Structural basis for chaperone-guided assembly of RNA-induced silencing complex

Abstract The RNA-induced silencing complex (RISC), comprising an Argonaute (AGO) protein and a small RNA, is the central effector in RNA silencing. Small RNAs are loaded onto AGO as bulky duplexes in an HSP70- and HSP90-dependent process1,2,3, but the molecular mechanism remains poorly understood. Here we identify the human AGO–HSP90–p23 complex, which captures AGO in an RNA-free state, termed the AGO maturation complex (AMC). The purified AMC enables RNA loading and AGO folding, faithfully recapitulating de novo RISC assembly. Using cryogenic electron microscopy, we determined the structure of AMC bound to a microRNA duplex. In contrast to its conformation in the RISC, AGO adopts a highly open conformation in the AMC: the N domain and the RNA-binding module (PAZ–MID–PIWI) are fully detached and anchored to opposite sides of the HSP90 dimer, connected solely by the unfolded L1 linker. This arrangement exposes a positively charged cleft that accommodates an RNA duplex. AGO folding is facilitated by a small RNA duplex containing a 5′-terminal phosphate—but not by single-stranded RNAs—revealing a role for the RNA duplex as a chaperone-like cofactor that directs AGO domain assembly. These findings elucidate the RISC assembly mechanism and establish the AMC as a molecular tool for probing optimal RNA features and chemical modifications for the rational design of small interfering RNA therapeutics. Our study also sheds light on how chaperones, together with ligands, can guide the folding of client proteins. This is a preview of subscription content, access via your institution Access options Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription £17.99 / 30 days cancel any time Subscribe to this journal Receive 52 print issues and online access £199.00 per year only £3.83 per issue Buy this article - Purchase on SpringerLink - Instant access to the full article PDF. £ 29.95 Prices may be subject to local taxes which are calculated during checkout Data availability The cryo-EM structure of AMC–let-7a-1 has been deposited in the PDB under accession code 9W5I. The composite map and consensus map of AMC–let-7a-1 have been deposited in the EMDB under accession codes EMD-65663 and EMD-65662, respectively. The partial maps of local refinement focused on MID/PIWI, N-domain and HSP90–p23 have been deposited in the EMDB under accession codes EMD-65661, EMD-65660 and EMD-65659, respectively. 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Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019). Acknowledgements Use of cryo-EM facilities of NEXUS and CELINE consortium was supported by the National Research Foundation of Korea. Cryo-EM data were collected at the Center for Macromolecular and Cell Imaging (CMCI) and the Research Solution Center at IBS, and were processed at the Global Science Experimental Data Hub Center (GSDC) of the Korea Institute of Science and Technology. We thank J. Woo, Y.-G. Choi and S. Ji for the general mammalian cell transfection protocol; J. Yang, D.-E. Choi, S. Bang and E. Kim for technical assistance; and H. Kim, K. Kim, M. Kim, Y. Park and C. Lee for discussions. Funding This research was supported by the Institute for Basic Science from the Ministry of Science and ICT of Korea (IBS-R008-D1 to Y.-Y.L., M.J., D.L. and V.N.K.); BK21 research fellowships from the Ministry of Education of Korea (to M.J., H.L. and D.L.); a grant funded by the Songwon Kim Young Hwan Foundation (to M.J.); a Presidential Science Fellowship from the Ministry of Science and ICT of Korea (to M.J.); the National Research Foundation of Korea (RS-2021-NR056571, RS-2020-NR049538, RS-2020-NF000307, RS-2024-00344154, RS-2024-00440289, RS-2025-00559184) and the SUHF Foundation (to S.-H.R.); and the National Research Foundation of Korea (RS-2025-02653992 to J.P.; RS-2023-00246343 and RS-2026-25476692 to H.L.). Author information Authors and Affiliations Contributions Y.-Y.L. conceived the project. Y.-Y.L. and M.J. designed the study, established AMC purification methods and prepared cryo-EM samples. H.L. and S.-H.R. carried out overall structural studies, with assistance from J.L. and J.P.; M.J. developed and performed the biochemical experiments with assistance from D.L.; M.J. and D.L. purified proteins for biochemical experiments. Y.-Y.L., M.J., H.L., V.N.K. and S.-H.R. wrote the manuscript. V.N.K. and S.