Skip to main content

Metabolic Labeling of Newly Synthesized RNA with 4sU to in Parallel Assess RNA Transcription and Decay

  • Protocol
  • First Online:
mRNA Decay

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1720))

Abstract

The development of genome-wide RNA profiling technologies greatly facilitates the global analysis of gene expression. However, such technologies alone could not distinguish the contribution to cellular RNA abundance by transcription versus decay. To overcome such limitation, metabolic labeling of newly synthesized RNA with 4-thiouridine (4sU) combined with genome-wide RNA profiling was used to in parallel measure RNA transcription and decay. Here, we describe the detailed protocol for using metabolic labeling with 4sU to separate newly synthesized RNA from the preexisting RNA in mammalian cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Institutional subscriptions

Similar content being viewed by others

References

  1. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Brown PO, Botstein D (1999) Exploring the new world of the genome with DNA microarrays. Nat Genet 21:33–37

    Article  CAS  PubMed  Google Scholar 

  3. Dölken L, Ruzsics Z, Rädle B et al (2008) High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay high-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14:1–13

    Article  Google Scholar 

  4. Schwanhäusser B, Busse D, Li N et al (2011) Global quantification of mammalian gene expression control. Nature 473:337–342

    Article  PubMed  Google Scholar 

  5. Rabani M, Levin JZ, Fan L et al (2011) Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat Biotechnol 29:436–442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Windhager L, Bonfert T, Burger K et al (2012) Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Genome Res 22:2031–2042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rabani M, Raychowdhury R, Jovanovic M et al (2014) High-resolution sequencing and modeling identifies distinct dynamic RNA regulatory strategies. Cell 159:1698–1710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hafner M, Landthaler M, Burger L et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141:129–141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Burger K, Mühl B, Kellner M et al (2013) 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response. RNA Biol 10:1623–1630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Wei Chen .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sun, W., Chen, W. (2018). Metabolic Labeling of Newly Synthesized RNA with 4sU to in Parallel Assess RNA Transcription and Decay. In: Lamandé, S. (eds) mRNA Decay. Methods in Molecular Biology, vol 1720. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7540-2_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7540-2_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7539-6

  • Online ISBN: 978-1-4939-7540-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics