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A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans

Nature Methods volume 7, pages 837–842 (2010)Cite this article

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An Erratum to this article was published on 29 November 2010

This article has been updated

Abstract

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.

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Figure 1: Strategy and workflow for quantification of potential C. elegans let-7–interacting genes.
Figure 2: Identification of proteins regulated by let-7.
Figure 3: Genes displaying protein changes in let-7(n2853) mutants are enriched in let-7 suppressors.
Figure 4: Comparison of let-7–dependent changes in protein and transcript amounts of candidate let-7 miRNA targets.
Figure 5: ztf-7 (F46B6.7) is a bona fide let-7 target gene.
Figure 6: Predicted miR-58 targets are significantly upregulated in mir-58(n4640) mutants.

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Change history

  • 09 November 2010

    In the version of this article initially published, the reported P values were incorrectly written and an incorrect wording change was inadvertently made to the Figure 1 legend. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank R.F. Ketting (Hubrecht Institute, Utrecht) and B.B. Tops (Utrecht University, Utrecht) for providing the metabolically labeled C. elegans sample, A. Stark (Research Institute of Molecular Pathology, Vienna) for sharing miRNA target predictions for C. elegans, M. Moser for the assistance with the reverse transcription–quantitative PCR assays, B. Roschitzky and B. Gerrits for technical support, H. Rehrauer for statistical support, R. Schlapbach for access to the Functional Genomics Center Zurich, members of the Hengartner, Aebersold, Grosshans and Miska laboratories and E. Brunner and the whole Quantitative Model Organism Proteomics team for insightful discussion and comments on the manuscript. This work was funded in part by the University of Zurich Research Priority Program in Systems Biology/Functional Genomics, the Swiss National Science Foundation, the Gerbert R¨f Foundation, the Swiss initiative for systems biology, SystemsX, the Ernst Hadorn Foundation and the ETH Zurich. R.A. was supported by the European Research Council grant ERC-2008-AdG 233226. M.J. and L.R. were supported by a grant from the Research Foundation of the University of Zurich. M.J. was also supported by a fellowship from the Roche Research Foundation. P.P. was supported by the Marie Curie Intra-European fellowship. V.L. was supported by a grant from F. Hoffmann-La Roche Ltd. to the Competence Center for Systems Physiology and Metabolic Diseases. H.G. was supported by the Swiss National Research Foundation, the Novartis Research Foundation and by an ERC Starting Investigator grant (miRTurn). X.C.D. was supported by a Boehringer Ingelheim Funds PhD Student fellowship. C.B., N.J.L. and E.A.M. were supported by a Cancer Research UK Programme grant to E.A.M.

Author information

Author notes

  1. Lukas Reiter, Vinzenz Lange & Cherie Blenkiron

    Present address: Present addresses: Biognosys AG, Zurich, Switzerland (L.R.), DKMS Life Science Lab, Dresden, Germany (V.L.) and Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand (C.B.).,

  2. Marko Jovanovic and Lukas Reiter: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

    Marko Jovanovic, Lukas Reiter, Erica Bogan, Manuel Weiss, Sabine P Schrimpf & Michael O Hengartner

  2. Doctorate Program in Molecular Life Sciences Zurich, Zurich, Switzerland

    Marko Jovanovic, Lukas Reiter, Erica Bogan & Manuel Weiss

  3. Quantitative Model Organism Proteomics, University of Zurich, Zurich, Switzerland

    Marko Jovanovic, Lukas Reiter, Manuel Weiss, Sabine P Schrimpf & Michael O Hengartner

  4. Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland

    Paola Picotti, Vinzenz Lange & Ruedi Aebersold

  5. Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland

    Vinzenz Lange & Ruedi Aebersold

  6. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Benjamin A Hurschler, Xavier C Ding & Helge Großhans

  7. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Cherie Blenkiron, Nicolas J Lehrbach & Eric A Miska

  8. Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    Cherie Blenkiron, Nicolas J Lehrbach & Eric A Miska

  9. Faculty of Science, University of Zurich, Zurich, Switzerland

    Ruedi Aebersold

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  1. Marko Jovanovic
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Contributions

M.J., L.R., M.O.H. and R.A. designed the experiments and wrote the paper. L.R. and M.J. did the majority of the data analysis. M.J. did the majority of the experiments. P.P. and V.L. contributed to and supervised the SRM experiments. E.B. contributed to the RNAi and reverse transcription–quantitative PCR experiments. B.A.H. and X.C.D. performed the polysomal profiling experiments. C.B. and N.J.L. contributed to the reporter assays. S.P.S. and M.W. provided the C. elegans proteome atlas. H.G. and E.A.M. provided critical input on the manuscript, contributed to the experimental design and the data analysis. M.O.H. and R.A. supervised the project.

Corresponding authors

Correspondence to Ruedi Aebersold or Michael O Hengartner.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 2–7 and 10–12, Supplementary Results 1–2, Supplementary Discussion (PDF 1263 kb)

Supplementary Table 1

let-7 candidate list. (XLS 127 kb)

Supplementary Table 8

Transitions and input parameters for the SRM-ICAT quantifications of potential let-7 targets and neutral controls. (XLS 427 kb)

Supplementary Table 9

Transitions and input parameters for the SRM quantifications of predicted miR-58 targets and random negative controls. (XLS 794 kb)

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Jovanovic, M., Reiter, L., Picotti, P. et al. A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans. Nat Methods 7, 837–842 (2010). https://doi.org/10.1038/nmeth.1504

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