-H.R. collected financial support. Corresponding authors Ethics declarations Competing interests The authors declare no competing interests. Peer review Peer review information Nature thanks Yukihide Tomari, who co-reviewed with Kotaro Tsuboyama; Hong-Wei Wang; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Extended data figures and tables Extended Data Fig. 1 Purification and characterization of human AMC. a, AGO2 pulldown experiments. Strep pulldown was performed for the indicated time periods and analysed by SDS-PAGE and Coomassie staining. HSP90 and p23 were identified by Western blot and HSP70 was identified by mass spectrometry. b, AGO2 pulldown experiment using HEK293T cells, analysed by SDS-PAGE and silver staining. Strep-Flag-AGO2 was pulled down using the Strep-tag and detected with anti-Flag antibody. HSP90 and p23 were identified by Western blot. c, AMC purification scheme. d, SEC analysis of purified AMCs containing different AGO paralogs. e, AMCs analysed by SDS-PAGE and Coomassie staining. f, RNA analysis of AMCs by urea-PAGE and SYBR Gold staining. Synthetic let-7a-1 duplex was loaded at an equimolar amount to AMCs. g, RNA analysis of high- and low-MW fractions collected in Fig. 1e by urea-PAGE and SYBR Gold staining. h, Three complementary assays for AGO folding. i, Limited proteolysis assay. AMC was incubated with varying amounts of thermolysin in the presence or absence of let-7a-1 duplex and analysed by SDS-PAGE and Coomassie staining. The assay was performed directly on the reaction mixture. j, Sedimentation assay. AMC was incubated with or without let-7a-1 duplex for the indicated times, then centrifuged to separate supernatant and pellet fractions, which were analysed by SDS-PAGE and Coomassie staining. k, SEC-based folding analysis with let-7a-1. Left, AMC was incubated with or without let-7a-1 duplex for the indicated times and analysed by SEC. Shaded areas indicate regions used for AUC calculation. Top right, AGO folding yield is calculated by dividing AUC (280 nm) of low-MW fraction by that of high-MW fraction. Bottom right, analysis of high- and low-MW fractions collected from SEC by SDS-PAGE and silver staining. Bars indicate mean ± s.e.m. (n = 3, independent experiments). P values were determined by two-sided Student’s t-test. All gel images are representative of two independent experiments. Extended Data Fig. 2 AGO folding is independent of passenger strand removal. a, Time-course SEC-based folding analysis with perfectly matched let-7a-1 for the indicated times. b, AUC (280 nm) of low-MW fraction in a. c, Analysis of low-MW fractions in a by SDS-PAGE and silver staining. d, Sequence and secondary structure of let-7a-1 with perfect match (pm). The red arrowhead indicates the AGO2 slicing site. 5p, 5′ arm of the duplex; 3p, 3′ arm of the duplex. e, Quantitative PCR of low-MW fractions in a. f, SEC-based folding analysis with perfectly matched let-7a-1. Values indicate the AGO folding yield. g, Analysis of low-MW fractions in f by SDS-PAGE and silver staining. h, Analysis of low-MW fractions in f by native-PAGE and SYBR Gold staining. i, Quantitative PCR of low-MW fractions in f. j, Mechanistic model of the temporal relationship between AGO folding and passenger strand removal. For all graphs, data indicate mean ± s.e.m. (n = 3, independent experiments). P values were determined by two-sided Student’s t-test. All gel images are representative of two independent experiments. Extended Data Fig. 3 Cryo-EM image processing procedure for human pre-loading AMC. a, Overview of the image processing procedure (see Methods). b, Representative micrograph. c, 2D class averages of pre-loading AMC. The yellow arrow indicates AGO2 density. d, Gold-standard FSC at 0.143 of pre-loading AMC. e, Angular particle distribution heat map. f, Consensus map of pre-loading AMC. g, Close-up view of the HSP90 lumen and diffuse density of AMC (90° rotated). h, 3D classification of AMC showing multiple conformations near the lumenal exit. i, Local-resolution analysis of pre-loading AMC. Extended Data Fig. 4 Cryo-EM image processing procedure for human AMC bound to let-7a-1 miRNA duplex. a, Glutaraldehyde (GA) crosslinking of RNA-loaded AMC confirmed by SDS-PAGE and Coomassie staining (n = 2). b, Overview of the image processing procedure (see Methods). c, Representative micrograph. d, 2D class averages of RNA-loaded AMC. The yellow arrow indicates AGO2 density. e, Gold-standard FSC at 0.143 of RNA-loaded AMC. f, Angular particle distribution heat map. g, Composite map of RNA-loaded AMC. h, Local-resolution analysis of RNA-loaded AMC. i, Atomic model fitting to the map of RNA-loaded AMC. j, Motion analysis of N, MID, and PIWI domains of AMC using 3D variability analysis in