Regulation of gene expression and genome organization by small RNAs

项目概述：小RNA调节从酵母到 在人类中，控制RNA沉默的基本机制在大多数真核生物中是保守的。 术语“小RNA”包括一组不同的小调控RNA，范围为约18-30个核苷酸。 在长度上，全部由Argonaute蛋白家族的成员结合。第一个被发现的小RNA是 microRNA是在C. elegans作为发育时间的调节器（Ambros和Ruvkun实验室）。几 20年后，RNA干扰（RNAi）现象出现，引入双 在C. elegans（火和梅洛实验室）。从那时起，许多工作揭示了 一组不同的蛋白质在转录水平上通过小RNA调节基因的作用， 翻译和mRNA稳定性。通过调节内源性和外源性RNA，小RNA在细胞内起着关键的作用。 在发育、基因组稳定性和病毒防御中的作用。 这项研究计划的目标是调查RNA沉默途径的机制， 调节基因表达并维持基因组完整性。像Ambros，Ruvkun，Fire和Mello一样，我们将使用 线虫C.线虫作为研究RNA沉默的模型系统，因为蠕虫联合收割机结合了 遗传学、细胞学、分子学和生物化学工具。有许多基本的未解之谜， 我们对RNA沉默的理解，我们计划在这个提案中解决。 我们和其他人已经确定了RNA沉默途径中许多因子的定位， 对RNA和DNA成分的细胞学研究很少或根本没有。此外，该机制通过 哪些靶向转录物被识别并进入RNA沉默途径是未知的。我们的第一 我们的目标是定位RNA沉默靶向的mRNA，并确定其表达所需的蛋白质。 从细胞核转录到细胞质RNA沉默转换的物理运动 隔室，突变体病灶。我们将进一步研究RNA沉默是否会影响亚核 定位靶基因座，以促进mRNA定位和RNA沉默。 尽管在该领域做了很多工作，但仍存在归因于小RNA途径的蛋白质， 作用机制未知。我们还相信这条途径中还有一些组成部分尚未实现 鉴定我们的第二个目标是结合分子生物学和细胞学来检查 在RNA的募集和保留过程中， 突变灶和mRNA靶点的修饰。我们的第三个目标是识别和描述新的 RNA沉默复合物的组成部分。这项研究将揭示保护 调节基因表达的途径，对发育、抗病毒免疫和基因组至关重要 organization.

Project Summary: Small RNAs regulate gene expression in a wide range of organisms from yeasts to humans and the fundamental mechanisms governing RNA silencing are conserved across most eukaryotes. The term “small RNA” encompasses a diverse set of small regulatory RNAs, ranging from ~18-30 nucleotides in length, all bound by members of the Argonaute protein family. The first small RNAs identified were microRNAs, discovered in C. elegans as regulators of development timing (Ambros and Ruvkun labs). Several years later, the phenomenon of RNA interference (RNAi), gene silencing induced by the introduction of double stranded RNA, was also discovered in C. elegans (Fire and Mello labs). Much work since then has uncovered the roles of a diverse set of proteins that regulate genes by way of small RNAs at the level of transcription, translation, and mRNA stability. By regulating both endogenous and foreign RNAs, small RNAs play a critical role in development, genome stability, and viral defense. The goal of this research program is to investigate the mechanisms by which RNA silencing pathways modulate gene expression and maintain genome integrity. Like Ambros, Ruvkun, Fire, and Mello, we will use the nematode, C. elegans, as a model system to study RNA silencing because worms combine the best of genetic, cytological, molecular, and biochemical tools. There are many fundamental unanswered questions in our understanding of RNA silencing that we plan to address in this proposal. We and others have determined the localization for many factors in the RNA silencing pathway but there has been little to no cytological exploration the RNA and DNA components. Furthermore, the mechanism by which targeted transcripts are recognized and routed into the RNA silencing pathways is unknown. Our first goal is to localize the mRNAs targeted by RNA silencing and to determine the protein requirements for their physical movement from transcription in the nucleus to turnover in the cytoplasmic RNA silencing compartment, the Mutator foci. We will further examine whether RNA silencing affects the subnuclear positioning of the targeted gene loci, to promote mRNA localization and RNA silencing. Despite much work in the field, there are proteins attributed to small RNA pathway for which the mechanism of action is unknown. We also believe there are components of the pathway that have yet to be identified. Our second goal is to use a combination of molecular biology and cytology to examine uncharacterized components of the pathway in the processes of recruitment and retention of RNA in the Mutator foci and in modification of mRNA targets. Our third goal is to identify and characterize new components of the RNA silencing complex using proteomics. This research will shed light on conserved pathways that regulate gene expression and are critical for development, antiviral immunity, and genome organization.