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Updated: May 28, 2026

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)
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Structural basis for RISC assembly of human Argonaute2.

Huaqun Zhang1, Vishal Annasaheb Adhav1, Audrey C Kehling1

  • 1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.

Molecular Cell
|May 26, 2026
PubMed
Summary
This summary is machine-generated.

Argonaute proteins (AGOs) load small interfering RNA (siRNA) duplexes to form RNA-induced silencing complexes (RISC). This study reveals how AGO2 ejects passenger strands, with mRNAs aiding RISC assembly.

Keywords:
ArgonauteRISCRISC assemblyRNA-induced silencing complexRNAibiotechnologygene silencingmiRNAsiRNAtherapeutics

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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.