Molecular Mechanism of Small RNA-induced RNA Silencing

小RNA诱导RNA沉默的分子机制

• 批准号: 7128693
• 负责人: QINGHUA LIU
• 金额: $ 28.1万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7128693
• 关键词: Drosophilidae; RNA interference; arthropod genetics; chemical cleavage; chimeric proteins; crosslink; enzyme complex; genetic regulation; immunoprecipitation; microRNAs; molecular assembly /self assembly; nucleic acid biosynthesis; point mutation; protein binding; protein purification; radiotracer; recombinant proteins; ribonuclease III; small interfering RNA; yeast two hybrid system

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.

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，这是一种保守的转录后基因 - 反沉降机制，由microRNA（miRNA）（miRNA）和小型干扰RNA（siRNA）介导。这些微小的监管RNA在许多基本的生物学过程中起着至关重要的作用。特别是，miRNA的正调与癌症等人类疾病有关。此外，RNAi已被广泛用作强大的基因计算工具，以在多个模型系统中进行功能性基因组研究。 RNAi的特异性和效力突出了其发展新颖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果蝇中的两个dicer复合物，即DCR-1/R3D1和DCR-2/R2D2，为各自的miRNA-生成酶。此外，DCR-2/R2D2复合物结合了新生的siRNA并促进其掺入siRNA诱导的沉默复合物（Sirisc）。基于这些研究，该提案的目的是解决果蝇RNAi途径中的几个杰出问题，重点是miRNA途径。我们将研究miRNA生物发生的固有特异性和机制（AIM 1）。我们将检验我们的假设，即DCR-1/R3D1复合物感受到miRNA的不对称性，并促进miRNA负载到其效应子复合物Mirisc上（AIM 2）。最后，我们将开发一种重组测定法，以探索米里斯克大会的机制，并识别和表征米里斯克大会的新组成部分（AIM 3）。这些研究将大大提高我们对果蝇RNAi途径的理解。人类疾病（例如癌症）的起源通常归因于重要基因（肿瘤抑制基因）和/或病理基因（Oncogenes）功能的功能丧失。由于未知原因，人类细胞中的RNAi效率不如果蝇细胞中的RNAi。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，并希望可以优化人类细胞中的RNAi。这可能会导致新型和有效的小型基因基因计算方法的发展，从而通过专门关闭病理基因来治愈人类疾病。