Structure and Function of RNAi-related Nucleases

描述(申请人提供)：RNA干扰(RNAi)和相关途径触发对真核生物中基因表达的有效和特异的调节。基因沉默始于靶向与21核苷酸引导RNAs互补的信使RNAs，称为短干扰RNAs(SiRNAs)或microRNAs(MiRNAs)，最终导致靶向转录本的破坏。在这一途径的两端都有两种特殊的细胞质核糖核酸酶：DICER内切酶催化长度特异的双链RNA切割产生si-和miRNAs，而Xrn1外切酶在被RNA诱导的沉默复合体(RISC)最初切割后降解靶向mRNAs。本项目的中心目标是确定Dird和Xrn1酶识别和处理RNA的结构基础，并阐明这些酶各自的功能和与其他蛋白质的相互作用。迪格尔的研究建立在我们在过去项目期间在确定迪格尔的结构和生化活性方面取得的成功的基础上。对Xrn1的研究是整个项目的一个重要补充，旨在剖析靶向mRNAs被破坏的机制。我们的具体目标是以人和果蝇的Disher和Xrn1酶为重点，确定切割活性是如何调控的，测试双链RNA结合蛋白在Dester底物特异性和指导链选择中的作用，并解决和分析Xrn1的分子结构。RNAi作为真核生物基因调控的一种基本而广泛的机制的重要性，突显了对这些酶的基本了解的必要性。对控制RNAi的分子机制的了解将使这些活动能够用于治疗目的。 与公共卫生相关：RNA干扰(RNAi)和相关途径涉及与信使RNA结合的小RNA分子，以改变细胞中产生的蛋白质水平。我们关于这种调控RNA如何在人类细胞中生产和利用的发现，将使学术和工业实验室能够利用RNAi来增强或抑制用于治疗目的的特定蛋白质的生产。

DESCRIPTION (provided by applicant): RNA interference (RNAi) and related pathways trigger potent and specific regulation of gene expression in eukaryotes. Gene silencing begins with the targeting of messenger RNAs complementary to 21-nucleotide guide RNAs called short interfering RNAs (siRNAs) or microRNAs (miRNAs), ultimately leading to destruction of the targeted transcript. Two specialized cytoplasmic ribonucleases function at either end of this pathway: the Dicer endonuclease catalyzes length-specific double-strand RNA cleavage to produce si- and miRNAs, whereas the Xrn1 exonuclease degrades targeted mRNAs after initial cleavage by the RNA-induced silencing complex (RISC). The central objective of this project is to determine the structural basis for RNA recognition and processing by Dicer and Xrn1 enzymes, and to elucidate the respective functions and interactions of these enzymes with other proteins. The investigation of Dicer builds on our success during the past project period in determining Dicer structures and biochemical activities. The study of Xrn1 represents an important addition to the overall project, aimed at dissecting the mechanism by which targeted mRNAs are destroyed. Focusing on the human and Drosophila Dicer and Xrn1 enzymes, our specific aims are to determine how dicing activity is regulated, to test the roles of double-stranded RNA binding proteins in Dicer substrate specificity and guide strand selection, and to solve and analyze molecular structures of Xrn1. The significance of RNAi as an essential and widespread mechanism of gene regulation in eukaryotes underscores the need for a fundamental understanding of these enzymes. Knowledge of the molecular mechanisms that govern RNAi will enable the engineering of these activities for therapeutic purposes. PUBLIC HEALTH RELEVANCE: RNA interference (RNAi) and related pathways involve small RNA molecules that bind to messenger RNAs to alter the levels of proteins produced in cells. Our discoveries about how such regulatory RNAs are produced and utilized in human cells will enable academic and industrial laboratories to harness RNAi to enhance or repress production of specific proteins for therapeutic purposes.