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 拟议的研究的目的是定义和表征编码的长双链RNA（DSRNA） 并在包括人类在内的动物中表达，以及结合，修饰和处理该dsRNA的蛋白质。钥匙， 与健康相关的，重点是这种内源性dsRNA如何与长期识别的长病毒dsRNA区分开 陌生以引发先天的免疫响应。最近的研究表明，在脊椎动物和无脊椎动物中， RNA编辑酶称为腺苷死亡，作用于RNA或ADARS，脱氨酸内源性 dsRNA，因此不会引发异常的免疫反应。 Adars和编辑的机制 他们创建的站点，排除先天免疫反应的激活尚不清楚，拟议的研究是 旨在填补知识的差距。使用遗传筛选和分子方法，模型有机体。 秀丽隐杆线将用于发现RNA和蛋白质，导致缺乏ADAR的菌株的免疫响应。 生化方法将用于提供深入的机械见解。哺乳动物使用干扰素 安装抗病毒反应的途径，无脊椎动物缺乏此途径，而是使用RNA干扰 （RNAi）抗病毒防御。酶DICER是抗病毒RNAi途径的关键，对于分裂至关重要 无脊椎动物免疫反应期间的病毒dsRNA。我们先前的体外研究表明DICER的解旋酶结构域 将病毒dsRNA的末端识别为“非自然”或外国，而拟议的研究旨在测试这一点 体内。无脊椎动物夹子的解旋酶结构域是一种引人入胜的分子运动和生物化学，瞬态 动力学分析和结构生物学将用于了解它如何坐标DSRNA裂解和 最终将小的RN​​A产物传递到下游因子，从而使RNAi途径的基因沉默。 将研究通过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dicer和无脊椎动物夹子的解旋酶结构域活动的差异 了解这种酶如何演变为脊椎动物和无脊椎动物的免疫病变。