Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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

Nature Protocols volume 15, pages 1371–1398 (2020)Cite this article

Subjects

Abstract

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).

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Tanenbaum, M. E., Stern-Ginossar, N., Weissman, J. S. & Vale, R. D. Regulation of mRNA translation during mitosis. eLife 4, e07957 (2015).

  2. Trcek, T., Larson, D. R., Moldon, A., Query, C. C. & Singer, R. H. Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast. Cell 147, 1484–1497 (2011).

    Article  CAS  Google Scholar 

  3. Ephrussi, A. & Lehmann, R. Induction of germ cell formation by oskar. Nature 358, 387–392 (1992).

    Article  CAS  Google Scholar 

  4. Lasko, P. mRNA localization and translational control in Drosophila oogenesis. Cold Spring Harb. Perspect. Biol. 4, a012294 (2012).

  5. Holt, C. E. & Schuman, E. M. The central dogma decentralized: new perspectives on RNA function and local translation in neurons. Neuron 80, 648–657 (2013).

    Article  CAS  Google Scholar 

  6. Shoemaker, C. J. & Green, R. Translation drives mRNA quality control. Nat. Struct. Mol. Biol. 19, 594–601 (2012).

    Article  CAS  Google Scholar 

  7. Schwanhausser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

    Article  Google Scholar 

  8. Pelechano, V., Wei, W. & Steinmetz, L. M. Widespread co-translational RNA decay reveals ribosome dynamics. Cell 161, 1400–1412 (2015).

    Article  CAS  Google Scholar 

  9. Hu, W., Sweet, T. J., Chamnongpol, S., Baker, K. E. & Coller, J. Co-translational mRNA decay in Saccharomyces cerevisiae. Nature 461, 225–229 (2009).

    Article  CAS  Google Scholar 

  10. He, F. & Jacobson, A. Nonsense-mediated mRNA decay: degradation of defective transcripts is only part of the story. Annu. Rev. Genet. 49, 339–366 (2015).

    Article  CAS  Google Scholar 

  11. Yan, X., Hoek, T. A., Vale, R. D. & Tanenbaum, M. E. Dynamics of translation of single mRNA molecules in vivo. Cell 165, 976–989 (2016).

    Article  CAS  Google Scholar 

  12. Ruijtenberg, S., Hoek, T. A., Yan, X. & Tanenbaum, M. E. Imaging translation dynamics of single mRNA molecules in live cells. Methods Mol. Biol. 1649, 385–404 (2018).

    Article  CAS  Google Scholar 

  13. Morisaki, T. et al. Real-time quantification of single RNA translation dynamics in living cells. Science 352, 1425–1429 (2016).

    Article  CAS  Google Scholar 

  14. Wang, C., Han, B., Zhou, R. & Zhuang, X. Real-time imaging of translation on single mRNA transcripts in live cells. Cell 165, 990–1001 (2016).

    Article  CAS  Google Scholar 

  15. Wu, B., Eliscovich, C., Yoon, Y. J. & Singer, R. H. Translation dynamics of single mRNAs in live cells and neurons. Science 352, 1430–1435 (2016).

    Article  CAS  Google Scholar 

  16. Pichon, X. et al. Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells. J. Cell Biol. 214, 769–781 (2016).

    Article  CAS  Google Scholar 

  17. Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    Article  CAS  Google Scholar 

  18. Chao, J. A., Patskovsky, Y., Almo, S. C. & Singer, R. H. Structural basis for the coevolution of a viral RNA-protein complex. Nat. Struct. Mol. Biol. 15, 103–105 (2008).

    Article  CAS  Google Scholar 

  19. Atkins, J. F., Loughran, G., Bhatt, P. R., Firth, A. E. & Baranov, P. V. Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use. Nucleic Acids Res. 44, 7007–7078 (2016).

    PubMed  PubMed Central  Google Scholar 

  20. Calvo, S. E., Pagliarini, D. J. & Mootha, V. K. Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl Acad. Sci. USA 106, 7507–7512 (2009).

