RNA interference: Does R2D2 witness Dicing?

描述(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因素。这一调控体系确保了基因的稳定性。胞体中RNAi的缺失可能导致癌症；生殖系中的RNAi缺失可能导致出生缺陷。在果蝇中，DICER-2和R2D2蛋白对于RNA干扰(RNAi)途径是必不可少的。这些蛋白质在体外形成稳定的异源二聚体，而在体内没有Dever-2，R2D2是不稳定的。然而，体外分析表明，在没有R2D2的情况下，DICER-2可以执行其功能之一-切割dsRNA。本研究的目的是确定这两种蛋白质相互结合所需的氨基酸，以便设计突变果蝇，在这些突变果蝇中，我可以测试R2D2在切割过程中是否必须与DICER-2结合，或者更确切地说，必须只出现在生产活性RNAi酶复合体的后续步骤中。为此，我建议使用反向酵母双杂交筛选法来筛选不能与R2D2结合的Dice-2突变体。我将进行定量的生化测试，以确定一个保持其将长的双链RNA(DsRNA)切割成小干扰RNA(SiRNAs)的突变体，siRNAs是指导RNAi的指南。我将产生两种形式的转基因果蝇：一种是不再与R2D2结合但能够切割dsRNA的Dice-2的突变形式，另一种是现有的结合R2D2但切割dsRNA的能力减弱的突变体Dice-2。利用已建立的技术，我将研究这些果蝇，以确定当将dsRNA加工成siRNAs和将siRNAs装入功能复合体去耦合时，是否会在活的果蝇中发生RNAi。我还将使用包括定量RT-PCR和平铺微阵列在内的分子工具来监测通常被沉默的遗传元件的水平，并对这些苍蝇中的小RNA群体进行高通量测序，以确定分离这些过程在体内的后果。

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.