Structure and Function of Dicer Enzymes

切丁酶的结构和功能

• 批准号: 7118729
• 负责人: JENNIFER A DOUDNA
• 金额: $ 22.63万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118729
• 关键词: Giardia; RNA interference; Schizosaccharomyces pombe; X ray crystallography; catalyst; chemical cleavage; double stranded RNA; enzyme activity; enzyme mechanism; enzyme structure; fungal proteins; gene deletion mutation; gene induction /repression; immunoprecipitation; microfluidics; protein purification; ribonuclease III; site directed mutagenesis

DESCRIPTION (provided by applicant): Double stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms. This mode of gene silencing, called RNA interference (RNAi), acts at the transcriptional level through formation of heterochromatin, and at the post-transcriptional leve,l through mRNA degradation and translational suppression. In all cases, RNAi begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides in length by the enzyme Dicer. The central objective of this project is to determine how RNA is recognized and cleaved by Dicer during induction of the RNAi pathway. We aim to understand the structural and biochemical basis for Dicer activity, including its ability to recognize double stranded RNA targets, produce approximately 22- nucleotide duplex RNA products and deliver these products to downstream silencing pathways. Although miRNAs and siRNAs are the indispensable mediators of all RNAi gene silencing processes, the biochemical basis for their generation remains poorly understood. In order for Dicer to produce functional products from long duplex RNA precursors, the enzyme must coordinate two double stranded cleavages over a distance of 60 A. The structural mechanism underlying this coordination is entirely unknown. Well-diffracting crystals are in hand for an intact Dicer enzyme and will be the focus of initial structure determination efforts using X-ray crystallography. In parallel, a series of in vitro and in vivo experiments will be conducted to elucidate mechanisms of RNA recognition and catalysis by different kinds of Dicers. The proposed studies will reveal the molecular "ruler" used to determine siRNA length and may enable the design of pre-siRNAs targeted to specific gene silencing pathways.

描述（由申请人提供）：双链RNA在广泛的真核生物中诱导有效和特异性的基因沉默。这种基因沉默模式被称为RNA干扰（RNAi），它在转录水平上通过异染色质的形成起作用，在转录后水平上通过mRNA降解和翻译抑制起作用。在所有情况下，RNAi开始于内源性或引入的前体RNA被Dicer酶加工成长度为21-25个核苷酸的微RNA （miRNAs）和小干扰RNA （sirna）。该项目的中心目标是确定在RNAi途径诱导过程中，RNA是如何被Dicer识别和切割的。我们的目标是了解Dicer活性的结构和生化基础，包括其识别双链RNA靶标的能力，产生大约22个核苷酸的双链RNA产物并将这些产物传递到下游沉默途径。尽管mirna和sirna是所有RNAi基因沉默过程中不可或缺的介质，但它们产生的生化基础仍然知之甚少。为了使Dicer从长双链RNA前体中产生功能性产物，酶必须在60 a的距离上协调两个双链切割。这种协调背后的结构机制是完全未知的。完整的Dicer酶的衍射良好的晶体已经到手，这将是使用x射线晶体学确定初始结构的重点。同时，我们将进行一系列的体外和体内实验来阐明不同种类的Dicers对RNA的识别和催化机制。拟议的研究将揭示用于确定siRNA长度的分子“标尺”，并可能使设计针对特定基因沉默途径的pre-siRNA成为可能。