Structure and Function of Dicer Enzymes

描述(申请人提供)：双链RNA在广泛的真核生物中诱导有效和特定的基因沉默。这种被称为RNA干扰的基因沉默模式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基因降解和翻译抑制作用于L。在所有情况下，RNAi都是从内源性或引入的前体RNA被Dester酶加工成长度为21-25个核苷酸的微RNA(MiRNAs)和小干扰RNA(SiRNAs)开始的。该项目的中心目标是确定RNA在RNAi途径的诱导过程中是如何被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，包括它识别双链RNA靶标的能力，产生大约22个核苷酸的双链RNA产物，并将这些产物输送到下游的沉默途径。虽然miRNAs和siRNAs是所有RNAi基因沉默过程中不可或缺的中介，但它们产生的生化基础仍然知之甚少。为了让Dester从长的双链RNA前体中产生功能产物，酶必须在60A的距离上协调两个双链裂解。这种协调的结构机制是完全未知的。衍射性良好的晶体可用于完整的Dice酶，并将成为使用X射线结晶学进行初始结构确定的重点。同时，还将进行一系列体外和体内实验，以阐明不同类型的骰子对RNA的识别和催化机制。这项拟议的研究将揭示用于确定siRNA长度的分子“尺子”，并可能使针对特定基因沉默途径的前siRNA的设计成为可能。

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.