Abstract

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

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Acknowledgements

We thank J. Jude (Research Institute of Molecular Pathology, IMP) for generously providing a modified version of pLenti-CRISPR-v2-GFP, G. Krssakova and K. Mechtler (IMP, Vienna) for high-performance liquid chromatography analysis, IMP/Institute of Molecular Biotechnology (IMBA) Biooptics facility for FACS support, and all laboratory members for support and discussions. Mass spectrometry was performed at the Vienna Biocenter Core Facilities (VBCF) Metabolomics unit (http://www.vbcf.ac.at), funded by the City of Vienna through the Vienna Business Agency. High-throughput sequencing was performed at the VBCF NGS Unit (http://www.vbcf.ac.at). This work was supported by grants from the European Research Council to S.L.A. (ERC-StG-338252) and J.Z. (ERC-StG-336860) and the Austrian Science Fund to S.L.A (Y-733-B22 START, W-1207-B09 and SFB F43-22) and A.v.H. (W-1207-B09). The IMP is generously supported by Boehringer Ingelheim.

Author information

Affiliations

  1. Institute of Molecular Biotechnology, Vienna Biocenter Campus, Vienna, Austria.

    • Veronika A Herzog
    • , Brian Reichholf
    • , Pooja Bhat
    • , Thomas R Burkard
    • , Wiebke Wlotzka
    •  & Stefan L Ameres
  2. Research Institute of Molecular Pathology, Vienna Biocenter Campus, Vienna, Austria.

    • Tobias Neumann
    •  & Johannes Zuber
  3. Center for Integrative Bioinformatics Vienna, Max F Perutz Laboratories, Medical University of Vienna, University of Vienna, Vienna Biocenter Campus, Vienna, Austria.

    • Philipp Rescheneder
    •  & Arndt von Haeseler

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Contributions

V.A.H. and S.L.A. conceived the approach and wrote the paper. V.A.H., B.R. and S.L.A. developed the methods, performed the experiments and analyzed the data. W.W. performed initial s4U-alkylation experiments. T.N., P.R., V.A.H., J.Z., A.v.H. and S.L.A. developed SLAM-DUNK. P.B., T.R.B., V.A.H. and S.L.A. performed mRNA 3′-end annotation.

Competing interests

V.A.H., B.R. and S.L.A. declare competing financial interest. A patent application related to this work has been filed.

Corresponding author

Correspondence to Stefan L Ameres.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14 and Supplementary Table 3.

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    Transcriptional output for 7179 genes in mES cells.Table is attached as Excel file and reports the steady-state abundance and background-subtracted T>C conversion containing reads, as determined by SLAM-seq mRNA 3′ end sequencing of mES cells subjected to s4U metabolic labeling for 45 min.

  2. 2.

    Supplementary Table 2

    High-confidence half-life data for 6665 transcripts in mES cells.Table is attached as Excel file and reports for each transcript the chromosome, start and end of the counting windows identifying transcript 3′ ends, Name of the associated transcript, length of the counting window, half-life (h), decay rate (k; h-1), standard error of half-life and decay rate (error of single exponential fit), and accuracy of fit (rsquare).

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DOI

https://doi.org/10.1038/nmeth.4435

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