Article

TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding

  • Nature Methods volume 15, pages 221225 (2018)
  • doi:10.1038/nmeth.4582
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Abstract

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.

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References

  1. 1.

    et al. Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions. Cell 150, 279–290 (2012).

  2. 2.

    , , & Nascent-Seq reveals novel features of mouse circadian transcriptional regulation. eLife 1, e00011 (2012).

  3. 3.

    et al. A wave of nascent transcription on activated human genes. Proc. Natl. Acad. Sci. USA 106, 18357–18361 (2009).

  4. 4.

    , , & Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation. Nat. Biotechnol. 33, 722–729 (2015).

  5. 5.

    , , & Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).

  6. 6.

    & Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011).

  7. 7.

    et al. TT-seq maps the human transient transcriptome. Science 352, 1225–1228 (2016).

  8. 8.

    et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat. Biotechnol. 29, 436–442 (2011).

  9. 9.

    et al. Tracking distinct RNA populations using efficient and reversible covalent chemistry. Mol. Cell 59, 858–866 (2015).

  10. 10.

    , , , & Incorporation of 6-thioguanosine and 4-thiouridine into RNA. Application to isolation of newly synthesised RNA by affinity chromatography. Eur. J. Biochem. 92, 373–379 (1978).

  11. 11.

    et al. High-resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies. Cell 159, 1698–1710 (2014).

  12. 12.

    et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

  13. 13.

    & Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. EMBO J. 14, 2679–2687 (1995).

  14. 14.

    & Permanganate oxidation of 4-thiouracil derivatives. Isolation and properties of I-substituted 2-pyrimidone 4-sulfonates. Biochim. Biophys. Acta 199, 303–315 (1970).

  15. 15.

    & A method for locating 4-thiouridylate in the primary structure of transfer ribonucleic acids. Biochemistry 8, 3242–3248 (1969).

  16. 16.

    et al. Nm-seq maps 2′-O-methylation sites in human mRNA with base precision. Nat. Methods 14, 695–698 (2017).

  17. 17.

    et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

  18. 18.

    et al. Integrative classification of human coding and noncoding genes through RNA metabolism profiles. Nat. Struct. Mol. Biol. 24, 86–96 (2017).

  19. 19.

    , , , & The role of heat shock transcription factor 1 in the genome-wide regulation of the mammalian heat shock response. Mol. Biol. Cell 15, 1254–1261 (2004).

  20. 20.

    , , , & Mammalian heat shock response and mechanisms underlying its genome-wide transcriptional regulation. Mol. Cell 62, 63–78 (2016).

  21. 21.

    , , & Widespread inhibition of posttranscriptional splicing shapes the cellular transcriptome following heat shock. Cell Rep. 7, 1362–1370 (2014).

  22. 22.

    , , & Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability. Methods 120, 39–48 (2017).

  23. 23.

    , , , & Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Nucleic Acids Res. 37, e115 (2009).

  24. 24.

    et al. Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. Br. J. Haematol. 145, 788–800 (2009).

  25. 25.

    & RNA mis-splicing in disease. Nat. Rev. Genet. 17, 19–32 (2016).

  26. 26.

    et al. Thiol-linked alkylation of RNA to assess expression dynamics. Nat. Methods 14, 1198–1204 (2017).

  27. 27.

    et al. Protocol in Protocol Exchange DOI .

  28. 28.

    & Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

  29. 29.

    , , & Interpreting reverse transcriptase termination and mutation events for greater insight into the chemical probing of RNA. Biochemistry 56, 4713–4721 (2017).

  30. 30.

    , , , & RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat. Methods 11, 959–965 (2014).

  31. 31.

    & Package “corrplot”: visualization of a correlation matrix (Version 0.84) (2017).

  32. 32.

    et al. FastUniq: a fast de novo duplicates removal tool for paired short reads. PLoS One 7, e52249 (2012).

  33. 33.

    Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).

  34. 34.

    , & HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).

  35. 35.

    et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  36. 36.

    , & HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

  37. 37.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  38. 38.

    et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

  39. 39.

    et al. Dynamic remodelling of human 7SK snRNP controls the nuclear level of active P-TEFb. EMBO J. 26, 3570–3580 (2007).

  40. 40.

    et al. STAN: a probabilistic programming language. J. Stat. Softw. 76, 1–32 (2017).

  41. 41.

    et al. PANTHER: a library of protein families and subfamilies indexed by function. Genome Res. 13, 2129–2141 (2003).

  42. 42.

    & Controlling the false discovery rate - a practical and powerful approach to multiple testing. J. R. Stat. Soc. B Stat. Methodol. 57, 289–300 (1995).

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Acknowledgements

We thank J. Steitz, A. Schepartz, D. Söll, D. Canzio, and the Simon Lab for insightful comments; and we thank Y. Wang and A. Sexton for assistance and scripts used in mutational analysis of targeted sequencing data. This work was supported by the NIH NIGMS T32GM007223 (J.A.S. and E.E.D.); NSF Graduate Research Fellowship (E.E.D.); NIH New Innovator Award DP2 HD083992-01 (M.D.S.), and a Searle scholarship (M.D.S.).

Author information

Affiliations

  1. Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut, USA.

    • Jeremy A Schofield
    • , Erin E Duffy
    • , Lea Kiefer
    • , Meaghan C Sullivan
    •  & Matthew D Simon
  2. Chemical Biology Institute, Yale University, West Haven, Connecticut, USA.

    • Jeremy A Schofield
    • , Erin E Duffy
    • , Lea Kiefer
    • , Meaghan C Sullivan
    •  & Matthew D Simon

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Contributions

J.A.S. and M.D.S. designed experiments. J.A.S., E.E.D., and L.K. carried out experiments. J.A.S., M.C.S., and M.D.S. performed computational analyses of data. J.A.S. and M.D.S. wrote the manuscript with assistance from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthew D Simon.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Note 1

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    Supplementary Protocol

Excel files

  1. 1.

    Supplementary Table 1

    Primers and oligonucleotides used in this study.

  2. 2.

    Supplementary Table 2

    Transcript half-lives for individual replicates and combined data for MEF (1h s4 U treatment) and K562 cells (4h s4 U treatment).