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

Abstract

Summary Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1–4) 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 miRNA 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.

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…Structures of human AGO1 bound to endogenous co-purified RNAs or loaded with let-7 miRNA are strikingly similar to the structures of AGO2 (Faehnle et al, 2013;Nakanishi et al, 2013). Evolutionary changes that rendered hAGO1 inactive included a mutation of a catalytic tetrad residue and mutations on a loop near the actives site (Faehnle et al, 2013;Nakanishi et al, 2013).…”

“…The core of the structural reports is thus formed by six articles reporting crystal structures of human Argonaute proteins (Elkayam et al, 2012;Schirle and MacRae, 2012;Faehnle et al, 2013;Nakanishi et al, 2013;Schirle et al, 2014;Schirle et al, 2015). Four articles describe crystal structures of AGO2 (Elkayam et al, 2012;Schirle and MacRae, 2012;Schirle et al, 2014;Schirle et al, 2015), which is capable of cleaving cognate transcripts and two describe miRNA-pathway dedicated AGO1 (Faehnle et al, 2013;Nakanishi et al, 2013).…”

“…Evolutionary changes that rendered hAGO1 inactive included a mutation of a catalytic tetrad residue and mutations on a loop near the actives site (Faehnle et al, 2013;Nakanishi et al, 2013).…”

From guide to target: molecular insights into eukaryotic RNA-interference machinery

“…With the ability to reproducibly purify unbound hAgos [84,90] and load them with specific guides [84,90], it became apparent that this binding confers stability to not only to the guide [65,90,91,92] but also Ago itself [90]. Moreover, this binding holds the guide RNA in a conformation that is consistent in all observed structures; this is particularly evident and critical in the seed region [61].…”

“…Recent biochemical and structural studies of hAgos have identified determinants of Argonaute slicer activity that extend beyond the active site. These are most notably in an adjacent loop to the active site and in the N domain [84][85][86][87][88]. Moreover, the activity of Agos can be modulated through post-translational modifications including proline hydroxylation that increases slicing activity, sumoylation that increases protein stability, ADP-ribosylation that relieves both slicing and translation repression, and phosphorylation that can either enhance or inhibit silencing efficacy (reviewed in [89]).…”