The structural basis for RNA slicing by human Argonaute2

SUMMARY Argonaute (AGO) proteins associate with guide RNAs to form complexes that slice transcripts that pair to the guide. This slicing drives post-transcriptional gene-silencing pathways that are essential for many eukaryotes and the basis for new clinical therapies. Despite this importance, structural information on eukaryotic AGOs in a fully paired, slicing-competent conformation—hypothesized to be intrinsically unstable—has been lacking. Here we present the cryogenic-electron microscopy structure of a human AGO–guide complex bound to a fully paired target, revealing structural rearrangements that enable this conformation. Critically, the N domain of AGO rotates to allow the RNA full access to the central channel and forms contacts that license rapid slicing. Moreover, a conserved loop in the PIWI domain secures the RNA near the active site to enhance slicing rate and specificity. These results explain how AGO accommodates targets possessing the pairing specificity typically observed in biological and clinical slicing substrates.


Figure S1. Structural comparisons and purification of the HsAGO2−miR-7−target ternary complex.
(A) Crystal-packing contacts for HsAGO2 RISC in the two-helix conformation (PDB: 6N4O 31 ).N and MID domains form packing contacts between two copies of HsAGO2 RISC in the two-helix conformation (colored red and yellow).The fully paired HsAGO2 structure (cyan) is overlaid, showing that it is in a conformation that would disrupt these packing interactions.(B) Crystal contacts for TtAGO complex with a complementary target.At the top is the asymmetric unit of TtAGO, which contains two copies of TtAGO (colored yellow and purple) (PDB: 4NCB 35 ), which form contacts between the N and PAZ domains.At the bottom the fully paired HsAGO2 structure (cyan) is overlaid, showing that it would not accommodate the packing contacts.(C) Clash observed when the N domain from the HsAGO2 two-helix structure (PDB: 6N4O 31 ) is overlaid with a 21-bp RNA duplex model generated in ChimeraX bound within the central channel.(D) Clash observed with the N domain when the RNA duplex from the AtAGO10 2−16-paired structure (PDB: 7SWF 34 ) is extended to position 22. (E) MpAGO bound to an RNA−DNA hybrid fully paired to position 20 (PDB: 5UXO 38 ) (red).The fully paired HsAGO2 structure (cyan) is overlaid.(F) Purification scheme for the HsAGO2 D669A −miR-7−target ternary complex.(G) Size-exclusion chromatography of the ternary complex on a Superdex 200 3.1/200 column, followed by analysis on an SDS-polyacrylamide gel, visualizing protein with Imperial (Coomassie R-250) staining.

Figure S5. Analyses of the central channel expansion and the EI Loop.
(A) Frames 0−39 of 3DFlex movie (Movie S3).Gradient depicts contracted (red) to expanded (cyan) states.(B) A ~15 Å distance between the EI loop modeled in our HsAGO2 structure and the target RNA from the HsAGO2 two-helix structure (PDB: 6N4O 31 ).The EI loop is shown as a cartoon from residues 820−850, and the rest of HsAGO2 is shown as a surface.(C) Schematic of miRNA guide-target duplexes examined in target-dissociation assays.The 32 P radiolabel at the 5′ end of target is indicated as an orange star.Otherwise, this panel is as in Figure 1B.(D) In vitro dissociation-rate constant (koff) values for either wildtype (WT) HsAGO2 (dark grey) or HsAGO2 with phosphomimetic substitutions in the EI loop (red), for the two miRNA−target sets tested.Otherwise, this panel is as in Figure 2E.(E) Dissociation of target RNA from either wildtype (WT) HsAGO2 or HsAGO2 with phosphomimetic substitutions in the EI loop.Curves represent nonlinear best-fits to an exponential decay equation.Number of data points for each set is indicated as n.Time points beyond the limit of the x axes are not shown.

Figure S2 .
Figure S2.Cryo-EM data collection and processing.(A) Representative micrograph low-pass filtered to 10 Å.This micrograph is representative of 12,106 micrographs.Scale bar is shown at the bottom left.(B) Representative 2D classes.Scale bar is shown.(C) Classification tree for determining the structure of HsAGO2 in a slicing-competent conformation.

