Single‐Molecule mRNA Detection in Live Yeast

Tineke L. Lenstra1, Daniel R. Larson1

1 Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, Bethesda
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.24
DOI:  10.1002/0471142727.mb1424s113
Online Posting Date:  January, 2016
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Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub‐second temporal resolution and nanometer spatial resolution. Studies of transcription in single cells indicate that gene activity is heterogeneous between cells and exhibits random variability over time, even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed. In this unit, we describe an approach for visualizing mRNA and transcription in live yeast cells. The method is based on binding of fluorescently labeled MS2 and PP7 coat proteins to stem loop sequences that are introduced into the gene of interest. Detailed protocols are provided for the construction of the necessary yeast strains, for image acquisition, and for validation. © 2016 by John Wiley & Sons, Inc.

Keywords: single‐molecule; RNA; transcription; live‐cell; microscopy; fluorescence

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Visualizing Gene Expression in Live Yeast Using MS2 and PP7 Labeling
  • Support Protocol 1: Coating of Dishes with Concanavalin A
  • Commentary
  • Figures
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Basic Protocol 1: Visualizing Gene Expression in Live Yeast Using MS2 and PP7 Labeling

  • Plasmid containing repeats of stem loop sequences and a selectable integration marker flanked by loxP sites, such as pDZ415 (24MS2SL loxPKanMXloxP), pDZ416 (24PP7SL loxPKanMXloxP), available at Addgene (, or pTL030 (14MS2SL loxPKanMXloxP), pTL031 (14PP7SL loxPKanMXloxP), available from the authors
  • PCR primers for integration of stem loop repeats
  • PCR primers to check genomic integration of stem loop repeats and to check Cre‐mediated excision of selectable marker
  • Standard materials for PCR (Kramer and Coen, )
  • 0.7% to 1% Agarose gels (Voytas, )
  • PCR cleaning kit to purify the PCR product and remove primers, dNTPs, and enzymes
  • Selective yeast plates and media for growth and transformation (including YPD plates, YPGAL medium, synthetic media minus uracil plates and media, 5‐fluoroorotic acid plates); for recipes, see Guthrie and Fink ( ) and Lundblad and Struhl ( )
  • Standard materials for transformation (Becker and Lundblad, )
  • 50% glycerol
  • URA3 plasmid, such as pSH47‐encoding Cre recombinase under a GAL1 promoter
  • Plasmid encoding the coat protein fused to an NLS and a fluorescent protein, such as pTL041 (pURA P ADE3­‐PCP‐NLS‐2×‐yeGFP) or pTL042 (pLEU P MET17‐MCP‐NLS‐mKate2), available from the authors
  • Concanavalin A‐coated glass‐bottom culture dishes with a coverslip thickness which matches the objective used in the experiment (e.g., MatTek 35 mm, No. 1.5 or NEST 35 mm) (see the Support Protocol)
  • Cryogenic vials
  • Incubator at 30°C
  • Inverted wide‐field microscope (e.g., Zeiss AxioObserver) with:
    • >100 × objective, NA > 1.3 (e.g., Zeiss Plan‐Apochromat 150 × /1.35 Glyc DIC)
  • Laser excitation source (or equivalent strong narrowband excitation)(e.g., Spectra‐Physics Excelsior diode‐pumped solid state lasers)
  • Excitation and emission filters optimized for fluorophore (e.g., for GFP: ET470/30x excitation filter (for non‐laser illumination), T495lpxr dichroic beamsplitter and ET525/50m emission filter, Chroma)
  • EM‐CCD (e.g., Evolve 512, Photometrics)
  • Focusing device (e.g., Definite focus, Zeiss)
  • Microscope stage incubator (e.g., Tokai Hit, INUB‐LPS)

Support Protocol 1: Coating of Dishes with Concanavalin A

  • Concanavalin A from Canavalia ensiformis (Jack bean)
  • Phosphate‐buffered saline (PBS; e.g., Gibco PBS, pH 7.4)
  • 0.22‐μm filter
  • Glass‐bottom culture dishes with a coverslip thickness that matches the objective used in the experiment (e.g., MatTek 35 mm, no. 1.5 or NEST 35 mm)
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Internet Resources
  The Saccharomyces genome database (SGD) Web site is a great repository for information on all yeast genes.
  μManager is an open source microscopy software package.
  Fiji is an imaging‐processing software package.
  Software downloads for RNA tracking.
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