Abstract
Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5′ terminal oligopyrimidine (5′ TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation.
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Acknowledgements
We thank F.-C. Tsai, a former graduate student of the Meyer lab, for the original Matlab code that was modified and used here to track single cells in the movies. The tunable ecDHFR plasmid was provided by T. Wandless (Stanford University). K.H. was supported by Stanford Graduate Fellowship. The work was supported by US National Institutes of Health grant GM030179.
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K.H. designed the reporter and the experiments, performed the experiments and analyzed the data. A.J. performed RNA FISH and provided the Matlab script to analyze the RNA FISH data. G.D. contributed to the development of the reporter. N.S. and O.M. provided MEFs. K.H. and T.M. interpreted the results and wrote the manuscript. All authors discussed the results and the manuscript. T.M. supervised the study.
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Han, K., Jaimovich, A., Dey, G. et al. Parallel measurement of dynamic changes in translation rates in single cells. Nat Methods 11, 86–93 (2014). https://doi.org/10.1038/nmeth.2729
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DOI: https://doi.org/10.1038/nmeth.2729
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