Functional dissection of a plant Argonaute.

Abstract

RNA guided ribonuclease complexes play central role in RNA interference. Members of the evolutionarily conserved Argonaute protein family form the catalytic cores of these complexes. Unlike a number of other plant Argonautes, the role of AGO2 has been obscure until recently. Newer data, however, have indicated its involvement in various biotic and abiotic stress responses. Despite its suggested importance, there is no detailed characterization of this protein to date. Here we report cloning and molecular characterization of the AGO2 protein of the virological model plant Nicotiana benthamiana. We show that AGO2 can directly repress translation via various miRNA target site constellations (ORF, 3′ UTR). Interestingly, although AGO2 seems to be able to silence gene expression in a slicing independent fashion, its catalytic activity is still a prerequisite for efficient translational repression. Additionally, mismatches between the 3′ end of the miRNA guide strand and the 5′ end of the target site enhance gene silencing by AGO2. Several functionally important amino acid residues of AGO2 have been identified that affect its small RNA loading, cleavage activity, translational repression potential and antiviral activity. The data presented here help us to understand how AGO2 aids plants to deal with stress.