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Global profiling of stimulus-induced polyadenylation in cells using a poly(A) trap

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Abstract

Polyadenylation of mRNA leads to increased protein expression in response to diverse stimuli, but it is difficult to identify mRNAs that become polyadenylated in living cells. Here we describe a click chemistry–compatible nucleoside analog that is selectively incorporated into poly(A) tails of transcripts in cells. Next-generation sequencing of labeled mRNAs enables a transcriptome-wide profile of polyadenylation and provides insights into the mRNA sequence elements that are correlated with polyadenylation.

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Figure 1: 2-Ethynyl adenosine synthesis and incorporation.
Figure 2: Using 2-ethynyl adenosine to isolate newly polyadenylated transcripts.
Figure 3: Enrichment analysis of regulatory motifs in 3′ UTRs of 2-ethynyl adenosine–trapped transcripts.

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Acknowledgements

We thank members of the Jaffrey lab for helpful comments and suggestions. We thank J. Richter (University of Massachusetts Medical School) for the antibody to CPEB. We gratefully acknowledge C. Bracken and N. Svensen for assistance with NMR spectroscopy. This work was supported by US National Institutes of Health–National Institute on Drug Abuse grant T32DA007274 (D.C. and M.C.) and US National Institute of Neurological Disorders and Stroke grant R01NS56306 (S.R.J.).

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Author notes

  1. Irtisha Singh and Christopher E Slagle: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA

    Dusica Curanovic, Michael Cohen & Samie R Jaffrey

  2. Department of Pharmacology, Oregon Health Sciences University, Portland, Oregon, USA

    Michael Cohen

  3. Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Irtisha Singh & Christina S Leslie

  4. Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Christopher E Slagle

Authors
  1. Dusica Curanovic
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  2. Michael Cohen
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  3. Irtisha Singh
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  4. Christopher E Slagle
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  5. Christina S Leslie
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  6. Samie R Jaffrey
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Contributions

D.C. and S.R.J. designed experiments, analyzed the data and wrote the manuscript; D.C. performed the experiments; C.E.S. performed oocyte injection experiments; I.S. and C.S.L. performed bioinformatics analyses; M.C. designed and synthesized 2-ethynyl adenosine and EAMP and wrote portions of the manuscript.

Corresponding author

Correspondence to Samie R Jaffrey.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–16, Supplementary Note and Supplementary Tables 1–18. (PDF 34944 kb)

Supplementary Data Set 1

List of EA-trapped and EA-depleted transcripts (XLSX 87 kb)

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Curanovic, D., Cohen, M., Singh, I. et al. Global profiling of stimulus-induced polyadenylation in cells using a poly(A) trap. Nat Chem Biol 9, 671–673 (2013). https://doi.org/10.1038/nchembio.1334

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  • DOI: https://doi.org/10.1038/nchembio.1334

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