RNA Mediated Genetic Interference in C. elegans

描述（由申请人提供）： RNA干扰（RNAi）最初被描述为通过实验将双链（ds）RNA引入线虫C.秀丽隐杆线虫（Fire等人，1998年）。术语RNAi现在用于指具有共同特征的一组不同的基因调控机制，包括涉及短的21-30个核苷酸（nt）长的RNA和Argonaute（RNA酶H相关）蛋白家族的蛋白质辅因子。作为一种实验工具，RNAi与许多领域的基础医学研究具有广泛的相关性，并且RNAi疗法现在正在开发用于几种临床应用。此外，RNAi相关机制在保守的基因调控途径中发挥作用，这些途径对人类细胞和发育生物学具有基本和根本的重要性。拟议的遗传和生物化学研究将促进我们对RNAi及其相关途径的理解。联合收割机将经典遗传学与深度测序、功能基因组学和蛋白质组学等较新学科相结合的能力，使C。elegans是这些研究的理想系统。 在迄今为止研究的所有动物中，多种RNAi相关途径在细胞内共存。In C.在线虫中，三种AGO途径具有介导全基因组或全转录组监视的潜力。这些途径是：（i）WAGO途径，其靶向转座子、假基因和其它隐蔽基因座以及一些蛋白质编码基因;（ii）CSR-1途径，其靶向在生殖系中表达的大多数（如果不是全部）蛋白质编码mRNA;和（iii）PRG-1途径，其靶向至少一个转座子家族，但具有超过一万五千个额外的基因组-编码小RNA辅因子，其靶点未知。这项工作的一个重要目标是了解这些不同的途径如何识别目标和介导特定的调控结果。拟议的研究将调查这些途径的功能和相互关系，使用一系列的生物化学，分子和遗传学方法。介导RNAi的机制和蛋白质家族在动物中高度保守，因此，拟议研究的见解将与人类生物学和疾病直接相关。PHS 398/2590（2004年9月修订，2006年4月重新印发） 公共卫生相关性： RNA干扰（RNAi）是一种高度保守的基因沉默机制，与动植物的免疫、基因组维持和发育基因调控有关。该项目的目标是研究RNAi及其相关机制，以便我们更好地了解它们如何影响发育和人类健康。这些研究的见解可能直接导致新的RNA干扰技术，用于探测包括人类在内的各种生物体的基因功能，并可能最终导致新的诊断和治疗方法。

DESCRIPTION (provided by applicant): Project Summary/Abstract RNA interference (RNAi) was originally described as a gene silencing mechanism triggered by the experimental introduction of double stranded (ds)RNA into the nematode C. elegans (Fire et al., 1998). The term RNAi is now used to refer to a diverse set of gene regulatory mechanisms that share common features including the involvement of a short 21-30 nucleotide (nt) long RNA and a protein cofactor of the Argonaute (RNase H-related) protein family. As an experimental tool, RNAi is of broad relevance to basic medical research in numerous fields, and RNAi therapeutics are now under development for several clinical applications. Furthermore, RNAi-related mechanisms function in conserved gene-regulatory pathways that are of basic and fundamental importance to human cellular and developmental biology. The proposed genetic and biochemical studies will advance our understanding of RNAi and related pathways. The ability to combine classical genetics with the newer disciplines of deep-sequencing, functional genomics and proteomics, make C. elegans an ideal system for these studies. In all animals studied to date, multiple RNAi-related pathways co-exist within cells. In C. elegans, three AGO pathways have the potential to mediate genome-wide or transcriptome-wide surveillance. These pathways are: (i) the WAGO pathway, which targets transposons, pseudogenes and other cryptic loci, as well as some protein encoding genes; (ii) the CSR-1 pathway, which targets most, if not all, protein-encoding mRNAs expressed in the germ line; and (iii) the PRG-1 pathway, which targets at least one transposon family but has more than fifteen thousand additional genomically-encoded small RNA cofactors whose targets are not known. An important goal of this work is to understand how these distinct pathways identify targets and mediate specific regulatory outcomes. The proposed studies will investigate the function and interrelationship of these pathways, using an array of biochemical, molecular and genetic approaches. The mechanisms and protein families that mediate RNAi are highly conserved in animals, therefore, insights from the proposed studies will be directly relevant to human biology and disease. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Project Narrative RNA interference (RNAi) is a highly conserved gene-silencing mechanism implicated in immunity, genome maintenance and developmental gene regulation in both plants and animals. The goal of this project is to investigate RNAi and related mechanisms so that we can better understand how they impact on development and human health. Insights from these studies may lead directly to new RNA-interference technologies for probing gene function in a variety of organisms including humans, and may ultimately lead to new diagnostics and therapeutics.