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Live-Cell Imaging of mRNP–NPC Interactions in Budding Yeast

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Imaging Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2038))

Abstract

Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of interactions between mRNPs and nuclear pore complexes (NPCs) in the yeast S. cerevisiae, including mRNP export. We describe in detail the steps that allow for high-resolution live-cell mRNP imaging and measurement of mRNP interactions with NPCs using simultaneous two-color imaging. Our protocol discusses yeast strain construction, choice of marker proteins to label the nuclear pore complex, as well as imaging conditions that allow high signal-to-noise data acquisition. Moreover, we describe various aspects of postacquisition image analysis for single molecule tracking and image registration allowing for the characterization of mRNP–NPC interactions.

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References

  1. Kim SJ, Fernandez-Martinez J, Nudelman I et al (2018) Integrative structure and functional anatomy of a nuclear pore complex. Nature 555:475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Folkmann A, Noble K, Cole C (2011) Dbp5, Gle1-IP6, and Nup159: a working model for mRNP export. Nucleus 2(6):540–548

    Article  PubMed  PubMed Central  Google Scholar 

  3. Green DM, Johnson CP, Hagan H, Corbett AH (2003) The C-terminal domain of myosin-like protein 1 (Mlp1p) is a docking site for heterogeneous nuclear ribonucleoproteins that are required for mRNA export. Proc Natl Acad Sci U S A 100:1010–1015. https://doi.org/10.1073/pnas.0336594100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Saroufim M-A, Bensidoun P, Raymond P et al (2015) The nuclear basket mediates perinuclear mRNA scanning in budding yeast. J Cell Biol 211:1131–1140. https://doi.org/10.1083/jcb.201503070

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Grünwald D, Singer RH (2010) In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. Nature 467:604–607. https://doi.org/10.1038/nature09438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Smith C, Lari A, Derrer CP et al (2015) In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p. J Cell Biol 211:1121–1130. https://doi.org/10.1083/jcb.201503135

    Article  PubMed  PubMed Central  Google Scholar 

  7. Siebrasse JP, Kaminski T, Kubitscheck U (2012) Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy. Proc Natl Acad Sci U S A 109:9426–9431. https://doi.org/10.1073/pnas.1201781109

    Article  PubMed  PubMed Central  Google Scholar 

  8. Mor A, Suliman S, Ben-Yishay R et al (2010) Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nat Cell Biol 12:543–552. https://doi.org/10.1038/ncb2056

    Article  CAS  PubMed  Google Scholar 

  9. Niño CA, Hérissant L, Babour A, Dargemont C (2013) mRNA nuclear export in yeast. Chem Rev 113:8523–8545. https://doi.org/10.1021/cr400002g

    Article  CAS  PubMed  Google Scholar 

  10. Floch AG, Palancade B, Doye V (2014) Fifty years of nuclear pores and nucleocytoplasmic transport studies: multiple tools revealing complex rules. Methods Cell Biol 122C:1–40. https://doi.org/10.1016/B978-0-12-417160-2.00001-1

    Article  CAS  Google Scholar 

  11. Oeffinger M, Zenklusen D (2012) To the pore and through the pore: a story of mRNA export kinetics. Biochim Biophys Acta 1819:494–506. https://doi.org/10.1016/j.bbagrm.2012.02.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Heinrich S, Derrer CP, Lari A et al (2017) Temporal and spatial regulation of mRNA export: single particle RNA-imaging provides new tools and insights. BioEssays 39. https://doi.org/10.1002/bies.201600124

    Article  Google Scholar 

  13. 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 71:468–480. https://doi.org/10.1016/J.MOLCEL.2018.07.022

    Article  CAS  PubMed  Google Scholar 

  14. Hocine S, Raymond P, Zenklusen D et al (2013) Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat Methods 10:119–121. https://doi.org/10.1038/nmeth.2305

    Article  CAS  PubMed  Google Scholar 

  15. Bertrand E, Chartrand P, Schaefer M et al (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2:437–445. https://doi.org/10.1016/S1097-2765(00)80143-4

    Article  CAS  PubMed  Google Scholar 

  16. Larson DR, Zenklusen D, Wu B et al (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332:475–478. https://doi.org/10.1126/science.1202142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tutucci E, Vera M, Biswas J et al (2018) An improved MS2 system for accurate reporting of the mRNA life cycle. Nat Methods 15:81–89. https://doi.org/10.1038/nmeth.4502

