Argonaute-RNA Interactome in Arabidopsis

智力优势：该项目的总体目标是阐明小RNA(SmRNAs)在拟南芥中与几种ArgAerte(AGO)蛋白相结合的分子机制，并使用高通量方法识别smRNA引导的靶标。在大多数真核生物中，SmRNAs是许多生物过程的关键调节因子。它们与一个AGO结合，抑制靶基因的表达。利用下一代测序技术已在拟南芥中鉴定出大量的smRNAs。然而，smRNAs选择性地装载到不同的AtAGO中的机制仍然不清楚。此外，对这些单链RNA的功能分析在很大程度上受到识别其靶标的缓慢过程的限制。此外，初步数据显示，一些AGO基因在某些细胞系中特异表达或在特定的非生物胁迫下诱导表达，表明这些AGO在特定的生理条件下具有特殊的功能。为了阐明ATAGO/smRNA/靶RNA结合的机制，并了解这些AGO的生物学作用，本项目将使用生化方法纯化含有ATAGO的核糖核蛋白复合体，并直接从分离的ATAGO复合体中鉴定所有RNA组分。该项目的三个具体目标将是：1)鉴定和鉴定与AtAGO10相关的小分子RNA，2)从AtAGO1和含AtAGO10的复合体中鉴定靶mRNAs，3)在高盐度条件下对AtAGO2和AtAG03进行功能分析。该项目的结果不仅将阐明RNA沉默的新机制，而且还将为剖析Atago家族基因的功能特长或冗余提供一个基准。此外，全面研究atagos在非生物胁迫反应中的生理相关性和潜在机制也将对农业生产力产生重大影响。更广泛的影响：计划中的研究为本科生和研究生提供了从生物化学、分子生物学、遗传学到尖端基因组学和高通量测序/计算生物学等广泛领域的独特和高级培训机会。PI还将把他在实验室的经历融入到他在课堂上的教学任务中，从而为学生提供更鼓舞人心的教育体验。最后，这项研究将接触到不同背景的科学家，以便利用从拟南芥植物获得的RNA沉默知识来改良重要的经济作物，使整个社会受益。该项目产生的数据库、信息、材料和其他资源将通过网络服务器或可下载的源代码直接提供给公众和其他研究人员。

Intellectual Merit: The overall goal of this project is to elucidate the molecular mechanisms by which small RNAs (smRNAs) are assorted to several Argonaute (AGO) proteins in Arabidopsis, and to identify smRNA-guided targets using high throughput approaches. SmRNAs are critical regulators to many aspects of biological processes in most eukaryotes. They associate with an AGO to repress expression of target genes. A large number of smRNAs have been identified in Arabidopsis using next-generational sequencing techniques. However, the mechanisms by which the smRNAs are selectively loaded into different AtAGOs remain elusive. Furthermore, functional analysis of these smRNAs is substantially limited by slow processes in identification of their targets. In addition, preliminary data revealed that some AGO genes are specifically expressed in certain cell lineages or induced by particular abiotic stresses, suggesting that these AGOs are functionally specialized for particular physiological conditions. To elucidate mechanisms of AtAGO/smRNA/target RNA association, and to understand the biological roles of these AGOs, this project will employ biochemical methods to purify AtAGO-containing ribonucleoprotein complexes and to directly identify all RNA components from the isolated AtAGO complexes. Three specific objectives of this project will be: 1) Identification and characterization of AtAGO10-associated smRNAs, 2) Identification of target mRNAs from AtAGO1 and AtAGO10-containing complexes, 3) Functional analysis of AtAGO2 and AtAGO3 in high salinity conditions. The results from this project will not only shed light on new mechanisms of RNA silencing, but also provide a benchmark to dissect functional specialties or redundancies in AtAGO family genes. In addition, comprehensive investigation of the physiological relevance and underlying mechanism of AtAGOs in abiotic stress responses will also have a significant impact on agricultural productivity. Broader Impacts: The planned research provides unique and advanced training opportunities for undergraduates, and graduate students in broad areas from biochemistry, molecular biology, genetics to cutting-edge genomics and high-throughput sequencing / computational biology. The PI will also integrate his experiences in the lab into his teaching mission in the classroom and thereby provide a more inspiring educational experience to students. Finally, the research will reach out to scientists of diverse backgrounds so that the knowledge of RNA silencing gained from Arabidopsis plants will be utilized to improve economically important crops to the benefits of the whole of society. Databases, information, materials, and other resources generated from this project will be made directly available to the public and other researchers, either through a web server or through downloadable source codes.