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

项目摘要： 小RNA（sRNA）作为调节基因表达的一种方式， 这对生殖系维持的重要性已经得到证实，这强调了理解生殖系维持的必要性。 这些药物控制其mRNA靶点的机制。先前研究蛋白质定位的工作 在不同的发育模型中，这一过程所需的研究表明，sRNA的许多组分 机器定位于生殖系内的核周结构，通常称为nuage。In C.优雅， sRNA靶向所需的Argonaute蛋白和RNA依赖性RNA聚合酶等因子 已经观察到扩增sRNA信号所需的RdRPs在这些核周细胞中富集。 细胞器然而，这些结构的重要性以及它们如何促进sRNA相关过程， 它仍然是未知的，并已成为一个非常有趣的话题。虽然sRNA蛋白因子的细胞生物学 以前已经被描述过了，一个有据可查的关于RNA在被这种靶向时如何定位的研究。 这一进程仍有待进行。为了解决这一领域的差距，我们首次 在发育过程中被sRNA敲低的RNA。通过启动 siRNA介导的通过RNAi干扰（RNAi）的转录物敲低，我发现在RNAi后， 治疗后，靶向RNA在nuage中特异性富集，其中包含非常重要的因子 siRNA介导的沉默所必需的，如RdRPs。这一令人兴奋的观察现在打开了大门， 通过一个以前从未见过的细胞生物学透镜来研究sRNA通路。我假设招募 将靶向sRNA的RNA导入nuage有助于RdRP介导的针对转录物的sRNA扩增。到 为了研究这种新特征的细胞生物学现象的基础，我将首先探索分子生物学。 如我的第一个目标中所描述的，这种sRNA诱导的RNA募集所需的参与者。使用已知的RNAi 突变体，我将系统地评估每个突变体的能力，积累RNA内nuage细胞生物学 用RNAi诱导后的水平。此外，我将用一个无偏见的以RNA为中心的分析来补充这个分析。 蛋白质组学方法来鉴定可能负责这种RNAi诱导的募集的因素。在我 第二个目的，我将分析浓缩RNA在浓缩物中对RdRP活性的影响 通过对冷凝物驻留在RdRP动力学上的体外分析。总的来说，这项建议旨在 了解sRNA靶向的一个新发现的方面，这可能对人类健康产生深远的影响 和生育能力，因为在人类生殖系中nuage和小RNA途径是保守的。

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.