RNA interference: Does R2D2 witness Dicing?

描述（由申请人提供）：RNA干扰（RNAi）是一种进化保守的细胞防御外来或寄生遗传元件。这个调控系统确保了基因的稳定性。胞体中RNAi的缺失可能导致癌症；生殖系中RNAi的缺失可能导致出生缺陷。在黑腹果蝇中，Dicer-2和R2D2蛋白是RNA干扰（RNAi）途径所必需的。这些蛋白在体外形成稳定的异源二聚体，没有Dicer-2， R2D2在体内是不稳定的。然而，体外分析表明，Dicer-2可以在没有R2D2的情况下发挥其功能之一——切割dsrna。本研究的目标是确定这两种蛋白质相互结合所需的氨基酸，从而设计突变果蝇，我可以在其中测试R2D2是否必须在切割过程中与Dicer-2结合，或者必须仅在生产活性RNAi酶复合物的后续步骤中存在。为此，我建议使用反向酵母双杂交筛选筛选不能结合R2D2的Dicer-2突变体。我将进行定量生化测试，以确定一种突变体，它保留了将长双链RNA （dsRNA）切割成小干扰RNA （sirna）的能力，sirna是指导RNAi的向导。我将产生具有两种形式的Dicer-2的转基因果蝇：不再结合R2D2但能够切割dsRNA的突变形式的Dicer-2，以及现有的结合R2D2但切割dsRNA能力减弱的突变Dicer-2。使用已建立的技术，我将检查这些果蝇，以确定当dsRNA加工成sirna和sirna装载到功能复合物中去耦时，RNAi是否会在活的果蝇中发生。我还将使用分子工具，包括定量RT-PCR和平片微阵列，来监测通常沉默的遗传元素的水平，并对这些果蝇的小RNA种群进行高通量测序，以确定分离这些过程的体内后果。

DESCRIPTION (provided by applicant): RNA interference (RNAi) is an evolutionarily conserved cellular defense against foreign or parasitic genetic elements. This regulatory system ensures genetic stability. Loss of RNAi in the soma may lead to cancer; loss of RNAi in the germ line may cause birth defects. In Drosophila melanogaster, the proteins Dicer-2 and R2D2 are essential for the RNA interference (RNAi) pathway. These proteins form a stable heterodimer in vitro, and without Dicer-2, R2D2 is unstable in vivo. Yet in vitro analysis indicates that Dicer-2 can carry out one of its functions-cleaving dsRNA-in the absence of R2D2. The goal of this study is to identify the amino acids required for the binding of these two proteins to each other, so as to design mutant flies in which I can test whether R2D2 must be bound to Dicer-2 during dicing or, rather, must only be present at subsequent steps in the production of an active RNAi enzyme complex. To this end, I propose to use a reverse yeast two- hybrid screen to select mutants of Dicer-2 that are unable to bind R2D2. I will carry out quantitative biochemical tests to identify a mutant that retains its ability to cleave long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs), the guides that direct RNAi. I will generate transgenic flies that have two forms of Dicer-2: the mutant form of Dicer-2 that no longer binds R2D2 but is able to cleave dsRNA, as well as an existing mutant Dicer-2 that binds R2D2 but is diminished in its ability to cleave dsRNA. Using established techniques, I will examine these flies to determine whether RNAi can occur in the living fly when the processing dsRNA into siRNAs and the loading of the siRNAs into functional complexes are decoupled. I will also use molecular tools, including quantitative RT-PCR and tiling microarrays, to monitor the levels of genetic elements that are normally silenced, and high throughput sequencing of the small RNA populations in these flies to determine the in vivo consequences of separating these processes.