RNA Interference in Drosophila

描述(由申请人提供)：广泛的、长期的目标是从基因上剖析果蝇的RNA干扰，并识别参与这一重要过程的分子成分。双链RNA(Double-stranded RNA，dsRNA)能诱导强大的细胞免疫应答，对包括病毒、病毒复制中间体和转座元件在内的有害RNA具有重要的防御作用。在许多系统中，dsRNA触发RNA干扰(RNAi)；与dsRNA触发同源的转录本的戏剧性和序列特异性失稳。利用RNAi可以沉默正常的细胞基因，使这项技术成为阐明孤儿基因产物功能的重要新工具。尽管这种现象在不同的生物体中非常相似，但介导RNAi的确切机制并不完全清楚。遗传筛选已经确定了拟南芥、脉孢子虫和线虫中RNAi所需的几个重要成分。然而，这些筛查并没有饱和，只恢复了可行的突变。由于RNAi抑制的可变外显率，在果蝇中筛选RNAi缺陷突变体是不可能的。最近，我们用一种新的转基因设计解决了这个难题，这种设计可以有效地沉默成人组织中的基因。这些研究为这里提出的旨在阐明果蝇RNAi分子基础的遗传筛选奠定了基础。在特定的目标1中，我们将利用FLP/FRT重组系统分离出对眼睛颜色色素转运蛋白White的RNAi抑制有缺陷和增强的突变体。这种筛选的优点是：1.影响RNAi的突变很容易被鉴定为眼睛颜色突变，2.纯合子突变克隆将被限制在复眼，因此我们将恢复在其他系统中未发现的RNAi所需的活基因和潜在致命基因的突变，3.我们将恢复影响RNAi的抑制子和增强子突变。在特定的目标2中，我们将通过互补、缺失定位、突变候选基因的序列分析和生殖系转化拯救来确定我们所观察到的突变表型的相关基因。这些研究的完成将加强我们对果蝇RNAi的了解，并拓宽我们对RNAi的总体理解。RNAi突变体增强子的恢复可能为后基因组时代果蝇孤儿基因功能的研究提供更好的遗传背景。

DESCRIPTION (provided by applicant): The broad, long-term objective is to genetically dissect RNA interference in Drosophila and identify the molecular components mediating this important process. Double-stranded RNA (dsRNA) induces potent cellular responses that compose an essential defense against deleterious RNAs, including viruses, viral replication intermediates and transposable elements. In many systems, dsRNA triggers RNA interference (RNAi); a dramatic and sequence specific destabilization of transcripts homologous to the dsRNA trigger. Normal cellular genes can be silenced using RNAi making this technique an important new tool to elucidate function of orphan gene products. Although the phenomenon is remarkably similar in diverse organisms, the precise mechanisms mediating RNAi are not fully understood. Genetic screens have identified several important components required for RNAi in Arabidopsis, Neurospora and C. elegans. However, these screens have not been saturating and recovered only viable mutations. Mutant screens for RNAi defective mutants in Drosophila have not been possible because of variable penetrance of RNAi suppression. Recently we solved this difficulty with a novel transgene design that effectively silences genes in adult tissues. These studies set the groundwork for the genetic screen proposed here designed to elucidate the molecular basis for RNAi in Drosophila. In .Specific Aim 1 we will isolate mutants defective and enhanced for RNAi suppression of the eye color pigment transporter WHITE using the FLP/FRT recombination system. The advantages of this screen are: 1. mutants affecting RNAi will be easily identified as eye color mutants, 2. homozygous mutant clones will be restricted to the compound eye, therefore we will recover mutations in viable and in potentially lethal genes required for RNAi not identified in other systems, 3. We will recover both suppressor and enhancer mutations affecting RNAi. In Specific Aim 2 we will use complementation, deficiency mapping, sequence analysis of mutated candidate genes and germline transformation rescue to identify the genes responsible for the mutant phenotypes observed in our screen. Completion of the studies will enhance our understanding of RNAi in Drosophila and broaden our understanding of RNAi in general. Recovery of enhancer of RNAi mutants may provide enhanced genetic backgrounds to facilitate research on orphan gene function in Drosophila in the post-genomic era.