Regulation of gene expression and genome organization by small RNAs

项目摘要：小RNA调节从酵母菌到生物的广泛的基因表达 人类和控制RNA沉默的基本机制在大多数真核生物中都是保守的。 术语“小RNA”包括一组不同的小调节RNA，从18-30个核苷酸不等 在长度上，它们都由ArgAerte蛋白家族的成员结合。第一批被发现的小RNA是 在线虫中发现的调节发育时间的microRNAs(Ambros和Ruvkun实验室)。几个 几年后，RNA干扰(RNAi)现象，基因沉默导致的双链引入 在线虫(Fire和Mello实验室)中也发现了搁浅的RNA。从那时起，许多工作都揭示了 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， 翻译和信使核糖核酸的稳定性。通过调节内源和外源RNAs，小RNAs发挥了关键作用 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研究RNA沉默途径的机制 调节基因表达并维持基因组的完整性。像Ambros、Ruvkun、Fire和Mello一样，我们将使用 线虫，线虫，作为研究RNA沉默的模型系统，因为蠕虫结合了 遗传学、细胞学、分子和生化工具。有许多基本的悬而未决的问题 我们对RNA沉默的理解，我们计划在本提案中解决这一问题。 我们和其他人已经确定了RNA沉默途径中许多因素的定位，但 对RNA和DNA成分的细胞学探索很少，甚至没有。此外，该机制通过 目前尚不清楚哪些靶向转录本被识别并送入RNA沉默途径。我们的第一次 目的是定位RNA沉默所针对的mRNAs，并确定其蛋白质需求 细胞质RNA沉默中从核内转录到周转的物理运动 分隔舱，诱变者焦点。我们将进一步研究RNA沉默是否会影响亚核 定位靶基因座，促进mRNA定位和RNA沉默。 尽管在这一领域做了很多工作，但仍有一些蛋白质被认为是小RNA途径的 其作用机制尚不清楚。我们还认为，这条途径的某些组成部分尚未形成 已确认身份。我们的第二个目标是利用分子生物学和细胞学的结合来检查 在RNA在细胞中的招募和保留过程中的未知途径成分 突变子焦点和mRNA靶的修饰。我们的第三个目标是识别和描述新的 使用蛋白质组学的RNA沉默复合体的组成部分。这项研究将有助于我们更好地 调节基因表达并对发育、抗病毒免疫和基因组至关重要的途径 组织。

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.