Elucidating roles and mechanisms of double-stranded RNA-mediated pathways

阐明双链RNA介导途径的作用和机制

• 批准号: 10189022
• 负责人: Brenda L. Bass
• 金额: $ 55.23万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10189022
• 关键词: Animal Model; Animals; Antiviral Agents; Antiviral Response; Binding Proteins; Biochemical; Biochemistry; Caenorhabditis elegans; Cleaved cell; Dicer Enzyme; Double-Stranded RNA; Enzymes; Gene Silencing; Genetic Screening; Goals; Health; Human; Immune; Immune response; In Vitro; Innate Immune Response; Interferons; Invertebrates; Kinetics; Knowledge; Lead; Mammals; Mediating; Molecular; Molecular Motors; Pathway interactions; Process; Proteins; RNA; RNA Editing; RNA Interference; RNA Interference Pathway; Research; Role; Site; Small RNA; Vertebrates; Viral; design; dsRNA adenosine deaminase; fascinate; helicase; in vivo evaluation; insight; structural biology

SUMMARY/ABSTRACT The goal of the proposed research is to define and characterize the long double-stranded RNA (dsRNA) encoded and expressed in animals, including humans, and the proteins that bind, modify, and process this dsRNA. A key, health-related, focus is how this endogenous dsRNA is discriminated from the long viral dsRNA that is recognized as foreign to trigger an innate immune response. Recent studies show that in both vertebrates and invertebrates, the RNA editing enzymes called Adenosine deaminases that act on RNA, or ADARs, deaminate endogenous dsRNA so that it will not trigger an aberrant immune response. The mechanism by which ADARs, and the editing sites they create, preclude activation of an innate immune response is unclear, and the proposed research is designed to fill this gap in knowledge. Using genetic screens and molecular approaches, the model organism C. elegans will be used to discover RNAs and proteins that lead to an immune response in strains lacking ADARs; biochemical approaches will be used to provide in-depth mechanistic insights. While mammals use the interferon pathway to mount an antiviral response, invertebrates lack this pathway, and instead, use RNA interference (RNAi) in antiviral defense. The enzyme Dicer is key to the antiviral RNAi pathway and is essential for cleaving viral dsRNA during the invertebrate immune response. Our prior in vitro studies indicate Dicer's helicase domain recognizes the ends of viral dsRNA as “nonself”, or foreign, and the proposed studies are designed to test this in vivo. The helicase domain of invertebrate Dicers is a fascinating molecular motor, and biochemistry, transient kinetic analyses, and structural biology, will be used to understand how it coordinates dsRNA cleavage, and ultimately passes small RNA products to downstream factors that enable gene silencing by the RNAi pathway. Modulation of Dicer's activity by accessory factors that interact with the helicase domain will be investigated. Differences in activities of the helicase domain of human Dicer and invertebrate Dicers will be explored to understand how this enzyme evolved as the immune pathways of vertebrates and invertebrates diverged.

摘要/摘要 拟议研究的目标是定义和表征长双链 RNA (dsRNA) 编码的 并在包括人类在内的动物中表达，以及结合、修饰和加工该 dsRNA A key 的蛋白质。 与健康相关，重点是如何将这种内源性 dsRNA 与已识别的长病毒 dsRNA 区分开来 最近的研究表明，在脊椎动物和无脊椎动物中， RNA 编辑酶（称为腺苷脱氨酶）作用于 RNA（或 ADAR），使内源性脱氨 dsRNA，使其不会触发异常的免疫反应 ADAR 的机制以及编辑。 他们创建的位点，阻止先天免疫反应的激活尚不清楚，拟议的研究是 旨在利用遗传筛选和分子方法填补这一知识空白，模型生物 C. 线虫将被用来发现 RNA 和蛋白质，从而在缺乏 ADAR 的菌株中产生免疫反应； 当哺乳动物使用干扰素时，生化方法将用于提供深入的机制见解。 产生抗病毒反应的途径，无脊椎动物缺乏该途径，而是使用 RNA 干扰 (RNAi) 抗病毒防御中的酶 Dicer 是抗病毒 RNAi 途径的关键，对于裂解至关重要。 我们之前的体外研究表明 Dicer 的解旋酶结构域。 将病毒 dsRNA 的末端识别为“非自身”或外源，拟议的研究旨在测试这一点 无脊椎动物 Dicers 的解旋酶结构域是一种令人着迷的分子马达，并且在生物化学中是短暂的。 动力学分析和结构生物学将用于了解它如何协调 dsRNA 切割，以及 最终将小RNA产物传递给下游因子，从而通过RNAi途径实现基因沉默。 将研究与解旋酶结构域相互作用的辅助因子对 Dicer 活性的调节。 将探索人类 Dicer 和无脊椎动物 Dicer 解旋酶结构域活性的差异 了解这种酶如何随着脊椎动物和无脊椎动物的免疫途径的分歧而进化。