Abstract

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies aimed at linking miRNA function to the biology of distinct cell types within complex tissues remain challenging, partly because in vivo miRNA-profiling methods lack cellular resolution. We report microRNome by methylation-dependent sequencing (mime-seq), an in vivo enzymatic small-RNA-tagging approach that enables high-throughput sequencing of tissue- and cell-type-specific miRNAs in animals. The method combines cell-type-specific 3′-terminal 2′-O-methylation of animal miRNAs by a genetically encoded, plant-specific methyltransferase (HEN1), with chemoselective small-RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of specific cells within Caenorhabditis elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes current challenges in cell-type-specific small-RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings.

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Acknowledgements

We thank Cochella lab members for various fosmid reporters; F. Schnorrer (IBDM, Marseille, France) for muscle-specific Gal4 driver lines; and the CGC (NIH-P40-OD010440) for worm strains. This work was supported by FP7/2007-2013 grants from the European Research Council to L.C. (ERC-StG-337161) and S.L.A. (ERC-StG-338252) and the Austrian Science Fund to L.C. (W-1207-B09 and SFB-F43-23) and S.L.A. (Y-733-B22 START, W-1207-B09 and SFB-F43-22). R.A.M. is a recipient of a DOC fellowship of the Austrian Academy of Sciences at IMBA. Basic research at IMP is supported by Boehringer Ingelheim GmbH.

Author information

Author notes

    • Stefan L Ameres
    •  & Luisa Cochella

    These authors contributed equally to this work.

Affiliations

  1. Research Institute of Molecular Pathology (IMP), Vienna Biocenter Campus (VBC), Vienna, Austria.

    • Chiara Alberti
    • , Thomas R Burkard
    • , Jingkui Wang
    • , Katharina Mahofsky
    •  & Luisa Cochella
  2. Institute of Molecular Biotechnology (IMBA), Vienna Biocenter Campus (VBC), Vienna, Austria.

    • Raphael A Manzenreither
    • , Ivica Sowemimo
    • , Thomas R Burkard
    •  & Stefan L Ameres

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Contributions

S.L.A. conceived the strategy. L.C. and S.L.A. designed and supervised the study. C.A. performed and analyzed all C. elegans experiments. S.L.A. and R.A.M. did in vitro and cell-culture experiments. I.S. performed all whole fly experiments. K.M. generated fosmid reporters and imaged transgenic worms. T.R.B. and J.W. performed bioinformatics analyses. L.C., C.A. and S.L.A. wrote the paper and prepared the figures.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Stefan L Ameres or Luisa Cochella.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Tables 7–8

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    MicroRNome by methylation-dependent sequencing (mime-seq)

Excel files

  1. 1.

    Supplementary Table 1

    Synthetic dilution experiment to determine thesensitivity of mime-seq

  2. 2.

    Supplementary Table 2

    A few miRNAs are not efficiently methylated by AthHEN1

  3. 3.

    Supplementary Table 3

    Pan-neuronal miRNAs from C. elegans revealed by mime-seq and comparison with transcriptional reporters

  4. 4.

    Supplementary Table 4

    Tissue-specific miRNAs from C. elegans revealed by mime-seq

  5. 5.

    Supplementary Table 5

    The miRNome of a single neuron class in C. elegans revealed by mime-seq in wild-type and henn-1(0) backgrounds

  6. 6.

    Supplementary Table 6

    miRNAs endogenously methylated by Cel-HENN-1

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DOI

https://doi.org/10.1038/nmeth.4610

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