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The Eukaryotic RNA Exosome pp 169–189Cite as

Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2062))

Abstract

The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.

Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq). When coupled to cost-effective mRNA 3′ end sequencing approaches, SLAMseq determines the half-life of polyadenylated transcripts in a global and transcript-specific manner using untargeted or targeted cDNA library preparation protocols.

We provide a step-by-step instruction for time-resolved mRNA 3′ end sequencing, which augments traditional RNA-seq approaches to acquire the temporal resolution necessary to study the molecular principles that control gene expression.

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Acknowledgments

We thank Brian Reichholf (IMBA, Vienna Biocenter, Austria) and Tobias Neumann (IMP, Vienna Biocenter, Austria) for bioinformatics support. HTP sequencing was performed at the VBCF NGS Unit (www.vbcf.ac.at). This work was supported in part by the European Research Council grants ERC-StG-338252 and ERC-PoC-825710 to S.L.A.

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Author notes

  1. Veronika A. Herzog and Nina Fasching contributed equally to this work.

Authors and Affiliations

  1. IMBA—Institute of Molecular Biotechnology, Vienna Biocenter (VBC), Vienna, Austria

    Veronika A. Herzog, Nina Fasching & Stefan L. Ameres

Authors
  1. Veronika A. Herzog
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  2. Nina Fasching
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  3. Stefan L. Ameres
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Corresponding author

Correspondence to Stefan L. Ameres .

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Editors and Affiliations

  1. Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA

    John LaCava

  2. Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic

    Štěpánka Vaňáčová

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Herzog, V.A., Fasching, N., Ameres, S.L. (2020). Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion. In: LaCava, J., Vaňáčová, Š. (eds) The Eukaryotic RNA Exosome. Methods in Molecular Biology, vol 2062. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9822-7_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9822-7_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9821-0

  • Online ISBN: 978-1-4939-9822-7

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