Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs

  1. Markus Landthaler1,
  2. Dimos Gaidatzis2,3,
  3. Andrea Rothballer1,
  4. Po Yu Chen1,
  5. Steven Joseph Soll1,
  6. Lana Dinic1,
  7. Tolulope Ojo1,
  8. Markus Hafner1,
  9. Mihaela Zavolan2, and
  10. Thomas Tuschl1
  1. 1Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA
  2. 2Swiss Institute of Bioinformatics, Biozentrum, University of Basel, CH-4056 Basel, Switzerland

Abstract

microRNAs (miRNAs) regulate the expression of mRNAs in animals and plants through miRNA-containing ribonucleoprotein particles (RNPs). At the core of these miRNA silencing effector complexes are the Argonaute (AGO) proteins that bind miRNAs and mediate target mRNA recognition. We generated HEK293 cell lines stably expressing epitope-tagged human AGO proteins and other RNA silencing-related proteins and used these cells to purify miRNA-containing RNPs. Mass spectrometric analyses of the proteins associated with different AGO proteins revealed a common set of helicases and mRNA-binding proteins, among them the three trinucleotide repeat containing proteins 6 (TNRC6A,-B,-C). mRNA microarray analyses of these miRNA-associated RNPs revealed that AGO and TNRC6 proteins bind highly similar sets of transcripts enriched in binding sites for highly expressed endogenous miRNAs, indicating that the TNRC6 proteins are a component of the mRNA-targeting miRNA silencing complex. Together with the very similar proteomic composition of each AGO complex, this result suggests substantial functional redundancy within families of human AGO and TNRC6 proteins. Our results further demonstrate that we have developed an effective biochemical approach to identify physiologically relevant human miRNA targets.

Keywords

Footnotes

  • ↵3 Present address: Friedrich-Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland.

  • Reprint requests to: Thomas Tuschl, Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA; e-mail: ttuschl{at}rockefeller.edu; fax: (212) 327-7652; or Mihaela Zavolan, Swiss Institute of Bioinformatics, Biozentrum, University of Basel, CH-4056 Basel, Switzerland; e-mail: Mihaela.Zavolan{at}unibas.ch.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1351608.

    • Received September 5, 2008.
    • Accepted September 15, 2008.
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  1. Published in Advance October 31, 2008, doi: 10.1261/rna.1351608 RNA 14: 2580-2596 Copyright © 2008 RNA Society

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