Abstract
The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast. The procedure is divided into three stages: tagging of endogenous gene with MBSV6 (~2 weeks), a two-color single-molecule RNA fluorescent in situ hybridization (smFISH) procedure to quantitatively assess whether mRNAs tagged with MS2 and MS2-coat protein (MCP) behave like untagged mRNAs (2 d, plus additional time for quantification), and a procedure to quantify single mRNAs by live imaging using wide-field microscopy (1 d, plus additional time for quantification). With this method it is now possible to interrogate all phases of mRNA expression, from transcription through decay. The described protocol is designed for S. cerevisiae; however, we think that our approach and the considerations discussed here can be extended to Escherichia coli, Drosophila, Caenorhabditis elegans, and mammalian cells.
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Acknowledgements
We thank X. Meng and N. Ghazale for technical help, and E. Bertrand for critical reading of the manuscript. This work was supported by the NIH (grant GM57071 to R.H.S.; grant AG055083 to M.V.) and the Swiss National Science Foundation (Fellowships P2GEP3_155692 and P300PA_164717 to E.T.).
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E.T. designed and performed the experiments and analyzed the data. E.T., M.V., and R.H.S. conceived the MBSV6 system. E.T., M.V., and R.H.S. wrote the manuscript. R.H.S. supervised the research.
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The material in this manuscript is the subject of a provisional application to the US Patent and Trademark Office (no. 62/487058). It has not been licensed to any corporation, and the authors (R.H.S., E.T., and M.V.) are the sole inventors.
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Tutucci, E. et al. Nat. Methods 15, 81–89 (2018) https://doi.org/10.1038/nmeth.4502
Trcek, T. et al. Nat. Protoc. 7, 408–419 (2012) https://doi.org/10.1038/nprot.2011.451
Bertrand, E. et al. Mol. Cell 2, 437–445 (1998) https://doi.org/10.1016/S1097-2765(00)80143-4
Wu, B. et al. Genes Dev. 29, 876–886 (2015) https://doi.org/10.1101/gad.259358.115
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Tutucci, E., Vera, M. & Singer, R.H. Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system. Nat Protoc 13, 2268–2296 (2018). https://doi.org/10.1038/s41596-018-0037-2
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DOI: https://doi.org/10.1038/s41596-018-0037-2
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