNaseL介导的RNA衰退的结构和功能的理想选择。我们提出了三个独立但互补的特定目标。在这项建议的目标1中，我们将阐明OAS1和OAS3合成2-5A的分子机制，2-5A是核糖核酸酶L激活核糖核酸衰变所需的一种特殊辅助因子。这项工作很重要，因为它将为Rational 2-5A的合成机制和该机制在疾病中的靶向治疗研究。在目标2中，我们将确定RNaseL的晶体结构。这项工作将提供对人类细胞中驱动应激激活RNA衰退的独特蛋白激酶/RNase的详细分子理解。在目标3中，我们将设计2-5A的发光报告，这将使快速和特异地检测2-5A的合成在体外和体内。这项工作将消除该领域的一个重大障碍，因为缺乏工具来专门和非侵入性地监测RNASEL通路的激活。我们工作的长期目标将是通过了解核糖核酸酶L介导的核糖核酸衰变的结构、功能和调节这一途径中的分子成分来了解该途径的机制。

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.