Molecular Mechanism of Small RNA-induced RNA Silencing

我们的长期目标是了解 RNA 干扰 (RNAi) 的分子和生化机制， 由 microRNA (miRNA) 和小分子介导的保守转录后基因沉默机制 干扰RNA (siRNA)。这些微小的调节RNA在许多基础生物学中发挥着关键作用 流程。特别是，最近发现 miRNA 的失调与人类疾病有关，例如 癌症。此外，RNAi已被广泛用作强大的基因沉默工具来执行功能 多个模型系统中的基因组研究。 RNAi 的特异性和效力凸显了其潜在的应用潜力 开发治疗人类疾病的新颖有效的治疗途径。我们之前已经确定了 两个 Dicer 复合物 DCR-1/R3D1 和 DCR-2/R2D2，分别作为 miRNA 和 siRNA 生成 果蝇中的酶。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)。基于这些研究，本次研究的目标 提案旨在解决果蝇 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（Aim 1）。我们将检验我们的假设，即 DCR-1/R3D1 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（目标 2）。最后，我们将开发一种重构测定法 探索 miRISC 组装机制并识别和表征 miRISC 新组件 组装（目标 3）。这些研究将显着增进我们对果蝇 RNAi 的理解 途径。人类疾病（例如癌症）的起源通常可归因于 重要基因（抑癌基因）和/或病理基因（癌基因）的功能获得。对于 未知原因，人类细胞中的 RNAi 效率低于果蝇细胞中的 RNAi。因此，比较 果蝇和人类系统将使我们能够识别潜在的差异，并希望能够 优化人类细胞中的 RNAi。这可能会导致新型高效的基于小RNA的开发 基因沉默方法通过特异性关闭病理基因来治疗人类疾病。

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 performfunctional 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/R3D1and DCR-2/R2D2,as the respectivemiRNA- 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.