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Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates

  • Alexandra LusserEmail author
  • Catherina Gasser
  • Lukas Trixl
  • Paolo Piatti
  • Isabel Delazer
  • Dietmar Rieder
  • Jeffrey Bashin
  • Christian Riml
  • Thomas Amort
  • Ronald MicuraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)

Abstract

The study of RNA dynamics, specifically RNA transcription and decay rates, has gained increasing attention in recent years because various mechanisms have been discovered that affect mRNA half-life, thereby ultimately controlling protein output. Therefore, there is a need for methods enabling minimally invasive, simple and high-throughput determination of RNA stability that can be applied to determine RNA transcription and decay rates in cells and organisms. We have recently developed a protocol which we named TUC-seq to directly distinguish newly synthesized transcripts from the preexisting pool of transcripts by metabolic labeling of nascent RNAs with 4-thiouridine (4sU) followed by osmium tetroxide-mediated conversion of 4sU to cytidine (C) and direct sequencing. In contrast to other related methods (SLAM-seq, TimeLapse-seq), TUC-seq converts 4sU to a native C instead of an alkylated or otherwise modified nucleoside derivative. TUC-seq can be applied to any cell type that is amenable to 4sU labeling. By employing different labeling strategies (pulse or pulse-chase labeling), it is suitable for a broad field of applications and provides a fast and highly efficient means to determine mRNA transcription and decay rates.

Key words

TUC-seq 4-Thiouridine RNA stability RNA decay Transcription rate Metabolic labeling RNA modification 

Notes

Acknowledgments

Funding of this research was provided by the Austrian Science Foundation (FWF) P27024-BBL to A.L. and P27947 to R.M.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Alexandra Lusser
    • 1
    Email author
  • Catherina Gasser
    • 2
  • Lukas Trixl
    • 1
  • Paolo Piatti
    • 3
  • Isabel Delazer
    • 1
  • Dietmar Rieder
    • 4
  • Jeffrey Bashin
    • 3
  • Christian Riml
    • 2
  • Thomas Amort
    • 1
  • Ronald Micura
    • 2
    Email author
  1. 1.Division of Molecular Biology, BiocenterMedical University of InnsbruckInnsbruckAustria
  2. 2.Department of Chemistry and Pharmacy, Institute of Organic ChemistryLeopold Franzens UniversityInnsbruckAustria
  3. 3.Zymo Research Corp.IrvineUSA
  4. 4.Division of Bioinformatics, BiocenterMedical University of InnsbruckInnsbruckAustria

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