Argonaute-RNA Interactome in Arabidopsis

智力优势：该项目的总体目标是阐明拟南芥中小rna （smrna）与几种Argonaute （AGO）蛋白相匹配的分子机制，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，smrna是生物过程许多方面的关键调节因子。它们与AGO结合抑制靶基因的表达。利用下一代测序技术已经在拟南芥中发现了大量的smrna。然而，smrna选择性装载到不同AtAGOs的机制仍然难以捉摸。此外，这些smrna的功能分析在很大程度上受到鉴定其靶标过程缓慢的限制。此外，初步数据显示，一些AGO基因在某些细胞系中特异性表达或被特定的非生物胁迫诱导，这表明这些AGO基因在特定的生理条件下具有功能特异性。为了阐明AtAGO/smRNA/靶RNA的结合机制，了解这些AGOs的生物学作用，本项目将采用生化方法纯化含AtAGO的核糖核蛋白复合物，并从分离的AtAGO复合物中直接鉴定所有RNA组分。该项目的三个具体目标是：1)atago10相关smRNAs的鉴定和表征，2)AtAGO1和atago10复合物的目标mrna鉴定，3)AtAGO2和AtAGO3在高盐度条件下的功能分析。该项目的研究结果不仅将揭示RNA沉默的新机制，而且还将为剖析AtAGO家族基因的功能特征或冗余提供基准。此外，全面研究AtAGOs在非生物胁迫响应中的生理相关性和潜在机制也将对农业生产力产生重要影响。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先进的培训机会。PI还将把他在实验室的经验融入到课堂的教学任务中，从而为学生提供更鼓舞人心的教育体验。最后，该研究将接触到不同背景的科学家，以便从拟南芥植物中获得的RNA沉默知识将用于改善经济上重要的作物，以造福整个社会。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.