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Simultaneous Detection of mRNA and Protein in S. cerevisiae by Single-Molecule FISH and Immunofluorescence

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RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

Abstract

Single-molecule fluorescent in situ hybridization (smFISH) enables the detection and quantification of endogenous mRNAs within intact fixed cells. This method utilizes tens of singly labeled fluorescent DNA probes hybridized against the mRNA of interest, which can be detected by using standard wide-field fluorescence microscopy. This approach provides the means to generate absolute quantifications of gene expression within single cells, which can be used to link molecular fluctuations to phenotypes. To be able to correlate the expression of an mRNA and a protein of interest in individual cells, we combined smFISH with immunofluorescence (IF) in yeast cells. Here, we present our smFISH-IF protocol to visualize and quantify two cell cycle-controlled mRNAs (CLN2 and ASH1) and the cell cycle marker alpha-tubulin in S. cerevisiae. This protocol, which is performed over 2 days, can be used to visualize up to three colors at the time (i.e., two mRNAs, one protein). Even if the described protocol is designed for S. cerevisiae, we think that the considerations discussed here can be useful to develop and troubleshoot smFISH-IF protocols for other model organisms.

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References

  1. Bruggeman FJ, Teusink B (2018) Living with noise: On the propagation of noise from molecules to phenotype and fitness. Curr Opin Syst Biol 8:144–150

    Article  Google Scholar 

  2. Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590

    Article  CAS  PubMed  Google Scholar 

  3. Zenklusen D, Larson DR, Singer RH (2008) Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 15(12):1263–1271. https://doi.org/10.1038/nsmb.1514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4(10):e309. https://doi.org/10.1371/journal.pbio.0040309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Tutucci E, Livingston NM, Singer RH, Wu B (2018) Imaging mRNA in vivo, from birth to death. Annu Rev Biophys 47:85–106. https://doi.org/10.1146/annurev-biophys-070317-033037

    Article  CAS  PubMed  Google Scholar 

  6. Pichon X, Bastide A, Safieddine A, Chouaib R, Samacoits A, Basyuk E, Peter M, Mueller F, Bertrand E (2016) Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells. J Cell Biol 214(6):769–781. https://doi.org/10.1083/jcb.201605024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging technology. Nat Methods 8(4 Suppl):S12–S19. https://doi.org/10.1038/nmeth.1573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Farack L, Egozi A, Itzkovitz S (2018) Single molecule approaches for studying gene regulation in metabolic tissues. Diabetes Obes Metab 20(Suppl 2):145–156. https://doi.org/10.1111/dom.13390

    Article  CAS  PubMed  Google Scholar 

  9. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. https://doi.org/10.1038/nmeth.1253

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Long X, Colonell J, Wong AM, Singer RH, Lionnet T (2017) Quantitative mRNA imaging throughout the entire Drosophila brain. Nat Methods 14(7):703–706. https://doi.org/10.1038/nmeth.4309

    Article  CAS  PubMed  Google Scholar 

  11. Pichon X, Lagha M, Mueller F, Bertrand E (2018) A growing toolbox to image gene expression in single cells: sensitive approaches for demanding challenges. Mol Cell 71(3):468–480. https://doi.org/10.1016/j.molcel.2018.07.022

    Article  CAS  PubMed  Google Scholar 

  12. Vera M, Biswas J, Senecal A, Singer RH, Park HY (2016) Single-cell and single-molecule analysis of gene expression regulation. Annu Rev Genet 50:267–291. https://doi.org/10.1146/annurev-genet-120215-034854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Trcek T, Larson DR, Moldon A, Query CC, Singer RH (2011) Single-molecule mRNA decay measurements reveal promoter-regulated mrna stability in yeast. Cell 147(7):1484–1497. https://doi.org/10.1016/j.cell.2011.11.051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Long RM, Singer RH, Meng X, Gonzalez I, Nasmyth K, Jansen RP (1997) Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 277(5324):383–387

    Article  CAS  PubMed  Google Scholar 

  15. Moor AE, Golan M, Massasa EE, Lemze D, Weizman T, Shenhav R, Baydatch S, Mizrahi O, Winkler R, Golani O, Stern-Ginossar N, Itzkovitz S (2017) Global mRNA polarization regulates translation efficiency in the intestinal epithelium. Science 357:1299–1303. https://doi.org/10.1126/science.aan2399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Buxbaum AR, Wu B, Singer RH (2014) Single beta-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science 343(6169):419–422. https://doi.org/10.1126/science.1242939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Levsky JM, Shenoy SM, Pezo RC, Singer RH (2002) Single-cell gene expression profiling. Science 297(5582):836–840. https://doi.org/10.1126/science.1072241

