Quantification of mRNA translation in live cells using single-molecule imaging


mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3ʹ-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2–4 d to complete (after cell lines expressing all required transgenes have been generated).

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Fig. 1: An assay for visualization of translation using the SunTag system.
Fig. 2: Overview of the experimental flow.
Fig. 3: Reporter design.
Fig. 4: Representative imaging data for SunTag, MoonTag and MashTag reporters.
Fig. 5: Screenshot of TransTrack software.
Fig. 6: Fluorescence intensity profile of a single ribosome translating the SunTag reporter.
Fig. 7: Example output of RiboFitter.
Fig. 8: Representative imaging data and analyses for experiments on NMD.

Data availability

Example datasets for analysis are provided in Supplementary Data 1 and 2.

Code availability

The analysis software (RiboFitter and TransTrack) is included in Supplementary Data 1 and 2, and is also freely available through GitHub (http://github.com/TanenbaumLab/RiboFitter and http://github.com/TanenbaumLab/TransTrack, respectively). The code in this manuscript has been peer-reviewed.


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We thank the members of the Tanenbaum lab for helpful discussions. This work was financially supported by an ERC starting grant (ERC-STG 677936-RNAREG), two grants from the Netherlands Organization for Scientific Research (NWO; ALWOP.290 and NWO/016.VIDI.189.005), and the Howard Hughes Medical Institute through an International Research Scholar grant to M.E.T. (HHMI/IRS 55008747). All authors were supported by the Oncode Institute, which is partly funded by the Dutch Cancer Society (KWF). T.A.H. was supported by a PhD fellowship from the Boehringer Ingelheim Fonds.

Author information




D.K., T.A.H. and S.S. made the figures in the paper. D.K., T.A.H. and M.E.T. wrote the manuscript with input from S.S., B.M.P.V. and S.B.

Corresponding author

Correspondence to Marvin E. Tanenbaum.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Niels Gehring and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Boersma, S. et al. Cell 178, 458–472.e419 (2019): https://doi.org/10.1016/j.cell.2019.05.001

Hoek, T. et al. Mol. Cell 75, 324–339.e11 (2019): https://doi.org/10.1016/j.molcel.2019.05.008

Supplementary information

Reporting Summary

Supplementary Data 1

TransTrack software, example data, and a tutorial on how to use TransTrack.

Supplementary Data 2

RiboFitter software, example data, and expected outcomes of RiboFitter analysis.

Supplementary Data 3

Excel template for making a Kaplan–Meier plot.

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Khuperkar, D., Hoek, T.A., Sonneveld, S. et al. Quantification of mRNA translation in live cells using single-molecule imaging. Nat Protoc 15, 1371–1398 (2020). https://doi.org/10.1038/s41596-019-0284-x

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