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Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system

Nature Protocols volume 13, pages 2268–2296 (2018)Cite this article

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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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Fig. 1: Scheme of MBS constructs.
Fig. 2: Tagging endogenous mRNAs with the new MBS systems.
Fig. 3: Two-color smFISH of DOA1 mRNA tagged with 24×MBSV6, and quantifications.
Fig. 4: Preparation of yeast spheroplasts and hybridization chamber for smFISH.
Fig. 5: DOA1 24×MBSV6-MCP2xyeGFP live imaging and quantification using Airlocalize.
Fig. 6: Two-color smFISH and live imaging for the cell-cycle mRNA ASH1.

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References

  1. Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  Google Scholar 

  2. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  Google Scholar 

  3. Zenklusen, D., Larson, D. R. & Singer, R. H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  Google Scholar 

  4. Gandhi, S. J., Zenklusen, D., Lionnet, T. & Singer, R. H. Transcription of functionally related constitutive genes is not coordinated. Nat. Struct. Mol. Biol. 18, 27–34 (2011).

    Article  CAS  Google Scholar 

  5. 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 

  6. Hocine, S., Vera, M., Zenklusen, D. & Singer, R. H. Promoter-autonomous functioning in a controlled environment using single molecule FISH. Sci. Rep. 5, 9934 (2015).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Fusco, D. et al. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161–167 (2003).

    Article  CAS  Google Scholar 

  9. Hocine, S., Raymond, P., Zenklusen, D., Chao, J. A. & Singer, R. H. Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat. Methods 10, 119–121 (2013).

    Article  CAS  Google Scholar 

  10. Grunwald, D. & Singer, R. H. In vivo imaging of labelled endogenous beta-actin mRNA during nucleocytoplasmic transport. Nature 467, 604–607 (2010).

    Article  Google Scholar 

  11. Golding, I., Paulsson, J., Zawilski, S. M. & Cox, E. C. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005).

    Article  CAS  Google Scholar 

  12. Bothma, J. P. et al. Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. eLife 4, e07956 (2015).

  13. Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14, 796–806 (2007).

    Article  CAS  Google Scholar 

  14. Lionnet, T. et al. A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat. Methods 8, 165–170 (2011).

    Article  CAS  Google Scholar 

  15. Park, H. Y. et al. Visualization of dynamics of single endogenous mRNA labeled in live mouse. Science 343, 422–424 (2014).

    Article  CAS  Google Scholar 

  16. 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 

  17. Long, X., Colonell, J., Wong, A. M., Singer, R. H. & Lionnet, T. Quantitative mRNA imaging throughout the entire Drosophila brain. Nat. Methods 14, 703–706 (2017).

    Article  CAS  Google Scholar 

  18. Tutucci, E., Livingston, N. M., Singer, R. H. & Wu, B. Imaging mRNA in vivo, from birth to death. Annu. Rev. Biophys. 47, 85–106 (2018).

    Article  CAS  Google Scholar 

  19. Vera, M., Biswas, J., Senecal, A., Singer, R. H. & Park, H. Y. Single-cell and single-molecule analysis of gene expression regulation. Annu. Rev. Genet. 50, 267–291 (2016).

    Article  CAS  Google Scholar 

  20. Garcia, J. F. & Parker, R. MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA 21, 1393–1395 (2015).

    Article  CAS  Google Scholar 

  21. Garcia, J. F. & Parker, R. Ubiquitous accumulation of 3′ mRNA decay fragments in Saccharomyces cerevisiae mRNAs with chromosomally integrated MS2 arrays. RNA 22, 657–659 (2016).

    Article  CAS  Google Scholar 

  22. Haimovich, G. et al. Use of the MS2 aptamer and coat protein for RNA localization in yeast: a response to “MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system”. RNA 22, 660–666 (2016).

    Article  CAS  Google Scholar 

  23. Heinrich, S., Sidler, C. L., Azzalin, C. M. & Weis, K. Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. RNA 23, 134–141 (2017).

    Article  CAS  Google Scholar 

  24. Tutucci, E. et al. An improved MS2 system for accurate reporting of the mRNA life cycle. Nat. Methods 15, 81–89 (2018).

