Structural basis for chaperone-guided assembly of RNA-induced silencing complex revealed
Researchers have determined the cryo-EM structure of the human AGO2–HSP70–HSP90–p23 chaperone complex (AMC) bound to a microRNA duplex, revealing the atomic-level mechanism by which molecular chaperones load small RNAs into the RNA-induced silencing complex (RISC). The study, published in Nature, shows that HSP90 holds AGO2 in an open, RNA-receptive conformation by engaging its L1 linker within the chaperone lumen, while p23 contacts the AGO2 N-terminal domain, and that duplex RNA bearing a 5′-phosphate and a rigid seed region is required for productive RISC assembly. These findings clarify a long-sought mechanistic step in gene silencing and have direct implications for the rational design of therapeutic siRNA and miRNA-based drugs.
A team led by V. Narry Kim and Soung-Hun Roh at Seoul National University and the Institute for Basic Science has solved cryo-EM structures of the human AGO2–HSP70–HSP90–p23 complex (AMC) in both RNA-free and let-7a-1 miRNA duplex-loaded states, deposited as PDB 9W5I and EMDB entries EMD-65663/65662. The structures show that HSP90 cradles the AGO2 L1 linker inside its lumen, keeping the protein in an expanded, open conformation that is incompatible with mature RISC but fully accessible to incoming double-stranded RNA. The co-chaperone p23 simultaneously contacts the AGO2 N-terminal domain, and together these interactions position the MID, PIWI, and PAZ domains on one face of the HSP90 dimer while the N domain sits on the opposite side, creating a wide RNA-binding cleft. Biochemical experiments with mutant AMCs and chemically modified RNA substrates demonstrate that a 5′-phosphate on the guide strand and a duplex length sufficient to span the MID-to-PAZ distance are essential for AGO2 folding and subsequent target slicing activity. The study also shows that AGO folding precedes passenger-strand removal, and that chemical modifications used in approved siRNA therapeutics such as inclisiran are compatible with chaperone-mediated loading, providing a structural rationale for optimizing next-generation RNAi drugs.
Limitations & open questions
The study characterizes the human AMC in vitro using purified components and HEK293T cell-derived complexes; whether the same structural intermediates exist at physiologically relevant concentrations and kinetics inside intact cells, or whether additional cellular factors modulate the process in vivo, is not addressed. The authors also note conformational heterogeneity in the PAZ domain and diffuse density outside the HSP90 lumen in the RNA-free state, indicating that some structural details of the pre-loading complex remain unresolved. The functional consequences of the observed chaperone interactions for AGO1, AGO3, and AGO4 paralogs are inferred from sequence conservation but not directly tested structurally.
What different sources said
- Nature NewsCenter
Structural basis for chaperone-guided assembly of RNA-induced silencing complex
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