Structure and Function of RNAi-related Nucleases

描述（由申请人提供）：RNA干扰（RNAi）和相关途径触发真核生物中基因表达的有效和特异性调节。基因沉默开始于靶向与21个核苷酸的引导RNA（称为短干扰RNA（siRNA）或微小RNA（miRNA））互补的信使RNA，最终导致靶向转录物的破坏。两种专门的细胞质核糖核酸酶在该途径的两端起作用：Dicer内切核酸酶催化长度特异性双链RNA切割以产生si和miRNA，而Xrn1外切核酸酶在RNA诱导沉默复合物（RISC）初始切割后降解靶向mRNA。该项目的中心目标是确定Dicer和Xrn1酶识别和加工RNA的结构基础，并阐明这些酶与其他蛋白质的各自功能和相互作用。Dicer的研究建立在我们在过去的项目期间在确定Dicer结构和生物化学活性方面取得的成功的基础上。Xrn1的研究是整个项目的重要补充，旨在剖析靶向mRNA被破坏的机制。专注于人类和果蝇Dicer和Xrn1酶，我们的具体目标是确定切割活性是如何调节的，测试双链RNA结合蛋白在Dicer底物特异性和引导链选择中的作用，并解决和分析Xrn1的分子结构。RNAi作为真核生物中基因调控的基本和广泛机制的重要性强调了对这些酶的基本理解的必要性。控制RNAi的分子机制的知识将使这些活性的工程化用于治疗目的。 公共卫生相关性：RNA干扰（RNAi）和相关途径涉及与信使RNA结合以改变细胞中产生的蛋白质水平的小RNA分子。我们关于这些调控RNA如何在人类细胞中产生和利用的发现将使学术和工业实验室能够利用RNAi来增强或抑制特定蛋白质的生产以用于治疗目的。

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.