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，这是一种由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，最近发现mirna的失调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应用于多种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它在开发新的和有效的治疗途径来治疗人类疾病方面的潜力。我们之前已经确定了两个Dicer复合物，DCR-1/R3D1和DCR-2/R2D2，作为果蝇中各自的miRNA和sirna生成酶。此外，DCR-2/R2D2复合体结合新生siRNA并促进其结合到siRNA诱导的沉默复合体（siRISC）中。基于这些研究，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生物发生的内在特异性和机制（目的1）。我们将验证我们的假设，即DCR-1/R3D1复合体感知miRNA的不对称性，并促进miRNA装载到其效应复合体miRISC上（目的2）。最后，我们将开发一种重组实验来探索miRISC组装的机制，并识别和表征miRISC组装的新组分（目标3）。这些研究将显著促进我们对果蝇RNAi通路的理解。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（致癌基因）的功能获得。由于未知的原因，人类细胞中的RNAi比果蝇细胞中的RNAi效率低。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，并有希望优化人类细胞中的RNAi。这可能导致新的和有效的基于小rna的基因沉默方法的发展，通过特异性关闭病理基因来治愈人类疾病。

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.