Structural basis for RNA slicing by a plant Argonaute

Argonaute (AGO) proteins use small RNAs to recognize transcripts targeted for silencing in plants and animals. Many AGOs possess an endoribonuclease activity termed RNA slicing, which catalyzes rapid turnover of target RNAs. The nuclease activity of Thermus thermophilus AGO (TtAgo) 1,2 is often used as a model for RNA slicing, but how well DNA-guided slicing by this bacterial thermophile resembles RNA-guided slicing by eukaryotic AGOs is not known. We present cryo-EM structures of the Arabidopsis thaliana Argonaute10 (AtAgo10)-guide RNA complex with and without a target RNA representing a slicing substrate. Like TtAgo, AtAgo10 conformation expands in response to target binding. However, the AtAgo10-guide-target complex adopts slicing-competent and -incompetent conformations that are distinct from structures of TtAgo. AtAgo10 slicing activity is licensed by docking target (t) nucleotides t9-t13 into a surface channel containing the AGO endoribonuclease active site. A ß-hairpin, conserved in the L1 domain of eukaryotic AGOs, secures the t9-t13 segment and coordinates t9-t13 docking with extended guide-target pairing, preventing the complex from becoming trapped in slicing-incompetent conformations. Results show the mechanism for achieving RNA slicing in eukaryotes is distinct from that of bacteria and provide insights for controlling small interfering RNA (siRNA) potency.