cryoSPARC. The first and the last volumes from two different principal components were overlaid. k, Preferred orientation rebalancing by discarding redundant views. Extended Data Fig. 5 Comparison of AMC with previously known HSP90–client structures. a, Resolved segments of client proteins accommodated within HSP90 lumen across two distinct chaperone systems. Left, HSP90–p23–client complexes: AGO2 (this study), glucocorticoid receptor (GR) (PDB: 7KRJ)12, and aryl hydrocarbon receptor (AHR) (PDB: 7ZUB)24. Right, HSP90–CDC37–client complexes: CDK4 (PDB: 5FWL; resolved regions based on 5FWK)18, RAF1 (PDB: 7Z37)25, and BRAF (PDB: 7ZR0)26. For each client, domain organization of the full-length construct, the protein construct used for structural determination, the region resolved within the HSP90–client complex, and lumen-interacting residues of each client are shown. Right panels show clients colour-coded as in the schematics. b, Pairwise per-residue Cα distance comparison of AMC and RISC structures. Left, AMC structure with Cα distances colour-coded from blue to red compared to miRNA-loaded RISC structure (PDB: 4W5N)6. Right, pairwise comparisons of AGO2 in the AMC with miRNA-loaded (PDB: 4W5N)6, seed-paired (PDB: 4W5O)28, and fully paired (PDB: 9CMP)29 RISC structures. Grey denotes regions absent in the compared models. c, AlphaFold3 prediction of RNA-bound AMC structure. Left, predicted structure of RNA-bound AMC. Right, pairwise comparisons of the predicted structure of AGO2 in the RNA-bound AMC with experimentally determined miRNA-loaded RISC (PDB: 4W5N)6 and AGO2 of RNA-bound AMC (this study). Extended Data Fig. 6 Chaperone interactions are required for productive RISC assembly. a, Limited proteolysis assay of WT and mutant AMCs analysed by SDS-PAGE and silver staining. b, Time-course target slicing assays using WT and mutant AMCs. c, HSP90-I–N interaction in RNA-loaded AMC. d, SEC-based folding analysis with let-7a-1. Values indicate the AGO folding yield. e, Limited proteolysis assay of WT and mutant AMCs. Top, SDS-PAGE followed by silver staining. Bottom, quantification of thermolysin-resistant AGO. Bars indicate mean ± s.e.m. (n = 3, independent experiments). P values were determined by two-sided Student’s t-test. f, Limited proteolysis assay of WT and mutant AMCs analysed by SDS-PAGE and silver staining. g, HSP90 lumen–L1 interaction in RNA-loaded AMC. h, Sequence alignment of L1 linkers of AGO1–4. i, Lumen-interacting residues of HSP90 in HSP90–client complexes. j, Superposition of lumen-interacting segments from multiple client complexes. HSP90 lumen residues repeatedly involved in client interaction are indicated and categorized by physicochemical properties. Dark grey, non-polar; blue, positively charged; red, negatively charged; green, polar. k, Logo plot summarizing residue frequencies at positions contacting the HSP90β lumen across clients (AGO2, AHR, CDK4, and RAF1). l, L1 linker mutants used in this study. m, SEC-based folding analysis with let-7a-1. Values indicate the AGO folding yield. n, Target slicing assays using WT and mutant AMCs. o, Quantification of target slicing assays in n. Data were fitted with a single-exponential model. All gel images are representative of three independent experiments. Extended Data Fig. 7 Duplex RNA containing 5′-phosphate and rigid seed region is critical for AGO folding. a, Limited proteolysis assay of WT and mutant AMCs. b, SEC-based folding analysis with duplex RNAs bearing either a canonical 5′-phosphate (grey dot) or an artificial 5′-hydroxyl (no dot) at the guide (light green) or passenger (light blue) termini. c, Target slicing assays using AMC assembled with duplex RNAs bearing 5′-terminal modifications. d, Quantification of target slicing assays in c. Data were fitted with a single-exponential model. e, Limited proteolysis assay of AMC incubated with duplex RNAs bearing 5′-terminal modifications. f, Limited proteolysis assay of AMC incubated with no RNA, dsRNA, and ssRNA. g, Target slicing assays using AMC assembled with no RNA, dsRNA, or ssRNA. h, Sequence and secondary structures of RNA substrates with perfect match (pm), 3′-single-stranded region (3′-ss), and 5′-single-stranded region (5′-ss). i, SEC-based folding analysis with RNA substrates in h. Values indicate the AGO folding yield. j, AGO folding yield in i. Data indicate mean ± s.e.m. (n = 3, independent experiments). P values were determined by two-sided Student’s t-test. All gel images are representative of three independent experiments. Extended Data Fig. 8 Impact of duplex length and chemical modification on AGO