Figure S3 .
Figure S3.Quality and resolution of cryo-EM data.(A) Estimate of average resolution.Dotted lines indicate Fourier shell correlation (FSC) of 0.5 and 0.143.Solid lines indicate FSC between half-maps of the reconstruction.(B) Angular distribution plot for particles used to reconstruct Map 1. Shading from blue to yellow indicates the number of particles at a given orientation.(C) Reconstruction of the fully paired HsAGO2 complex, colored by local resolution (Map 1).(D) Model of the RNA duplex in density from Map 2. (E) Model of the N domain in density from Map 2. (F) Model of the L1 domain in density from Map 2. (G) Model of the PAZ domain in density from Map 2. (H) Model of the L2 domain in density from Map 2. (I) Model of the MID domain in density from Map 2. (J) Model of the PIWI domain in density from Map 2. (K) Model of PIWI-domain residues 672−695 in density from Map 2. (L) Model of active site in density from Map 2.

Figure S4 .
Figure S4.Residues in N and PIWI domains regulate HsAGO2 slicing activity.(A) On the left is a multiple sequence alignment of select AGO and PIWI homologs at the proposed Ndomain contacts.Residues identical to HsAGO2 are shaded in teal.Residues that changed from HsAGO2 but remained basic are shaded in yellow.An H56L substitution in HsAGO3 is shaded in pink.Species codes are as follows: Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Gg, Gallus gallus (chicken); Dm, Drosophila melanogaster (fruit fly); Bm, Bombyx mori (silkmoth); Ce, Caenorhabditis elegans (nematode); Sm, Schmidtea mediterranea (flatworm); Nv, Nematostella vectensis (starlet sea anemone); Ef, Ephydatia fluviatilis (river sponge); Ol, Oscarella lobularis (sea sponge); Dr, Danio rerio (zebrafish); Ca, Candida albicans; Kp, Kuyveromyces polysporus; Nc, Naumovozyma castellii; Sp, Schizosaccharomyces pombe (fission yeast); At, Arabidopsis thaliana; Os, Oryza sativa (rice); Bo, Brassica oleracea (wild cabbage); Nt, Nicotiana tabacum (tobacco); Aa, Aquifex aeolicus; Mj, Methanocaldococcus jannaschii; Pf, Pyrococcus furiosus; Tt, Thermus thermophilus.On the right is a multiple sequence alignment of select AGO and PIWI homologs at the central loop.Residues identical to HsAGO2 are shaded in green; otherwise, as in the left panel.(B) Fraction of target RNA sliced over time by either wildtype (WT) or N-domain mutants of HsAGO2−miR-7, across different RISC concentrations (gray gradient).Solid lines represent best-fit lines from fitting to the ordinary differential equation system.The extrapolated reaction curve at infinite RISC concentration, which represents a reaction rate determined by only kslice and not kon, is plotted in black.Magenta dashed lines indicate simulated results that would have occurred if there were a substantial (10fold) defect in elementary rate constant for target association (kon).Number of data points for each set is indicated as n.Time points beyond the limits of the x axes are not shown.(C) Analysis of major-groove width deviation from A-form of the RNA duplex in HsAGO2 in the slicingcompetent conformation and the AtAGO10 2−16-paired conformation (PDB: 7SWF 34 ).(D) Central loop in the AtAGO10 2−16-paired conformation (PDB: 7SWF 34 ).Colors are as in Figure 4D.(E) Glutamate finger, central loop, and active site of HsAGO2 in the fully paired conformation.Otherwise, as in D. (F) Fraction of target RNA sliced over time by either wildtype (WT) or central-loop mutants, across different RISC concentrations.Dashed lines indicate simulated results that would have occurred if there were a substantial (10-fold) defect in elementary rate constant for target association (kon).Colors are as in Figure 4F; otherwise, as in B. Values with WT HsAGO2 are replotted from B for reference.(G) Fraction of centrally mismatched target RNAs sliced over time by either wildtype (WT) or central-loop mutants, across different RISC concentrations; otherwise, as in F.