Article | Published:

RESA identifies mRNA-regulatory sequences at high resolution

Nature Methods volume 14, pages 201207 (2017) | Download Citation

Abstract

Gene expression is extensively regulated at the levels of mRNA stability, localization and translation. However, decoding functional RNA-regulatory features remains a limitation to understanding post-transcriptional regulation in vivo. Here, we developed RNA-element selection assay (RESA), a method that selects RNA elements on the basis of their activity in vivo and uses high-throughput sequencing to provide a quantitative measurement of their regulatory functions at near-nucleotide resolution. We implemented RESA to identify sequence elements modulating mRNA stability during zebrafish embryogenesis. RESA provides a sensitive and quantitative measure of microRNA activity in vivo and also identifies novel regulatory sequences. To uncover specific sequence requirements within regulatory elements, we developed a bisulfite-mediated nucleotide-conversion strategy for large-scale mutational analysis (RESA–bisulfite). Finally, we used the versatile RESA platform to map candidate protein–RNA interactions in vivo (RESA–CLIP).

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Acknowledgements

We thank K. Bilguvar, S. Mane, C. Castaldi and I. Tikhonova for sequencing support; H. Codore for technical assistance; A. Bazzini, P. Oikonomou and S. Tavazoie for discussions; and all the members of the Giraldez laboratory for intellectual and technical support. This research was supported by the US National Institutes of Health (R01 HD074078, GM103789, GM102251, GM101108 and GM081602), the Pew Scholars Program in the Biomedical Sciences, the March of Dimes (1-FY12-230), the Yale Scholars Program, the HHMI Faculty Scholars Program, Whitman fellowship funds provided by E.E. Just, Lucy B. Lemann, and Evelyn and Melvin Spiegel, and the H. Keffer Hartline and Edward F. MacNichol, Jr. Fellowship Fund of the Marine Biological Laboratory (Woods Hole, Massachusetts, USA) to A.J.G.; the Swiss National Science Foundation (grant P2GEP3_148600) to C.E.V.; NIH Fellowship F32 HD061194 to C.M.T.; NIH Fellowship F32 HD071697 and start-up funds from the University of Pittsburgh to M.T.L.; and NIH Training Grants T32 GM007223 and T32 HD007149, an Edward L. Tatum Fellowship (Yale University) and the Yale MRSP to V.Y.

Author information

Author notes

    • Valeria Yartseva
    •  & Carter M Takacs

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Valeria Yartseva
    • , Carter M Takacs
    • , Charles E Vejnar
    • , Miler T Lee
    •  & Antonio J Giraldez
  2. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

    • Miler T Lee
  3. Yale Stem Cell Center, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Antonio J Giraldez
  4. Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA.

    • Antonio J Giraldez

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Contributions

V.Y., C.M.T. and A.J.G. designed and conceived the project. V.Y. generated the RESA UTR, RESA-B and RESA–CLIP libraries, and performed the validation experiments. V.Y. and M.T.L. developed the RESA and RESA-B analysis. M.T.L. and C.E.V. performed the computational analyses. All authors interpreted and analyzed the data. V.Y., M.T.L. and A.J.G. wrote the manuscript with input from the other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Miler T Lee or Antonio J Giraldez.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Tables 1–3

Text files

  1. 1.

    Supplementary Data 1

    List of primer pairs used to amplify the RESA library.

  2. 2.

    Supplementary Data 2

    BED file of the UTR regions covered by the RESA library.

  3. 3.

    Supplementary Data 3

    Regulatory regions identified by RESA.

  4. 4.

    Supplementary Data 4

    Positions with significant RESA-bisulfite induced allele biases.

  5. 5.

    Supplementary Data 5

    Ago2 bound regions identified by RESA-CLIP at ≥ 50X coverage.

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DOI

https://doi.org/10.1038/nmeth.4121

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