Structural basis for RISC assembly of human Argonaute2

Argonaute proteins (AGOs) load small RNA duplexes and select one strand to form the RNA-induced silencing complex (RISC), but the mechanism of assembly remains unclear. We report four cryogenic-electron microscopy structures of human AGO2 bound to a small interfering RNA (siRNA) duplex, which captures previously unanticipated intermediates. Unexpectedly, only the MID-PIWI lobe secures one duplex end, while α-helix 14 probes its stability. When the N domain reaches the opposite end, the L1 hairpin and Stalk wedge into the duplex and the PAZ domain engages the guide 3′ end. This configuration peels the passenger from its 5′ end while leaving it paired to the guide seed. AGO2 completely ejects the passenger by replacing it even with partially complementary target RNAs. This target-assisted passenger ejection (TAPE) also operates for the cleaved passenger strand. Our study uncovers that mRNAs are bifunctional molecules that not only serve as targets of RISCs but also help drive RISC assembly.