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Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH

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mRNA Decay

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

Abstract

Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells. Here, we provide a step-by-step protocol that allows quantification of mRNA decay in single Saccharomyces cerevisiae using smFISH. Our approach relies on fluorescent labeling of single cytoplasmic mRNAs and nascent mRNAs found at active sites of transcription, coupled with mathematical modeling to derive mRNA half-lives. Commercially available, single-stranded smFISH DNA oligonucleotides (smFISH probes) are used to fluorescently label mRNAs followed by the quantification of cellular and nascent mRNAs using freely available spot detection algorithms. Our method enables quantification of mRNA decay of any mRNA in single, unperturbed yeast cells and can be implemented to quantify mRNA turnover in a variety of cell types as well as tissues.

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Acknowledgments

We thank Daniel R. Larson for helpful comments on our manuscript. T.T. is an HHMI fellow of the Jane Coffin Childs Memorial Fund. D.Z. is supported by the Canadian Institute for Health Research (Project Grant-366682), Natural Sciences and Engineering Research Council of Canada and the Canadian Foundation for Innovation.

Author contributions: T.T. wrote the manuscript. S.R. and D.Z. provided the figures, and all three authors worked on the final design of the figures and manuscript text. All the authors approved the final version of the manuscript. The authors declare no competing financial interests.

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Authors and Affiliations

  1. Department of Cell Biology, Skirball Institute of Biomolecular Medicine, NYU School of Medicine, New York, NY, USA

    Tatjana Trcek

  2. Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC, Canada

    Samir Rahman & Daniel Zenklusen

Authors
  1. Tatjana Trcek
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  2. Samir Rahman
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  3. Daniel Zenklusen
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Corresponding author

Correspondence to Tatjana Trcek .

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Editors and Affiliations

  1. Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Victoria, Australia

    Shireen R. Lamandé

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Trcek, T., Rahman, S., Zenklusen, D. (2018). Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH. 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_4

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  • DOI: https://doi.org/10.1007/978-1-4939-7540-2_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7539-6

  • Online ISBN: 978-1-4939-7540-2

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