RNA Mediated Genetic Interference in C. elegans

 描述(申请人提供)：RNA干扰(RNAi)最初被描述为通过实验将双链(DS)RNA引入线虫而触发的基因沉默机制(Fire等人，1998)。术语RNAi现在被用来指一套不同的基因调控机制，这些机制具有共同的特征，包括参与一个短的21-30个核苷酸(NT)的长RNA和ArgAerte(RNase H相关)蛋白家族的一个蛋白质辅因子。作为一种实验工具，RNAi在许多领域的基础医学研究中具有广泛的相关性，RNAi疗法目前正在开发中，用于几个临床应用。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基本的重要性。值得注意的是，线虫中的RNAi相关机制保留了所有mRNAs的清单，并许可了生殖系中的基因表达，通过卵子和精子将这一信息从一代传给下一代。在构成CSR-1“自我”/保护性(RNAa途径)和WAGO“非自我”/沉默(RNAe途径)的小RNA信号的跨代遗传中，不同的ArgAerte途径发挥作用。第三种途径是PrG-1/Piwi途径，它扫描胚系mRNA中的外源序列。在目标1中，我们探索这些ArgAerte途径如何识别它们的目标并招募下游因素。在目标2中，我们研究了这些ArgAerte通路如何相互作用来调节全基因组转录监测。最后，在目标3中，我们描述了基因筛选将使我们 确定RNAi所需的新基因以及相关的RNAe和RNAa途径。将经典遗传学与技术相结合的能力，包括深度测序、CRISPR介导的基因组编辑和蛋白质组学，使线虫成为这些研究的理想系统。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。这些研究将揭示一种古老的基因调控机制，其在人类中的相关性可能在保护人类基因组和维持干细胞方面发挥重要的保守作用。

 DESCRIPTION (provided by applicant): 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. Remarkably, RNAi-related mechanisms in C. elegans keep inventory of all mRNAs and license gene expression in the germline, passing this information via the egg and sperm from one generation to the next. Distinct Argonaute pathways function in the transgenerational inheritance of small-RNA signals that constitute the CSR-1 "self"/protective (RNAa pathway) and the WAGO "non-self"/silencing (RNAe pathway). A third Argonaute pathway, the PRG-1/Piwi pathway scans germline mRNA for foreign sequences. In Aim 1, we explore how these Argonaute pathways identify their targets and recruit downstream factors. In Aim 2, we investigate how these Argonaute pathways interact to mediate genome-wide transcriptional surveillance. And finally, in Aim 3, we describe genetic screens that will allow us to identify new genes required for RNAi and the related RNAe and RNAa pathways. The ability to combine classical genetics with technology, including deep-sequencing, CRISPR-mediated genome editing, and proteomics, make C. elegans an ideal system for these studies. The mechanisms and protein families that mediate RNAi are highly conserved in animals and therefore insights from the proposed studies will be directly relevant to human biology and disease. These studies will shed light on an ancient gene-regulatory mechanism whose correlates in humans are likely to play important conserved roles in the protection of the human genome and the maintenance of stem cells.