Advertisement

Labeling and Purification of Temporally Expressed RNAs During the S-Phase of the Cell Cycle in Living Cells

  • Matthieu Meryet-FiguiereEmail author
  • Mohamad Moustafa Ali
  • Santhilal Subhash
  • Chandrasekhar KanduriEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2254)

Abstract

From high-throughput DNA and RNA sequencing technologies, it is evident that more than two-thirds of the mammalian genome is transcribed and nearly 98% of the transcriptional output in humans constitute noncoding RNA, comprising tens of thousands of small and long noncoding RNAs. These observations have put the study of RNA expression levels at the center of molecular biology research. The transcriptional output of cells changes temporally throughout different cell cycle phases, or in response to a large panel of stimuli. In such instances, the measure of induced RNA transcripts might be obscured by the presence of steady-state RNA levels in the total transcriptome. With this protocol, we provide a method for labeling and purification of the nascent RNAs transcribed over short periods of time in cultured cells. The supplementation of cell culture medium with a chemically modified analog of uridine, ethynyl-uridine, allows for the subsequent biotinylation of ethynyl-uridine residues with a click-chemistry reaction. The labeled RNA is then purified on streptavidin beads and eluted. The purified RNA is suitable for use in RT-qPCR assays as well as in deep sequencing applications.

Key words

RNA labeling Nascent RNA Cell cycle S-phase Next generation sequencing Temporal gene expression Nascent RNA sequencing 

Notes

Acknowledgments

This work was supported by the grants from Knut and Alice Wallenberg Foundation [KAW2014.0057]; Swedish Foundation for Strategic Research [RB13-0204]; Swedish Cancer Research foundation [Cancerfonden: Kontrakt no. CAN2018/591]; Swedish Research Council [2017-02834]; Barncancerfonden [PR2018-0090]; Ingabritt Och Arne Lundbergs forskningsstiftelse and LUA/ALF (to C.K.).

References

  1. 1.
    Bremer K, Moyes CD (2014) mRNA degradation: an underestimated factor in steady-state transcript levels of cytochrome c oxidase subunits? J Exp Biol 217(Pt 12):2212–2220CrossRefGoogle Scholar
  2. 2.
    Molin C, Jauhiainen A, Warringer J et al (2009) mRNA stability changes precede changes in steady-state mRNA amounts during hyperosmotic stress. RNA 15(4):600–614CrossRefGoogle Scholar
  3. 3.
    Chu T, Rice EJ, Booth GT et al (2018) Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme. Nat Genet 50(11):1553–1564CrossRefGoogle Scholar
  4. 4.
    Mahat DB, Kwak H, Booth GT et al (2016) Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc 11(8):1455–1476CrossRefGoogle Scholar
  5. 5.
    Fan J, Zhan M, Shen J et al (2006) En masse nascent transcription analysis to elucidate regulatory transcription factors. Nucleic Acids Res 34(5):1492–1500CrossRefGoogle Scholar
  6. 6.
    Liu Y, Chen S, Wang S et al (2017) Transcriptional landscape of the human cell cycle. Proc Natl Acad Sci U S A 114(13):3473–3478CrossRefGoogle Scholar
  7. 7.
    Gariglio P, Buss J, Green MH (1974) Sarkosyl activation of RNA polymerase activity in mitotic mouse cells. FEBS Lett 44(3):330–333CrossRefGoogle Scholar
  8. 8.
    Marzluff WF Jr (1978) Transcription of RNA in isolated nuclei. Methods Cell Biol 19:317–332CrossRefGoogle Scholar
  9. 9.
    Core LJ, Waterfall JJ, Lis JT (2008) Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322(5909):1845–1848CrossRefGoogle Scholar
  10. 10.
    McKinlay A, Araya CL, Fields S (2011) Genome-wide analysis of nascent transcription in Saccharomyces cerevisiae. G3 (Bethesda) 1(7):549–558CrossRefGoogle Scholar
  11. 11.
    Kwak H, Fuda NJ, Core LJ et al (2013) Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339(6122):950–953CrossRefGoogle Scholar
  12. 12.
    Nojima T, Gomes T, Grosso ARF et al (2015) Mammalian NET-Seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161(3):526–540CrossRefGoogle Scholar
  13. 13.
    Meryet-Figuiere M, Alaei-Mahabadi B, Ali MM et al (2014) Temporal separation of replication and transcription during S-phase progression. Cell Cycle 13(20):3241–3248CrossRefGoogle Scholar
  14. 14.
    Ali MM, Akhade VS, Kosalai ST et al (2018) PAN-cancer analysis of S-phase enriched lncRNAs identifies oncogenic drivers and biomarkers. Nat Commun 9(1):883CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska AcademyUniversity of GothenburgGothenburgSweden
  2. 2.Normandie Univ, UNICAEN, INSERM U1086 “ANTICIPE” (Interdisciplinary Research Unit for Cancers Prevention and Treatment, Axis BioTICLA “Biology and Innovative Therapeutics for Ovarian Cancers”)CaenFrance
  3. 3.Comprehensive Cancer Centre François Baclesse, UNICANCERCaenFrance

Personalised recommendations