The Making of a Slicer: Activation of Human Argonaute-1

Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1–hAgo4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 microRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad, combined with a mutation on a loop adjacent to the active site of hAgo1, results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2 without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme. [Display omitted] •Structure of human Argonaute 1 (hAgo1) bound to let-7 miRNA•hAgo1 becomes an active slicer by using elements from the N and PIWI domains of hAgo2•hAgo3 is an active slicer upon swapping of the hAgo2 N domain•Mechanistic insight into the essential elements of human Argonaute slicing Unlike human Argonaute 2 (hAgo2), hAgo1 and hAgo3 are not active slicers. To bring light to the molecular mechanisms underlying this long-standing conundrum, Joshua-Tor and colleagues solved the structure of hAgo1 in complex with let-7 microRNA. By combining structural analysis and mutagenesis, they show that two point mutations in the PIWI domain can activate hAgo1. Furthermore, they demonstrate that the hAgo2 N domain is optimized for slicing, given that it enhances slicing by activated hAgo1 and alone imparts slicing activity to hAgo3.