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

Marko Jovanovic; Lukas Reiter; Alejandra Clark; Manuel Weiss; Paola Picotti; Hubert Rehrauer; Andreas Frei; Lukas J. Neukomm; Ethan Kaufman; Bernd Wollscheid; Martin J. Simard; Eric A. Miska; Ruedi Aebersold; André P. Gerber; Michael O. Hengartner

doi:10.1101/gr.133330.111

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

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

↵16 These authors contributed equally to this work.

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

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

↵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.