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

描述（申请人提供）：RNA衰变是人类细胞基因调控的关键机制，控制RNA丰度和应激反应。本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），并建立工具来克服该途径研究中相当大的实验限制（目标3）。RNase L激活干扰素和细胞因子的产生，并抑制广泛的人类病毒，因此大量的研究集中在它的抗病毒作用上。RNase L是前列腺癌中的肿瘤抑制基因，是脂肪细胞分化和凋亡的重要介质，使其成为许多人类疾病诊断和治疗的一个有吸引力的靶点。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，是我们对控制这一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。本研究的重点是RNase L和激活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。在我们最近发表的文章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个晶体结构。这一进展和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RNase l介导的RNA衰变的结构和功能分析的理想场所。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，因为它将奠定理性的基础

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.