Argonaute-RNA Interactome in Arabidopsis

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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.