Molecular Mechanism of Small RNA-induced RNA Silencing

描述(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发挥着关键作用。特别是，miRNAs的错误调控最近被认为与癌症等人类疾病有关。此外，RNAi作为一种强大的基因沉默工具已被广泛用于在多个模型系统中进行功能基因组研究。RNAi的特异性和有效性突出了它在开发治疗人类疾病的新的和有效的治疗途径方面的潜力。我们先前已经鉴定出两个DICER复合体，DCR-1/R3D1和DCR-2/R2D2，分别是果蝇中miRNA和siRNA产生酶。此外，DCR-2/R2D2复合体与新生的siRNA结合，并促进其进入siRNA诱导的沉默复合体(SiRISC)。在这些研究的基础上，本提案的目标是解决果蝇RNAi途径中的几个悬而未决的问题，重点是miRNA途径。我们将研究miRNA生物发生的内在特异性和机制(目标1)。我们将检验我们的假设，即DCR-1/R3D1复合体感知miRNA的不对称，并促进miRNA加载到其效应器复合体miRISC上(目标2)。最后，我们将建立一种重建实验来探索miRISC组装的机制，并鉴定和表征miRISC组装的新成分(目标3)。这些研究将极大地促进我们对果蝇RNAi途径的理解。人类疾病的起源，如癌症，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，在人类细胞中的RNAi不如在果蝇细胞中的RNAi有效。因此，对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、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.