    Article  CAS  PubMed  Google Scholar 

  18. Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X (2015) RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348(6233):aaa6090. https://doi.org/10.1126/science.aaa6090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lubeck E, Cai L (2012) Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat Methods 9(7):743–748. https://doi.org/10.1038/nmeth.2069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lubeck E, Coskun AF, Zhiyentayev T, Ahmad M, Cai L (2014) Single-cell in situ RNA profiling by sequential hybridization. Nat Methods 11(4):360–361. https://doi.org/10.1038/nmeth.2892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Moffitt JR, Zhuang X (2016) RNA imaging with multiplexed error-robust fluorescence in situ hybridization (MERFISH). Methods Enzymol 572:1–49. https://doi.org/10.1016/bs.mie.2016.03.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wittenberg C, Sugimoto K, Reed SI (1990) G1-specific cyclins of S. cerevisiae: cell cycle periodicity, regulation by mating pheromone, and association with the p34CDC28 protein kinase. Cell 62(2):225–237

    Article  CAS  PubMed  Google Scholar 

  23. Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2(4):437–445

    Article  CAS  PubMed  Google Scholar 

  24. Jansen RP, Dowzer C, Michaelis C, Galova M, Nasmyth K (1996) Mother cell-specific HO expression in budding yeast depends on the unconventional myosin myo4p and other cytoplasmic proteins. Cell 84(5):687–697

    Article  CAS  PubMed  Google Scholar 

  25. Long RM, Chartrand P, Gu W, Meng XH, Schaefer MR, Singer RH (1997) Characterization of transport and localization of ASH1 mRNA in yeast. Mol Biol Cell 8:2060–2060

    Google Scholar 

  26. Tutucci E, Vera M, Biswas J, Garcia J, Parker R, Singer RH (2018) An improved MS2 system for accurate reporting of the mRNA life cycle. Nat Methods 15(1):81–89. https://doi.org/10.1038/nmeth.4502

    Article  CAS  PubMed  Google Scholar 

  27. Cosma MP (2004) Daughter-specific repression of Saccharomyces cerevisiae HO: Ash1 is the commander. EMBO Rep 5(10):953–957. https://doi.org/10.1038/sj.embor.7400251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pereira G, Schiebel E (2001) The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events. Curr Opin Cell Biol 13(6):762–769

    Article  CAS  PubMed  Google Scholar 

  29. Bayer LV, Batish M, Formel SK, Bratu DP (2015) Single-molecule RNA in situ hybridization (smFISH) and Immunofluorescence (IF) in the Drosophila egg chamber. Methods Mol Biol 1328:125–136. https://doi.org/10.1007/978-1-4939-2851-4_9

    Article  CAS  PubMed  Google Scholar 

  30. Eliscovich C, Shenoy SM, Singer RH (2017) Imaging mRNA and protein interactions within neurons. Proc Natl Acad Sci U S A 114(10):E1875–E1884. https://doi.org/10.1073/pnas.1621440114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mueller F, Senecal A, Tantale K, Marie-Nelly H, Ly N, Collin O, Basyuk E, Bertrand E, Darzacq X, Zimmer C (2013) FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods 10(4):277–278. https://doi.org/10.1038/nmeth.2406

    Article  CAS  PubMed  Google Scholar 

  32. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  33. Maekiniemi A, Singer RH, Tutucci E (2020) Single molecule mRNA fluorescent in situ hybridization combined with immunofluorescence in S. cerevisiae: dataset and quantification. Data in Brief. https://doi.org/10.1016/j.dib.2020.105511

  34. Tutucci E, Maekiniemi A, Singer RH (2020) Single molecule mRNA fluorescent in situ hybridization combined to immunofluorescence in S. cerevisiae: dataset and quantification. Mendeley Data, v4, https://doi.org/10.17632/bcmn9cxyzs.4

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Acknowledgments

We would like to thank Carolina Eliscovich and Noura Ghazale for helpful discussion during the protocol development. This work was supported by NIH Grant GM57071 to R.H.S. E.T. was supported by Swiss National Science Foundation Fellowships P2GEP3_155692 and P300PA_164717.

Contributions: E.T. designed the protocol, performed the experiments, and analyzed the data. E.T. and R.H.S. wrote the manuscript.

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Author notes

  1. Evelina Tutucci

    Present address: Systems Biology Lab, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

Authors and Affiliations

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

    Evelina Tutucci & Robert H. Singer

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

    Robert H. Singer

Authors
  1. Evelina Tutucci
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  2. Robert H. Singer
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Corresponding authors

Correspondence to Evelina Tutucci or Robert H. Singer .

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Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

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Tutucci, E., Singer, R.H. (2020). Simultaneous Detection of mRNA and Protein in S. cerevisiae by Single-Molecule FISH and Immunofluorescence. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

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