    Article  CAS  Google Scholar 

  25. Mueller, F. et al. FISH-quant: automatic counting of transcripts in 3D FISH images. Nat. Methods 10, 277–278 (2013).

    Article  CAS  Google Scholar 

  26. Bernardi, A. & Spahr, P. F. Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17. Proc. Natl. Acad. Sci. USA 69, 3033–3037 (1972).

    Article  CAS  Google Scholar 

  27. Lowary, P. T. & Uhlenbeck, O. C. An RNA mutation that increases the affinity of an RNA-protein interaction. Nucleic Acids Res. 15, 10483–10493 (1987).

    Article  CAS  Google Scholar 

  28. Valegard, K., Murray, J. B., Stockley, P. G., Stonehouse, N. J. & Liljas, L. Crystal structure of an RNA bacteriophage coat protein–operator complex. Nature 371, 623–626 (1994).

    Article  CAS  Google Scholar 

  29. Valegard, K. et al. The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions. J. Mol. Biol. 270, 724–738 (1997).

    Article  CAS  Google Scholar 

  30. Peabody, D. S. The RNA binding site of bacteriophage MS2 coat protein. EMBO J. 12, 595–600 (1993).

    Article  CAS  Google Scholar 

  31. Peabody, D. S. & Ely, K. R. Control of translational repression by protein-protein interactions. Nucleic Acids Res. 20, 1649–1655 (1992).

    Article  CAS  Google Scholar 

  32. Wu, B. et al. Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev. 29, 876–886 (2015).

    Article  CAS  Google Scholar 

  33. Tantale, K. et al. A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting. Nat. Commun. 7, 12248 (2016).

    Article  CAS  Google Scholar 

  34. Martin, R. M., Rino, J., Carvalho, C., Kirchhausen, T. & Carmo-Fonseca, M. Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity. Cell Rep. 4, 1144–1155 (2013).

    Article  CAS  Google Scholar 

  35. Slobodin, B. & Gerst, J. E. RaPID: an aptamer-based mRNA affinity purification technique for the identification of RNA and protein factors present in ribonucleoprotein complexes. Methods Mol. Biol. 714, 387–406 (2011).

    Article  CAS  Google Scholar 

  36. Yoon, J. H. & Gorospe, M. Identification of mRNA-interacting factors by MS2-TRAP (MS2-tagged RNA affinity purification). Methods Mol. Biol. 1421, 15–22 (2016).

    Article  CAS  Google Scholar 

  37. 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 

  38. Larson, D. R., Zenklusen, D., Wu, B., Chao, J. A. & Singer, R. H. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332, 475–478 (2011).

    Article  CAS  Google Scholar 

  39. Smith, C. et al. In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p. J. Cell Biol. 211, 1121–1130 (2015).

    Article  Google Scholar 

  40. Saroufim, M. A. et al. The nuclear basket mediates perinuclear mRNA scanning in budding yeast. J. Cell Biol. 211, 1131–1140 (2015).

    Article  CAS  Google Scholar 

  41. Wu, B., Chao, J. A. & Singer, R. H. Fluorescence fluctuation spectroscopy enables quantitative imaging of single mRNAs in living cells. Biophys. J. 102, 2936–2944 (2012).

    Article  CAS  Google Scholar 

  42. Brodsky, A. S. & Silver, P. A. Identifying proteins that affect mRNA localization in living cells. Methods 26, 151–155 (2002).

    Article  CAS  Google Scholar 

  43. Chung, S. & Takizawa, P. A. In vivo visualization of RNA using the U1A-based tagged RNA system. Methods Mol. Biol. 714, 221–235 (2011).

    Article  CAS  Google Scholar 

  44. Lange, S. et al. Simultaneous transport of different localized mRNA species revealed by live-cell imaging. Traffic 9, 1256–1267 (2008).

    Article  CAS  Google Scholar 

  45. Urbanek, M. O., Galka-Marciniak, P., Olejniczak, M. & Krzyzosiak, W. J. RNA imaging in living cells—methods and applications. RNA Biol. 11, 1083–1095 (2014).

    Article  Google Scholar 

  46. Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).

