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性地将双链（ds）RNA引入线虫C中而触发的基因沉默机制。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多种基因调控机制，包括涉及短的21-30个核苷酸（nt）长的RNA和Argonaute（RNase 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。值得注意的是，C.秀丽线虫保存所有的mRNA清单，并在种系中许可基因表达，通过卵子和精子将这些信息从一代传递到下一代。不同的Argonaute途径在小RNA信号的跨代遗传中起作用，所述小RNA信号构成CSR-1“自我”/保护（RNAa途径）和WAGO“非自我”/沉默（RNAe途径）。第三个Argonaute途径，PRG-1/Piwi途径扫描生殖系mRNA的外源序列。在目标1中，我们探索这些Argonaute途径如何识别它们的靶点并招募下游因子。在目标2中，我们研究了这些Argonaute途径如何相互作用以介导全基因组转录监视。最后，在目标3中，我们描述了基因筛选， 以鉴定RNAi所需的新基因以及相关的RNAe和RNAa途径。将联合收割机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C. elegans是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.