Structure of yeast Argonaute with guide RNA

Abstract

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.

Figure 1. Cleavage activity of budding-yeast AGO

a, Domain architectures of AGO proteins. b, RNAi reconstituted in S. cerevisiae using K. polysporus genes. Median GFP intensity is plotted as a fraction of GFP-only control. Error bars, quartiles; dashed line, background fluorescence. c, Cleavage activity of KpAGO. RNAs labeled at a cap phosphate (red) and matching the guide (either perfectly or with mismatches to positions 10–11) were incubated with/without (+/−) KpAGO that had been pre-incubated in the presence/absence (+/−) of synthetic guide RNA. Product was resolved on a denaturing polyacrylamide gel alongside cap-labeled synthetic product (left) and RNA standards (migration shown on right).

Figure 2. KpAGO architecture and co-purifying RNA

a, KpAGO protein structure, with N (cyan), linker L1 (yellow), PAZ (violet), linker L2 (grey), MID (orange), and PIWI (green) domains in ribbon representation. Constant (cS) and variable (vS) insertion segments, blue and slate, respectively; disordered regions, dotted lines. b, Fo–Fc map (blue) contoured at 2.8 σ before modeling RNA. c, Simulated-annealing omit map (blue) contoured at 3.5 σ around final RNA model (red). d, Nuclease sensitivity of co-purifying nucleic acid. End-labeled polynucleotides extracted from the indicated KpAGO samples were either untreated (−) or incubated with RNase (R) or DNase (D) before analysis on a denaturing gel. e, Nucleotide composition and origin of co-purifying RNA. Sequences were analyzed for enriched or depleted nucleotides (positive or negative bits, respectively) at each of the first eight positions (top). Numbers of sequencing reads mapping along each strand of the KpAGO expression plasmid are indicated (bottom, log scale). f, Cleavage activity guided by co-purifying RNA. As in Fig. 1c, except labeled RNAs were designed to match the indicated co-purifying RNA. g, Autonomous duplex loading and passenger-strand cleavage. Labeled ssRNA or siRNA duplex was incubated +/− KpAGO. h, Autonomous duplex loading and target cleavage. As in Fig. 1c, except KpAGO and RNA concentrations were reduced by 90% and 99.95%, respectively.

Figure 3. Organization of the guide RNA

a–b, The 5′-nucleotide–binding pockets of KpAGO (a) and TtAGO (b). Colors, as in Fig. 2a; protein, ribbon representation; highlighted residues and RNA, stick representation; O2′, O4′ and phosphate, white, cyan and yellow, respectively; hydrogen bonds, dotted lines. c–d, Interactions involving bases and either phosphates (c) or 2′-OH groups (d) of the seed region. Intermolecular (black) and intramolecular (blue) hydrogen bonds, dotted lines; hydrophobic interactions, van der Waals radii. e, Effects of guide-strand modifications on duplex loading and passenger-strand cleavage. KpAGO was incubated with siRNA duplexes with the indicated guide strands; p, 5′ monophosphate; upper case, 2′-OH; lower case, 2′-deoxy. The fraction of labeled passenger strand cleaved is plotted (average of three independent replicates; error bars, standard deviations; points connected by smooth curves). f, Superposition of guide-RNA nucleotides 2–8 (red) on A-form RNA (cyan and blue). Dihedral angles (θ) between guide-RNA bases and those of A-form RNA are in parenthesis. g, Solvent-exposed seed nucleotides (red). KpAGO surface is rendered, domains colored as in Fig. 2a.

Figure 4. An extended, potentially unobstructed nucleic acid–binding channel in KpAGO

a–b, Position of N domain relative to L1-linker domain in TtAGO (a) and KpAGO (b). Domains are oriented based on their N-terminal beta strands (dashed line connects strand termini). Colors as in Fig. 2a. c–d, Channels of TtAGO ternary complex (c) and KpAGO with modeled A-form duplex (d). Protein surfaces are rendered, highlighting distances between the N and PAZ domains (parallel lines) and the cS1/3/10 cluster (blue), which fills a cavity (dashed circle).

Figure 5. A plugged-in glutamate finger at the active site

a, Superposition of α25 and β34 of KpAGO (green) on counterparts of NcQDE-2 MID-PIWI lobe (grey), highlighting the extended loop L2 (dark green). b, Hydrogen-bond network stabilizing the plugged-in loop L2. Loop L2, dark green; otherwise, as in Fig. 3a. c, Closed (left) and open (right) configurations of the loop L1 gate (purple) in NcQDE-2 MID-PIWI lobe and KpAGO, respectively. cS11, blue; otherwise, as in panel b. d, Superposition of the region flanking loop L2 in the unplugged (grey) and plugged-in (green) conformations of TtAGO, depicted as in panel a. e, Hydrogen-bond network stabilizing the plugged-in loop L2 in TtAGO, depicted as in panel b. f, Closed and open configurations of the loop L1 gate in the unplugged and plugged-in conformations of TtAGO, respectively, depicted as in panel c. g, RNAi reconstituted in S. cerevisiae using wild-type K. polysporus AGO1 WT or genes with the indicated substitutions. Silencing was monitored under either permissive (induced hairpin, blue bars) or stringent (repressed hairpin, open bars) conditions. Q1052 and Y902, conserved residues insensitive and sensitive to substitution, respectively, were included as controls. Dashed lines (blue and black), background fluorescence in permissive and stringent conditions, respectively; otherwise, as in Fig. 1b. h, Stereoview of KpAGO catalytic residues (green) superpositioned with catalytic residues, divalent cations, scissile phosphate and adjacent nucleoside in TtAGO (blue) and BhRNase H1 (yellow) ternary complexes.