Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient ‘eudicot-type’ DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5′ phosphates and 3′ overhangs, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1–10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5′ nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5′ adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5′ A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.

The model plantArabidopsis thalianaencodes as many as ten Argonaute proteins (AGO1–10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5′ nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5′ adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5′ A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.