Structure and Function of Dicer Enzymes

描述（由申请人提供）：双链RNA在广泛的真核生物中诱导强效和特异性基因沉默。这种基因沉默的模式，称为RNA干扰（RNAi），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开始于通过酶Dicer将内源性或引入的前体RNA加工成长度为21-25个核苷酸的微小RNA（miRNA）和小干扰RNA（siRNA）。该项目的中心目标是确定在RNAi途径的诱导过程中，RNA如何被Dicer识别和切割。我们的目标是了解Dicer活性的结构和生物化学基础，包括其识别双链RNA靶标，产生约22个核苷酸的双链RNA产物并将这些产物递送到下游沉默途径的能力。虽然miRNAs和siRNAs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前体产生功能性产物，该酶必须在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。衍射良好的晶体是一个完整的切丁酶在手，将是使用X射线晶体学的初步结构测定工作的重点。与此同时，将进行一系列的体外和体内实验，以阐明不同种类的Dicers识别和催化RNA的机制。拟议的研究将揭示用于确定siRNA长度的分子“标尺”，并可能使针对特定基因沉默途径的pre-siRNA设计成为可能。

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.