Understanding the Mechanism of RNA Interference (RNAi)

描述(申请人提供)：十年前才发现的RNA干扰(RNAi)很可能是真核生物生命中最早的创新之一，早于真菌、植物和动物的分化。RNAi包括细胞对外源双链RNA(DsRNA)的反应--通过实验传递或在病毒感染期间产生--以及对寄生遗传元件的反应，如转座子和高度重复的序列。我们试图了解RNAi机制的生化细节如何反映RNAi的这些功能。我们的目标是以果蝇和哺乳动物细胞为模型，了解RNAi在人类中的生化基础和生物学逻辑。当然，更好地理解RNAi的生化基础将导致更好的RNAi实验工具。这些工具为有效的体细胞遗传学提供了基础，并已成为学术和药物基因发现的重要组成部分。了解RNAi的分子细节也将有助于揭示在自私的遗传元件存在的情况下，基因组是如何保持稳定的，而自私的遗传元件约占人类基因组的一半。体细胞遗传稳定性的丧失会直接导致癌症，在生殖系中，还会导致出生缺陷。我们实验策略的核心是使用黑腹果蝇作为模型系统，因为果蝇结合了出色的遗传、生化和表型工具，我们将使用培养的小鼠和人类细胞将我们在果蝇身上的发现扩展到哺乳动物。在果蝇和哺乳动物中，RNAi都是由双链RNA特异内切酶DICER家族的成员将长的双链RNA转化为小干扰RNA(SiRNAs)而启动的。我们将使用定量生化和分子工具来破译不同的Dird酶催化siRNA合成的机制。我们将努力理解为什么一些Dier酶的活性需要ATP，而另一些不需要，为什么只有一些似乎是过程性的，以及为什么特定的Dier酶被限制为产生离散的小RNA类别。DICER产生的siRNAs在RISC中发挥作用，RISC是介导RNAi功能的蛋白质-siRNA复合体。我们将致力于了解细胞如何将siRNAs加载到RISC的核心成分ArgAerte蛋白质中。一种复杂的RISC组装细胞途径开始从生化和遗传学实验中浮现出来。这条途径的生物学目的是什么？小RNA和ArgAerte蛋白的功能多样化是否反映了这些分子的不同生物学作用？我们还将继续努力定义RISC是如何工作的，特别强调它作为RNA指导的RNA内切酶的作用。最后，我们将把注意力转向了解重复相关小RNA(RasiRNAs)的起源、机制和功能，这是一类知之甚少的小RNA，负责沉默转座子和重复序列，从而确保基因组的稳定性。

DESCRIPTION (provided by applicant): Discovered just a decade ago, RNA interference (RNAi) is likely one of the earliest innovations of eukaryotic life, pre-dating the divergence of fungi, plants, and animals. RNAi encompasses the cellular response to exogenous double-stranded RNA (dsRNA) - delivered experimentally or generated during a viral infection - and the response to parasitic genetic elements, such as transposons and highly repeated sequences. We seek to understand how the biochemical details of the RNAi mechanism reflect these functions of RNAi. Our goal is to understand the biochemical basis and biological logic of RNAi in humans, using Drosophila and mammalian cells as models. A better understanding of the biochemical basis for RNAi will, of course, lead to better experimental RNAi tools. Such tools provide the underpinnings of effective somatic genetics and have become an essential part of academic and pharmaceutical gene discovery. Understanding the molecular details of RNAi will also help reveal how genomic stability is maintained in the presence of selfish genetic elements, which compose about half the human genome. Loss of genetic stability in the soma can lead directly to cancer, and, in the germ line, to birth defects. The core of our experimental strategy is to use Drosophila melanogaster as a model system, because flies combine outstanding genetic, biochemical, and phenotypic tools, and we will extend our findings in flies to mammals, using cultured mouse and human cells. In both flies and mammals, RNAi is initiated by the conversion of long double-stranded RNA into small interfering RNAs (siRNAs) by members of the Dicer family of double-stranded RNA-specific endonucleases. We will use quantitative biochemical and molecular tools to decipher the mechanism by which distinct Dicer enzymes catalyze siRNA synthesis. We will work to understand why some Dicer enzymes require ATP for activity whereas others do not, why only some appear to be processive, and why specific Dicer enzymes are restricted to produce discrete classes of small RNAs. The siRNAs produced by Dicer function in RISC, the protein-siRNA complex that mediates the functions of RNAi. We will work to understand how cells load siRNAs into Argonaute proteins, the core constituents of RISC. An elaborate cellular pathway for RISC assembly is beginning to emerge from biochemical and genetic experiments. What is the biological purpose of this pathway? Does functional diversification of small RNAs and of Argonaute proteins reflect discrete biological roles for these molecules? We will also continue our efforts to define how RISC works, with particular emphasis on its role as an RNA-directed RNA endonuclease. Finally, we will turn our attention to understanding the origins, mechanisms, and functions of repeat-associated small RNAs (rasiRNAs), the poorly understood, small RNA class responsible for silencing transposons and repetitive sequences, thereby ensuring genomic stability.