Helix‐7 in Argonaute2 shapes the microRNA seed region for rapid target recognition

• Published in: The EMBO journal Vol. 37; no. 1; pp. 75 - 88
• Main Authors: Klum, Shannon M, Chandradoss, Stanley D, Schirle, Nicole T, Joo, Chirlmin, MacRae, Ian J
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 04.01.2018; Blackwell Publishing Ltd; John Wiley and Sons Inc
• Subjects: Argonaute; Argonaute 2 protein; Argonaute Proteins - chemistry; Argonaute Proteins - genetics; Argonaute Proteins - metabolism; Base pairs; Binding; Biochemistry; Complementarity; Crystallography, X-Ray; Diffusion rate; EMBO36; EMBO40; Gene silencing; Humans; microRNA; MicroRNAs - genetics; MicroRNAs - metabolism; miRNA; Models, Biological; Post-transcription; Protein Binding; Protein Conformation; Proteins; Ribonucleic acid; RNA; RNA silencing; RNA, Guide, CRISPR-Cas Systems; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-induced silencing complex; RNA-mediated interference; Searching; seed; Shape recognition; Target recognition; target search; microRNA; RNA silencing; target search; seed; Argonaute
• ISSN: 0261-4189; 1460-2075
• DOI: 10.15252/embj.201796474

Argonaute proteins use microRNAs (miRNAs) to identify mRNAs targeted for post‐transcriptional repression. Biochemical assays have demonstrated that Argonaute functions by modulating the binding properties of its miRNA guide so that pairing to the seed region is exquisitely fast and accurate. However, the mechanisms used by Argonaute to reshape the binding properties of its small RNA guide remain poorly understood. Here, we identify a structural element, α‐helix‐7, in human Argonaute2 (Ago2) that is required for speed and fidelity in binding target RNAs. Biochemical, structural, and single‐molecule data indicate that helix‐7 acts as a molecular wedge that pivots to enforce rapid making and breaking of miRNA:target base pairs in the 3′ half of the seed region. These activities allow Ago2 to rapidly dismiss off‐targets and dynamically search for seed‐matched sites at a rate approaching the limit of diffusion. Synopsis The RNA‐Induced Silencing Complex (RISC) is guided to target mRNAs via complementarity to the Argonaute‐bound miRNA. Argonaute2 controls target pairing in the miRNA seed region using a structural element (helix‐7) and thereby enables fast and accurate target searches. Structural and biochemical data reveal how Ago2 mediates miRNA target search and binding. The miRNA seed region is composed of two functionally distinct domains. The seed 5′ domain is immobile and the 3′ domain is flexible and dynamic. Dynamics of the seed 3′ domain are controlled by helix‐7 in Ago2. Helix‐7 enables efficient miRNA target searches by accelerating the making and breaking of miRNA:target base pairs and minimizing time spent on off‐targets. Graphical Abstract Argonaute2, the key effector of microRNA‐mediated gene silencing, controls base‐pairing in the miRNA seed region to facilitate fast and accurate binding of target mRNAs.