Molecular Mechanism of Small RNA-induced RNA Silencing

描述（由申请人提供）：我们的长期目标是了解RNA干扰（RNAi）的分子和生化机制，RNAi是一种由microRNA（miRNA）和小干扰RNA（siRNA）介导的保守的转录后基因沉默机制。这些微小的调控RNA在许多基本的生物过程中起着关键作用。特别是，miRNAs的错误调节最近与人类疾病如癌症有关。此外，RNAi已被广泛用作一种强大的基因沉默工具，在多个模型系统中进行功能基因组研究。RNAi的特异性和效力突出了其开发新的和有效的治疗途径来治疗人类疾病的潜力。我们以前已经确定了两个Dicer复合物，DCR-1/R3 D1和DCR-2/R2D2，作为果蝇中各自的miRNA和siRNA生成酶。此外，DCR-2/R2D2复合物结合新生siRNA并促进其掺入siRNA诱导的沉默复合物（siRISC）中。在这些研究的基础上，本提案的目标是解决果蝇RNAi途径中的几个突出问题，重点是miRNA途径。我们将研究miRNA生物合成的内在特异性和机制（目的1）。我们将测试我们的假设，即DCR-1/R3 D1复合物感知miRNA的不对称性，并促进miRNA加载到其效应复合物miRISC上（Aim 2）。最后，我们将开发一种重建试验，以探索miRISC组装的机制，并鉴定和表征miRISC组装的新组分（目的3）。这些研究将大大推进我们对果蝇RNAi途径的理解。人类疾病如癌症的起源通常可归因于重要基因（肿瘤抑制基因）的功能丧失和/或病理基因（癌基因）的功能获得。由于未知的原因，RNAi在人类细胞中的效率低于RNAi在果蝇细胞中的效率。因此，果蝇和人类系统的比较将使我们能够识别潜在的差异，并有望优化人类细胞中的RNAi。这可能导致开发新的和有效的基于小RNA的基因沉默方法，通过特异性关闭病理基因来治疗人类疾病。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular and biochemical mechanisms of RNA interference (RNAi), a conserved post-transcriptional gene-silencing mechanism mediated by microRNA (miRNA) and small interfering RNA (siRNA). These tiny regulatory RNAs play critical roles in many fundamental biological processes. In particular, misregulation of miRNAs has recently been linked to human diseases, such as cancer. Moreover, RNAi has been widely used as a powerful gene-silencing tool to perform functional genomic studies in multiple model systems. The specificity and potency of RNAi highlight its potential for developing novel and efficient therapeutic avenues to treat human diseases. We have previously identified two Dicer complexes, DCR-1/R3D1 and DCR-2/R2D2, as the respective miRNA- and siRNA-generating enzymes in Drosophila. In addition, the DCR-2/R2D2 complex binds nascent siRNA and facilitates its incorporation into the siRNA-induced silencing complex (siRISC). Based on these studies, the goal of this proposal is to address several outstanding questions in the Drosophila RNAi pathway with an emphasis on the miRNA pathway. We will investigate the inherent specificity and mechanism of miRNA biogenesis (Aim 1). We will test our hypothesis that the DCR-1/R3D1 complex senses asymmetry of miRNA and facilitates miRNA loading onto its effector complex, miRISC (Aim 2). Finally, we will develop a reconstitution assay to explore the mechanism of miRISC assembly and to identify and characterize new components of miRISC assembly (Aim 3). These studies will significantly advance our understanding of the Drosophila RNAi pathway. The origins of human diseases, such as cancer, can be generally attributed to loss-of-function of important genes (tumor suppressor genes) and/or gain-of-function of pathological genes (oncogenes). For unknown reasons, RNAi in human cells is less efficient than RNAi in Drosophila cells. Thus, comparison of the Drosophila and human systems will allow us to identify the underlying differences and, hopefully, to optimize RNAi in human cells. This could lead to the development of novel and efficient small RNAs-based gene-silencing methods to cure human diseases by specifically shutting down pathological genes.