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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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.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Niels Gehring and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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
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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
Trends in Cell Biology (2020)