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: 10594483
• 负责人: Brenda L. Bass
• 金额: $ 60.39万
• 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/10594483
• 关键词: Animals; Antiviral Response; Binding Proteins; Biochemical; Biochemistry; Caenorhabditis elegans; Deamination; Dicer Enzyme; Double-Stranded RNA; Enabling Factors; Enzymes; Gene Silencing; Genetic Screening; Goals; Health; Human; Immune; Immune response; In Vitro; Innate Immune Response; Interferons; Invertebrates; Kinetics; Knowledge; 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; model organism; structural biology; viral RNA

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 这项拟议研究的目标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、修饰和处理这种dsRNA的蛋白质。一把钥匙， 与健康相关，焦点是这种内源性dsRNA如何与公认的长病毒dsRNA区分开来 因为是外来的才能触发先天免疫反应。最近的研究表明，在脊椎动物和无脊椎动物中， RNA编辑酶称为腺苷脱氨酶，作用于RNA或ADAR，使内源脱氨 DsRNA，这样它就不会引发异常的免疫反应。ADAR的机制，以及编辑 他们创建的阻止激活先天免疫反应的部位尚不清楚，拟议的研究是 旨在填补这一知识空白。利用遗传筛选和分子方法，模式生物C. 秀丽基因将被用来发现在缺乏ADAR的菌株中导致免疫反应的RNA和蛋白质； 生物化学方法将被用来提供深入的机械性见解。当哺乳动物使用干扰素时 无脊椎动物缺乏这条途径，而是使用RNA干扰 (RNAi)在抗病毒防御中。Dester酶是抗病毒RNAi途径的关键，对切割是必不可少的 病毒dsRNA在无脊椎动物免疫应答中的作用。我们先前的体外研究表明，迪格尔的解旋酶结构域 识别病毒dsrna的末端是“非我”或外来的，拟议的研究旨在测试这一点。 在活体内。无脊椎动物骰子的解旋酶结构域是一个迷人的分子马达，生物化学，瞬间 动力学分析和结构生物学将被用来理解它是如何协调dsRNA切割的，以及 最终将小RNA产物传递给下游因子，使RNAi途径能够使基因沉默。 将研究与解旋酶结构域相互作用的辅助因子对迪格尔活性的调节。 我们将探索人类DICER和无脊椎动物DICR解旋酶区域活性的差异 了解这种酶是如何随着脊椎动物和无脊椎动物免疫途径的分化而进化的。