    Article  CAS  Google Scholar 

  21. Boersma, S. et al. Multi-color single-molecule imaging uncovers extensive heterogeneity in mRNA decoding. Cell 178, 458–472.e419 (2019).

    Article  CAS  Google Scholar 

  22. Lyon, K. et al. Live-cell single RNA imaging reveals bursts of translational frameshifting. Mol. Cell 75, 172–183.e9 (2019).

    Article  CAS  Google Scholar 

  23. Hoek, T. A. et al. Single-molecule imaging uncovers rules governing nonsense-mediated mRNA decay. Mol. Cell 75, 324-339.e11 (2019).

  24. He, F. et al. Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5ʹ to 3ʹ mRNA decay pathways in yeast. Mol. Cell 12, 1439–1452 (2003).

    Article  CAS  Google Scholar 

  25. Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).

    Article  CAS  Google Scholar 

  26. Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).

    Article  CAS  Google Scholar 

  27. Brittis, P. A., Lu, Q. & Flanagan, J. G. Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target. Cell 110, 223–235 (2002).

    Article  CAS  Google Scholar 

  28. Aakalu, G., Smith, W. B., Nguyen, N., Jiang, C. & Schuman, E. M. Dynamic visualization of local protein synthesis in hippocampal neurons. Neuron 30, 489–502 (2001).

    Article  CAS  Google Scholar 

  29. Han, K. et al. Parallel measurement of dynamic changes in translation rates in single cells. Nat. Methods 11, 86–93 (2014).

    Article  CAS  Google Scholar 

  30. Leung, K. M. et al. Asymmetrical β-actin mRNA translation in growth cones mediates attractive turning to netrin-1. Nat. Neurosci. 9, 1247–1256 (2006).

    Article  CAS  Google Scholar 

  31. Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X. S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006).

    Article  CAS  Google Scholar 

  32. Rodriguez, A. J., Shenoy, S. M., Singer, R. H. & Condeelis, J. Visualization of mRNA translation in living cells. J. Cell Biol. 175, 67–76 (2006).

    Article  CAS  Google Scholar 

  33. Halstead, J. M. et al. Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science 347, 1367–1671 (2015).

    Article  CAS  Google Scholar 

  34. Katz, Z. B. et al. Mapping translation ‘hot-spots’ in live cells by tracking single molecules of mRNA and ribosomes. eLife 5, e10415 (2016).

  35. Wu, B., Buxbaum, A. R., Katz, Z. B., Yoon, Y. J. & Singer, R. H. Quantifying protein-mRNA interactions in single live cells. Cell 162, 211–220 (2015).

    Article  CAS  Google Scholar 

  36. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    Article  CAS  Google Scholar 

  37. Dunn, J. G., Foo, C. K., Belletier, N. G., Gavis, E. R. & Weissman, J. S. Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster. eLife 2, e01179 (2013).

    Article  Google Scholar 

  38. Young, D. J., Guydosh, N. R., Zhang, F., Hinnebusch, A. G. & Green, R. Rli1/ABCE1 recycles terminating ribosomes and controls translation reinitiation in 3ʹUTRs in vivo. Cell 162, 872–884 (2015).

    Article  CAS  Google Scholar 

  39. Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769 (1995).

    Article  CAS  Google Scholar 

  40. Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using µManager. Curr. Protoc. Mol. Biol., 92, 14.20 (2010).

  41. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

Author notes

  1. These authors contributed equally: Deepak Khuperkar, Tim A. Hoek.

Authors and Affiliations

  1. Oncode Institute, Hubrecht Institute—KNAW and University Medical Center Utrecht, Utrecht, the Netherlands

    Deepak Khuperkar, Tim A. Hoek, Stijn Sonneveld, Bram M. P. Verhagen, Sanne Boersma & Marvin E. Tanenbaum

Authors
  1. Deepak Khuperkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tim A. Hoek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stijn Sonneveld
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bram M. P. Verhagen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sanne Boersma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marvin E. Tanenbaum
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

Related links

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-019-0284-x

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing