High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down

Xiaoyi Li, Yuri Pritykin, Carla P. Concepcion, Yuheng Lu, Gaspare La Rocca, Minsi Zhang, Bryan King, Peter J. Cook, Yu Wah Au, Olesja Popow, Joao A. Paulo, Hannah G. Otis, Chiara Mastroleo, Paul Ogrodowski, Ryan Schreiner, Kevin M. Haigis, Doron Betel, Christina S. Leslie, Andrea Ventura

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Original languageEnglish (US)
Pages (from-to)167-179.e11
JournalMolecular Cell
Volume79
Issue number1
DOIs
StatePublished - Jul 2 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Keywords

  • Ago2
  • CLIP
  • HaloTag
  • conditional
  • microRNAs
  • mouse
  • non-coding RNAs
  • targets

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