Argonaute PIWI domain and microRNA duplex structure contribute to small RNA sorting in Arabidopsis.

[EN] Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5¿-terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis ¿AGO1 and ¿AGO2. ¿AGO2 favours miRNA duplexes with no middle mismatches, whereas ¿AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of ¿AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ¿ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to ¿AGO2.