Characterization of the role of biomolecular condensates in gene regulation by small RNAs

生物分子缩合物在小 RNA 基因调控中作用的表征

• 批准号: 10242701
• 负责人: John Paul Tsu Ouyang
• 金额: $ 1.17万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-09-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242701
• 关键词: Address; Behavior; Binding; Biological; Biological Assay; Biological Phenomena; Caenorhabditis elegans; Cell Maintenance; Cells; Cellular biology; Complement; Complex; Defect; Development; Drosophila genus; Ensure; Enzymes; Fertility; Gene Expression; Gene Expression Regulation; Genetic Transcription; Germ Cells; Health; Homeostasis; Human; In Vitro; Kinetics; Knowledge; Lead; Length; Life; Liquid substance; Maintenance; Mediating; Messenger RNA; Modeling; Molecular; Nucleotides; Organelles; Organism; Pathway interactions; Process; Production; Protein Family; Proteins; Proteomics; RNA; RNA Interference; RNA Stability; RNA amplification; RNA chemical synthesis; RNA-Directed RNA Polymerase; Regulator Genes; Residencies; Role; Secondary to; Signal Transduction; Small Interfering RNA; Small RNA; Specificity; Structure; System; Time; Transcript; Translations; Work; base; in vitro activity; insight; interest; knock-down; lens; mutant; novel; piRNA; recruit

Project Summary: The near-universal conservation of small RNAs (sRNAs) as a way of regulating gene expression and the demonstrated importance of this for germline maintenance underscores the need for understanding the mechanisms by which these agents control their mRNA targets. Prior work studying the localization of proteins required for this process in different developmental models has shown that many components of the sRNA machinery localize to perinuclear structures within the germline, known generally as nuage. In C. elegans, factors such as Argonaute proteins required for sRNA targeting and RNA-dependent RNA polymerases (RdRPs) needed for amplifying sRNA signals have been observed to be enriched within these perinuclear organelles. The significance of these structures and how they facilitate sRNA related processes, however, remains unknown and has become a topic of intense interest. While the cell biology of sRNA protein factors has been previously characterized, a well-documented study of how RNAs are localized when targeted by this process remains to be conducted. In an effort to address this gap in the field, we have for the first time visualized RNAs in the process of being knocked down by sRNAs in a developmental context. By initiating the sRNA-mediated knockdown of transcripts though RNAi interference (RNAi), I have found that upon RNAi treatment, targeted RNAs become specifically enriched within the nuage, which houses the very factors required for sRNA-mediated silencing, such as RdRPs. This exciting observation now opens the doors into studying the sRNA pathway through a previously uncharted cell biological lens. I hypothesize that recruitment of sRNA-targeted RNAs into nuage aids in RdRP-mediated amplification of sRNAs against the transcript. To study the basis for this newly characterized cell biological phenomenon, I will first explore the molecular players required for this sRNA-induced RNA recruitment as described in my first aim. Using known RNAi mutants, I will systematically assess each mutant's ability to accumulate RNAs within nuage on a cell biological level upon induction with RNAi. Furthermore, I will complement this analysis with an unbiased RNA-centric proteomics approach to identify factors that may be responsible for this RNAi-induced recruitment. In my second aim, I will assay the effects that concentrating RNAs within condensates could have on RdRP activity through an in vitro analysis of condensate residency on RdRP kinetics. Altogether, this proposal seeks to understand a newly identified aspect of sRNA targeting that could have profound implications in human health and fertility given the conservation of nuage and small RNA pathways in the human germline.

项目摘要： 小型RNA（SRNA）的近乎全世界的保存是调节基因表达和的一种方式 这对种系维护的重要性表明了理解的需求 这些试剂控制其mRNA靶标的机制。先前研究蛋白质定位的工作 在不同的发展模型中，此过程需要的是SRNA的许多组成部分 机械位于种系内的核周结构，通常称为nuage。在秀丽隐杆线虫中， 诸如SRNA靶向和RNA依赖性RNA聚合酶所需的ARGONAUTE蛋白等因素 （RDRP）已观察到放大SRNA信号所需的（在这些核周内富集） 细胞器。但是，这些结构的意义及其如何促进与SRNA相关的过程 仍然未知，已经成为一个激烈的兴趣的话题。而SRNA蛋白因子的细胞生物学 以前已经表征了一项有据可查的研究，该研究是针对RNA的定位 过程仍有待进行。为了解决该领域的这一差距，我们第一次有 在发育环境中被SRNA击倒的过程中可视化的RNA。通过发起 SRNA介导的转录本通过RNAi干扰（RNAi）的敲低，我发现在RNAi上 治疗，有针对性的RNA被特异性地富集在nuage中，这是一个因素 SRNA介导的沉默所必需的，例如RDRP。现在，这种令人兴奋的观察将门打开 通过以前未知的细胞生物学晶状体研究SRNA途径。我假设该招聘 SRNA靶向的RNA中的RNA辅助辅助RDRP介导的SRNA针对转录本的扩增。到 研究这种新特征的细胞生物学现象的基础，我将首先探索分子 如我的第一个目标所述，该SRNA诱导的RNA招募所需的玩家。使用已知的RNAi 突变体，我将系统地评估每个突变体在细胞生物学上累积RNA的能力 RNAi诱导后水平。此外，我将以公正以RNA为中心的分析来补充该分析 蛋白质组学方法是识别可能导致RNAI诱导募集的因素。在我的 第二个目的，我将测定将RNA集中在冷凝水中可能对RDRP活动的影响 通过对RDRP动力学的冷凝水居留的体外分析。总之，该提议试图 了解SRNA靶向的新鉴定的方面，可能对人类健康产生深远的影响 鉴于人类种系中的细微差异和小RNA途径的保存。