Structural basis for differential targeting properties of small RNAs in plants and animals

microRNAs (miRNAs) regulate gene expression in plants and animals. Animals use miRNAs to sculpt the transcriptome, with each miRNA modestly repressing hundreds of otherwise unrelated targets. By contrast, each plant miRNA potently silences a small number of physiologically related targets. Here, we show that this major functional distinction depends on a minor structural difference between plant and animal Argonaute (AGO) proteins. A 7-amino-acid loop in the PIWI domain of Arabidopsis Argonaute10 (AtAGO10) reduces the affinity of the miRNA seed region for target RNAs. Swapping the PIWI-loop from human Argonaute2 (HsAGO2) into AtAGO10 increases seed strength and target-binding promiscuity, resembling animal miRNA targeting. Conversely, swapping the plant PIWI-loop into HsAGO2 increases targeting stringency and elevates target cleavage rates. The loop-swapped HsAGO2-siRNA complex silences targets more potently, with reduced off-targeting, than wild-type HsAGO2 and small interfering RNA (siRNA) duplexes in mammalian cells. Thus, tiny structural differences can tune the targeting properties of AGO proteins for distinct biological roles and HsAGO2 can be engineered for potential applications in siRNA therapeutics.