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

总结/摘要 这项研究的目的是定义和表征编码长双链RNA（dsRNA）的基因。 并在包括人类在内的动物中表达，以及结合、修饰和加工这种dsRNA的蛋白质。一把钥匙 与健康相关的，重点是如何将这种内源性dsRNA与被识别的长病毒dsRNA区分开来， 来引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， dsRNA，使其不会引发异常的免疫反应。ADAR的机制，以及编辑 他们创建的网站，阻止先天免疫反应的激活尚不清楚，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生物学方法，模式生物C。 elegans将用于发现在缺乏ADAR的菌株中导致免疫应答的RNA和蛋白质; 生物化学方法将用于提供深入的机制见解。哺乳动物使用干扰素 无脊椎动物缺乏这种途径，而是使用RNA干扰 （RNAi）在抗病毒防御中的作用。酶Dicer是抗病毒RNAi途径的关键， 无脊椎动物免疫反应期间的病毒双链RNA。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“非自身”或外来的，并且所提出的研究旨在测试这一点。 in vivo.无脊椎动物Dicers的解旋酶结构域是一个迷人的分子马达， 动力学分析和结构生物学将用于了解它如何协调dsRNA切割， 最终将小RNA产物传递给下游因子，从而通过RNAi途径使基因沉默。 将研究与解旋酶结构域相互作用的辅助因子对Dicer活性的调节。 将探索人类Dicer和无脊椎动物Dicer的解旋酶结构域活性的差异， 了解这种酶是如何随着脊椎动物和无脊椎动物的免疫途径的分化而进化的。

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.