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Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes

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  • Yanli Wang

    (Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA)

  • Stefan Juranek

    (Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA)

  • Haitao Li

    (Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA)

  • Gang Sheng

    (Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA)

  • Greg S. Wardle

    (Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA)

  • Thomas Tuschl

    (Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA)

  • Dinshaw J. Patel

    (Structural Biology Program, Memorial-Sloan Kettering Cancer Center, New York, New York 10065, USA)

Abstract

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5′-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg2+-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide–target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3′-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

Suggested Citation

  • Yanli Wang & Stefan Juranek & Haitao Li & Gang Sheng & Greg S. Wardle & Thomas Tuschl & Dinshaw J. Patel, 2009. "Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes," Nature, Nature, vol. 461(7265), pages 754-761, October.
    • Handle: RePEc:nat:nature:v:461:y:2009:i:7265:d:10.1038_nature08434
    DOI: 10.1038/nature08434
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    Cited by:

    1. Yuvaraj Bhoobalan-Chitty & Shuanshuan Xu & Laura Martinez-Alvarez & Svetlana Karamycheva & Kira S. Makarova & Eugene V. Koonin & Xu Peng, 2024. "Regulatory sequence-based discovery of anti-defense genes in archaeal viruses," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    2. Lidiya Lisitskaya & Yeonoh Shin & Aleksei Agapov & Anna Olina & Ekaterina Kropocheva & Sergei Ryazansky & Alexei A. Aravin & Daria Esyunina & Katsuhiko S. Murakami & Andrey Kulbachinskiy, 2022. "Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    3. Jithesh Kottur & Radhika Malik & Aneel K. Aggarwal, 2024. "Nucleic acid mediated activation of a short prokaryotic Argonaute immune system," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    4. Sarah Willkomm & Leonhard Jakob & Kevin Kramm & Veronika Graus & Julia Neumeier & Gunter Meister & Dina Grohmann, 2022. "Single-molecule FRET uncovers hidden conformations and dynamics of human Argonaute 2," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    5. Yonghua Wang & Yan Li & Zhi Ma & Wei Yang & Chunzhi Ai, 2010. "Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis," PLOS Computational Biology, Public Library of Science, vol. 6(7), pages 1-19, July.
    6. Hanlun Jiang & Fu Kit Sheong & Lizhe Zhu & Xin Gao & Julie Bernauer & Xuhui Huang, 2015. "Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement," PLOS Computational Biology, Public Library of Science, vol. 11(7), pages 1-21, July.

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