Structural basis for RISC assembly of human Argonaute2

Area of Science:

• Molecular Biology
• Structural Biology
• RNA Interference

Background:

• Argonaute proteins (AGOs) are central to RNA interference (RNAi) pathways.
• The precise mechanism of small RNA duplex loading and strand selection by AGOs to form the RNA-induced silencing complex (RISC) remains incompletely understood.

Purpose of the Study:

• To elucidate the step-by-step mechanism of human AGO2 assembly with small interfering RNA (siRNA) duplexes.
• To identify key protein-RNA interactions and conformational changes during RISC assembly.
• To investigate the role of target RNAs in passenger strand ejection.

Main Methods:

• Cryogenic-electron microscopy (cryo-EM) was used to determine four high-resolution structures of human AGO2 bound to an siRNA duplex.
• Structural analysis focused on intermediate states of RISC assembly.

Main Results:

• Cryo-EM structures revealed novel intermediate states of AGO2-siRNA binding.
• The MID-PIWI lobe initially secures one end of the siRNA duplex, with α-helix 14 probing its stability.
• The N domain, L1 hairpin, and Stalk domain facilitate passenger strand separation, while the PAZ domain anchors the guide 3' end.
• Target-assisted passenger ejection (TAPE) was identified as a mechanism for passenger strand removal, even with partial target complementarity, and also applies to cleaved passenger strands.

Conclusions:

• mRNA molecules function bifunctionally, acting as both targets for RISC and facilitators of RISC assembly.
• The study provides unprecedented structural insights into the dynamic process of RISC formation and siRNA loading.
• Target RNA engagement is crucial for efficient passenger strand ejection and completion of the RISC assembly process.