Mechanism and Function of Argonaute 4 Subcellular Localization

智力优势：基因表达的调控对从发育到环境反应的所有细胞过程都至关重要。一种沉默基因表达的方法是通过阻止转录的染色质修饰。这种转录基因沉默可以由与Argonaute蛋白相关的小rna诱导。这提出了一个难题，因为根据定义，染色质修饰必须发生在细胞核中，但许多Argonaute蛋白是在细胞质中发现的。核作用Argonaute蛋白在细胞质积累中的作用尚不清楚，但可能反映了Argonaute作用的调节，或与细胞间小rna的运输有关。该项目将利用模式植物拟南芥来了解调节小RNA介导的转录基因沉默的亚细胞定位的分子机制，特别是ARGONAUTE 4和RNA聚合酶iv依赖的小RNA在细胞内的空间分布以及它们在细胞内和细胞间的功能之间的关系。由于小rna和ARGONAUTE的分子功能在许多物种中都是保守的，因此研究ARGONAUTE 4在蛋白质、细胞和有机体水平上的定位，本研究将为许多物种的基因沉默提供可应用的机制范例。更广泛的影响：这项研究为研究生和博士后提供了出色的培训，他们将利用先进的基因组和生物信息学工具以及传统的分子和遗传分析。本科生也将被纳入研究环境，同时推进他或她自己的教育，并为研究生和博士后创造指导机会。这项研究也将为我们对转录基因沉默的理解做出基础贡献，这是基因调控的一个重要方面，也是作物改良的一个有力途径。

Intellectual Merit: Regulation of gene expression is critical to all cellular processes, from development to environmental response. One way to silence gene expression is by chromatin modifications that prevent transcription. Such transcriptional gene silencing can be induced by small RNAs in association with Argonaute proteins. This presents a conundrum, because by definition, chromatin modification must occur in the nucleus, but many Argonaute proteins are found in the cytoplasm. The role of cytoplasmic accumulation of nuclear-acting Argonaute proteins is unclear, but might reflect regulation of Argonaute action, or relate to transport of small RNAs between cells. This project will exploit the model plant Arabidopsis thaliana to understand the molecular mechanisms that regulate the subcellular localization of small RNA-mediated transcriptional gene silencing, in particular how ARGONAUTE 4 and RNA Polymerase IV-dependent small RNAs are spatially distributed within a cell and how this relates to their functions within and between cells. Because the molecular function of small RNAs and Argonautes are conserved in many species, investigating the localization of ARGONAUTE 4 at the protein, cellular, and organismal levels, this research will create mechanistic paradigms that could be applicable to gene silencing in many species. Broader Impacts: This research offers outstanding training for a graduate student and a postdoctoral fellow, who will utilize advanced genomic and bioinformatics tools as well as traditional molecular and genetic analyses. An undergraduate student will also be included in the research environment, simultaneously advancing his or her own education and creating mentoring opportunities for the graduate student and postdoctoral fellow. This research will also make fundamental contributions to our understanding of transcriptional gene silencing, an important aspect of gene regulation and a powerful avenue for crop improvement.