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Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

Nature Structural & Molecular Biology volume 18, pages 237–244 (2011)Cite this article

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A Corrigendum to this article was published on 06 September 2011

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Abstract

MicroRNAs (miRNAs) are 19–22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer−/− mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3′ untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.

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Figure 1: Identification of miRNA-dependent and –independent motifs associated with Ago2.
Figure 2: Ago2-CLIP genes show a miRNA-dependent gene expression signature.
Figure 3: Ago2-CLIP–identified GCACUU motif–containing cluster is sufficient to confer miR-295–mediated repression.
Figure 4: G-rich motif modulates miRNA-mediated repression.

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Change history

  • 12 May 2011

    In the version of this article initially published, the blue curve in Figure 2c was mistakenly replaced with a duplicate of that in Figure 2a. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank C. Burge, J. Wilusz, A. Ravi and members of the Sharp laboratory for critical comments, M. Lindstrom for illustration, T. Cybulski for technical help, A. Leshinsky and R. Cook for running the Solexa sequencing samples in the KI Biopolymer & Proteomics Core Facility, M. Luo and L. Smeester for microarray technical assistance in the MIT Department of Biology Biomicrocenter and C. Whittaker for bioinformatics support in the Bioinformatics & Computing Core Facility at the Koch Institute. A.K.L.L. was supported by a special fellowship from the Leukemia and Lymphoma Society. A.G.Y. was partially supported by a David H. Koch graduate fellowship. This work was supported by United States Public Health Service grants R01-GM34277 and R01-CA133404 from the US National Institutes of Health, P01-CA42063 from the National Cancer Institute to P.A.S. and partially by Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute.

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Author notes

  1. Amanda G Young, Grace X Zheng & Cydney B Nielsen

    Present address: Present addresses: Salk Institute for Biological Studies, La Jolla, California, USA (A.G.Y.); Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA (G.X.Z.); Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada (C.B.N.).,

  2. Anthony K L Leung and Amanda G Young: These authors contributed equally to this work.

Authors and Affiliations

  1. The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Anthony K L Leung, Amanda G Young, Arjun Bhutkar, Grace X Zheng, Andrew D Bosson & Phillip A Sharp

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Amanda G Young, Andrew D Bosson, Cydney B Nielsen & Phillip A Sharp

  3. Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Grace X Zheng

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Contributions

A.K.L.L. and A.G.Y. designed and performed the experiments; A.K.L.L., A.G.Y. and P.A.S. wrote the paper; A.D.B. performed experiments; A.B., C.B.N. and G.X.Z. performed the bioinformatics analyses. All authors reviewed and approved the manuscript.

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Correspondence to Phillip A Sharp.

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Leung, A., Young, A., Bhutkar, A. et al. Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs. Nat Struct Mol Biol 18, 237–244 (2011). https://doi.org/10.1038/nsmb.1991

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