RIP-chip-SRM—a new combinatorial large-scale approach identifies a set of translationally regulated bantam/miR-58 targets in C. elegans

  1. Michael O. Hengartner1,17
  1. 1Institute of Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland;
  2. 2Ph.D. Program in Molecular Life Sciences Zurich, 8057 Zurich, Switzerland;
  3. 3Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom;
  4. 4Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, United Kingdom;
  5. 5Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland;
  6. 6Functional Genomics Center, ETH and University of Zurich, 8057 Zurich, Switzerland;
  7. 7Laval University Cancer Research Centre, Hôtel-Dieu de Québec (CHUQ), Québec City, Québec G1R 2J6, Canada;
  8. 8Competence Center for Systems Physiology and Metabolic Diseases, 8093 Zurich, Switzerland;
  9. 9Faculty of Science, University of Zurich, 8057 Zurich, Switzerland;
  10. 10Institute of Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland

    1. ↵16 These authors contributed equally to this work.

    • ↵11 Present addresses: Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA;

    Abstract

    MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression. As miRNAs are involved in a wide range of biological processes and diseases, much effort has been invested in identifying their mRNA targets. Here, we present a novel combinatorial approach, RIP-chip-SRM (RNA-binding protein immunopurification + microarray + targeted protein quantification via selected reaction monitoring), to identify de novo high-confidence miRNA targets in the nematode Caenorhabditis elegans. We used differential RIP-chip analysis of miRNA-induced silencing complexes from wild-type and miRNA mutant animals, followed by quantitative targeted proteomics via selected reaction monitoring to identify and validate mRNA targets of the C. elegans bantam homolog miR-58. Comparison of total mRNA and protein abundance changes in mir-58 mutant and wild-type animals indicated that the direct bantam/miR-58 targets identified here are mainly regulated at the level of protein abundance, not mRNA stability.

    Footnotes

    • ↵12 Biognosys AG, 8952 Zurich, Switzerland;

    • ↵13 Department of Biology, Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland;

    • ↵14 Neurobiology Department, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA;

    • ↵15 Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom.

    • ↵17 Corresponding authors

      E-mail michael.hengartner{at}imls.uzh.ch

      E-mail a.gerber{at}surrey.ac.uk

      E-mail aebersold{at}imsb.biol.ethz.ch

    • [Supplemental material is available for this article.]

    • Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.133330.111.

      Freely available online through the Genome Research Open Access option.

    • Received October 13, 2011.
    • Accepted March 19, 2012.

    This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.

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    1. Published in Advance March 27, 2012, doi: 10.1101/gr.133330.111 Genome Res. 22: 1360-1371 © 2012, Published by Cold Spring Harbor Laboratory Press

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