    Article  CAS  Google Scholar 

  47. Strack, R. L., Disney, M. D. & Jaffrey, S. R. A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat. Methods 10, 1219–1224 (2013).

    Article  CAS  Google Scholar 

  48. Filonov, G. S., Moon, J. D., Svensen, N. & Jaffrey, S. R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J. Am. Chem. Soc. 136, 16299–16308 (2014).

    Article  CAS  Google Scholar 

  49. Song, W. et al. Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex. Nat. Chem. Biol. 13, 1187–1194 (2017).

    Article  CAS  Google Scholar 

  50. Dolgosheina, E. V. et al. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem. Biol. 9, 2412–2420 (2014).

    Article  CAS  Google Scholar 

  51. Engelhart, A. E. RNA imaging: a tale of two G-quadruplexes. Nat. Chem. Biol. 13, 1140–1141 (2017).

    Article  CAS  Google Scholar 

  52. Pijlman, G. P. et al. A highly structured, nuclease-resistant, noncoding RNA produced by flaviviruses is required for pathogenicity. Cell Host Microbe 4, 579–591 (2008).

    Article  CAS  Google Scholar 

  53. MacFadden, A. et al. Mechanism and structural diversity of exoribonuclease-resistant RNA structures in flaviviral RNAs. Nat. Commun. 9, 119 (2018).

    Article  Google Scholar 

  54. Tyagi, S., Bratu, D. P. & Kramer, F. R. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49–53 (1998).

    Article  CAS  Google Scholar 

  55. Tyagi, S. & Kramer, F. R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).

    Article  CAS  Google Scholar 

  56. Chen, A. K., Behlke, M. A. & Tsourkas, A. Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells. Nucleic Acids Res. 35, e105 (2007).

    Article  Google Scholar 

  57. Mhlanga, M. M., Vargas, D. Y., Fung, C. W., Kramer, F. R. & Tyagi, S. tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells. Nucleic Acids Res. 33, 1902–1912 (2005).

    Article  CAS  Google Scholar 

  58. Chen, A. K., Davydenko, O., Behlke, M. A. & Tsourkas, A. Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells. Nucleic Acids Res. 38, e148 (2010).

    Article  Google Scholar 

  59. Zhao, D. et al. Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2′-O-methyl RNA molecular beacons. Biomaterials 100, 172–183 (2016).

    Article  CAS  Google Scholar 

  60. Chen, M. et al. A molecular beacon-based approach for live-cell imaging of RNA transcripts with minimal target engineering at the single-molecule level. Sci. Rep. 7, 1550 (2017).

    Article  Google Scholar 

  61. Nelles, D. A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).

    Article  CAS  Google Scholar 

  62. Lahtvee, P. J. et al. Absolute quantification of protein and mRNA abundances demonstrate variability in gene-specific translation efficiency in yeast. Cell Syst. 4, 495–504 (2017).

    Article  CAS  Google Scholar 

  63. Marguerat, S. et al. Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells. Cell 151, 671–683 (2012).

    Article  CAS  Google Scholar 

  64. Trcek, T., Lionnet, T., Shroff, H. & Lehmann, R. mRNA quantification using single-molecule FISH in Drosophila embryos. Nat. Protoc. 12, 1326–1348 (2017).

    Article  CAS  Google Scholar 

  65. Trcek, T. et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat. Protoc. 7, 408–419 (2012).

    Article  CAS  Google Scholar 

  66. 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 

  67. Grimm, J. B. et al. Bright photoactivatable fluorophores for single-molecule imaging. Nat. Methods 13, 985–988 (2016).

    Article  CAS  Google Scholar 

  68. Long, R. M. et al. Characterization of transport and localization of ASH1 mRNA in yeast. Mol. Biol. Cell 8, 2060 (1997).

    Google Scholar 

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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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Authors and Affiliations

  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Evelina Tutucci, Maria Vera & Robert H. Singer

  2. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA

    Robert H. Singer

  3. Janelia Research Campus of the HHMI, Ashburn, VA, USA

    Robert H. Singer

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  1. Evelina Tutucci
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  2. Maria Vera
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  3. Robert H. Singer
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Contributions

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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Correspondence to Robert H. Singer.

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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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Related links

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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