Poster abstracts
Poster number 49 submitted by Mi Seul Park
Structural and Functional study of human Argonaute3
Mi Seul Park (Department of Chemistry and Biochemistry, The Ohio State University), Hong-Duc Phan, Florian M. Busch, Kimberly Nguyen (Department of Chemistry and Biochemistry, The Ohio State University), Junan Li, Ming Poi (Division of Pharmacy Practice and Science, College of Pharmacy, The Ohio State University), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Kotaro Nakanishi (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
microRNAs cooperates with Argonaute proteins in the RNA-induced silencing complexes that post-transcriptionally regulate gene expression by degrading target mRNAs. In humans, there are four Argonaute paralogs: Argonaute1, Argonaute2, Argonaute3, and Argonaute4. To date, Argonaute2 has been reported to function as the only RNA slicer, but Argonaute3 retains the same set of the catalytic tetrad, Asp-Glu-Asp-His, that Argonaute2 possesses. Therefore, why Argonaute3 has retained the catalytic tetrad throughout its molecular evolution remains a long-standing question. Here we report that Argonaute3 indeed cleaves RNAs. To revisit this question, we purified recombinant Argonaute3 from insect cells and immunopurified FLAG-tagged Argonaute3 from HEK293T cells. Both proteins showed cleavage activities though the immunopurified protein showed much higher slicer activity than the recombinant protein. Intriguingly, our biochemical data revealed that the slicer efficiencies of AGO3 heavily depended on the guide sequence and the length of target RNAs. To understand the molecular basis for the target cleavage, we also determined the first crystal structure of Argonaute3 in complex with guide RNA. Comparison with the structure of Argonaute2 elucidated the molecular mechanism that the intrinsic slicer activity of Argonaute3 is restrained by the incompletion of the nucleic acid binding channel due to the flexible N domain and its unique insertion. Given the discrepancy in the observed activities between the recombinant and immunopurified samples, we surmise that unidentified binding factor(s) in the cells stabilizes the channel, thereby activating Argonaute3 for RNA cleavage.
References:
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Keywords: microRNA, Argonaute