folding. a, SEC-based folding analysis with RNA substrates of varying duplex lengths (16–28-nt). Sequence and secondary structures of RNA substrates are shown, with the variable-length region highlighted in grey. Values indicate the AGO folding yield. b, Heterogeneity analysis of PAZ domain in AMC. 3D classification identified multiple distinct conformations of PAZ, ranging from positions proximal to the chaperone core (grey) to more distal states (purple). The displacement of the PAZ domain spans ~14.0 Å (left view) and ~20.5 Å (right view), illustrating its substantial conformational range during the chaperone-guided RNA-loading process. c, SEC-based folding analysis with chemically modified and unmodified inclisiran shown in Fig. 4s. Values indicate the AGO folding yield. Extended Data Fig. 9 Model of chaperone-guided RISC assembly. a, Structural transitions of AGO2 during post-chaperone folding and RNA duplex accommodation. The AGO2 L1 linker, which is expanded and flexible in the AMC state (left, with MLP mapped on the L1 linker surface), becomes folded in mature RISC (top right), forming a hydrophobic core that stabilizes the spatial arrangement of the N domain and L2 region. This structural compaction is a hallmark of post-chaperone folding. A positively charged patch is formed across the N and L1 domains in mature RISC, which interacts electrostatically with the negatively charged phosphate backbone of the RNA duplex (bottom right). Electrostatic potential was calculated for the miRNA-loaded RISC structure (PDB: 4W5N)6 and overlaid on the fully paired RISC structure (PDB: 9CMP)29. b, Structural comparison of RNA duplexes in AMC and RISC. In AMC (top left), the guide–passenger RNA duplex is accommodated without distortion, while in the mature RISC (top right), pairing between the guide and target strands induces steric strain, promoting passenger release and target recognition. The superimposed RNA structures (bottom) reveal the distortion of the guide–target RNA duplex in the mature RISC, particularly near the 3′-end, highlighting strain induced during target binding. c, Modes of chaperone–client interaction in HSP90 complexes with AGO2, GR, and AHR. Cryo-EM structures of HSP90β–p23–AGO2, HSP90α–p23–GR (PDB: 7KRJ)12, and HSP90β–XAP2–AHR (PDB: 7ZUB)24 reveal distinct client-engagement modes. Insets highlight the clients’ proximity to conserved aromatic residues of HSP90 (W320 in HSP90α or W312 in HSP90β) or p23 C-terminal helix. Right panels show AlphaFold3 models of folded GR and AHR, indicating positions of the client segments resolved in structural studies. Supplementary information Supplementary Figs. 1–4 (download PDF ) Supplementary Figs. 1–4. Supplementary Table 1 (download XLSX ) Oligonucleotides used in this study for in vitro RISC assembly, target slicing and cryo-EM. Supplementary Video 1 (download MP4 ) Overview of the structures of AMC and AMC–miRNA complexes. In the RNA-free AMC, distinct extra density is observed within the HSP90 lumen (yellow), accompanied by diffuse and heterogeneous density outside the lumenal cavity (pink). In the duplex RNA-loading state, AGO2 forms a wide RNA-binding pocket, with the MID, PIWI and PAZ domains clustered on one side of the HSP90 dimer and the N domain positioned on the opposite side. The colouring of the maps and models is as defined in Fig. 1. Supplementary Video 2 (download MP4 ) Open conformation of AGO2 in the AMC accessible to dsRNA. Comparison of domain arrangement between AGO2 in the AMC and mature RISC reveals a similar positioning of the MID and PIWI domains, but substantial rearrangements of the N, L1 and PAZ domains. Duplex RNA is accommodated within the AGO2 RNA-binding cleft, with the 5′ end anchored in the MID domain pocket, magnesium ions coordinated at the catalytic site and the 3′-end positioned within the PAZ domain pocket. Supplementary Video 3 (download MP4 ) Interaction of AGO2 with chaperones within the AMC. The N-terminal domain of AGO2 engages the p23 helix, while the L1 linker is positioned within the HSP90 lumen. Three tryptophan-binding pockets (TRP1–3) of AGO2 are highlighted, including TRP1, which interacts with W312 of HSP90. These interactions stabilize the unstructured regions of AGO2 and maintain its domain arrangement. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Lee, YY., Jeong, M., Lee, H. et al. Structural basis for chaperone-guided assembly of RNA-induced silencing complex. Nature (2026). https://doi.org/10.1038/s41586-026-10640-2 Received: Accepted: Published: Version of record: DOI: https://doi.org/10.1038/s41586-026-10640-2