    Article  CAS  PubMed  Google Scholar 

  18. Güldener U, Heck S, Fielder T et al (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524

    Article  PubMed  PubMed Central  Google Scholar 

  19. Chan LY, Mugler CF, Heinrich S et al (2018) Non-invasive measurement of mRNA decay reveals translation initiation as the major determinant of mRNA stability. elife 7. https://doi.org/10.7554/eLife.32536

  20. Sherman BF, Sherman MF, Enzymol M (2003) Getting started with yeast. Contents 41:3–41

    Google Scholar 

  21. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96. https://doi.org/10.1016/S0076-6879(02)50957-5

    Article  CAS  PubMed  Google Scholar 

  22. Longtine MS, McKenzie A 3rd, Demarini DJ et al (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953–961. https://doi.org/10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U

    Article  CAS  PubMed  Google Scholar 

  23. Tutucci E, Vera M, Singer RH (2018) Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system. Nat Protoc 13:2268–2296. https://doi.org/10.1038/s41596-018-0037-2

    Article  CAS  PubMed  Google Scholar 

  24. Amberg DC, Burke DJ, Strathern JN (2006) Tetrad dissection. Cold Spring Harb Protoc 2006:pdb.prot4181. https://doi.org/10.1101/pdb.prot4181

    Article  Google Scholar 

  25. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  26. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25:1463–1465. https://doi.org/10.1093/bioinformatics/btp184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Colquhoun D, Hawkes AG (1982) On the stochastic properties of bursts of single ion channel openings and of clusters of bursts. Philos Trans R Soc Lond Ser B Biol Sci 300:1–59

    Article  CAS  Google Scholar 

  29. Kubitscheck U, Grünwald D, Hoekstra A et al (2005) Nuclear transport of single molecules. J Cell biol 168:233–243. https://doi.org/10.1083/jcb.200411005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shaner NC, Lambert GG, Chammas A et al (2013) A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10:407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shcherbo D, Merzlyak EM, Chepurnykh TV et al (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4:741

    Article  CAS  PubMed  Google Scholar 

  32. Ryan KJ, McCaffery JM, Wente SR (2003) The Ran GTPase cycle is required for yeast nuclear pore complex assembly. J Cell Biol 160:1041–1053. https://doi.org/10.1083/jcb.200209116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bensidoun P, Raymond P, Oeffinger M, Zenklusen D (2016) Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae. Methods 98:104–114. https://doi.org/10.1016/j.ymeth.2016.01.006

    Article  CAS  PubMed  Google Scholar 

  34. Trcek T, Rahman S, Zenklusen D (2018) Measuring mRNA decay in budding yeast using single molecule FISH. Methods Mol Biol 1720:35–54. https://doi.org/10.1007/978-1-4939-7540-2_4

    Article  CAS  PubMed  Google Scholar 

  35. Garcia JF, Parker R (2015) 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. https://doi.org/10.1261/rna.051797.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Heinrich S, Sidler CL, Azzalin CM, Weis K (2017) Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. RNA 23:134–141. https://doi.org/10.1261/rna.057786.116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sengupta SK, Kay SM (1995) Fundamentals of statistical signal processing: estimation theory. Technometrics 37:465. https://doi.org/10.2307/1269750

    Article  Google Scholar 

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Acknowledgments

We would like to acknowledge the laboratories of Drs. Robert Singer and Karsten Weis for reagents and support of previous works related to the methods described here. A.L. was supported by a Natural Sciences and Engineering Research Council Canada Graduate Scholarship; D.Z. is supported by the Canadian Institutes of Health (Project Grant-366682), Fonds de recherche du Québec—Santé (Chercheur-boursier Junior 2), Canada Foundation for Innovation, and the Natural Sciences and Engineering Research Council; D.G. by a National Institute of General Medical Sciences award (5R01GM123541); B.M. and D.G. by a National Institute of General Medical Sciences award (5R01GM124120). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Author notes

  1. Azra Lari, Farzin Farzam and Pierre Bensidoun contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, University of Alberta, Edmonton, Canada

    Azra Lari & Ben Montpetit

  2. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA

    Farzin Farzam & David Grunwald

  3. Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC, Canada

    Pierre Bensidoun, Marlene Oeffinger & Daniel Zenklusen

  4. Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada

    Pierre Bensidoun & Marlene Oeffinger

  5. Faculty of Medicine, Division of Experimental Medicine, McGill University, Montréal, QC, Canada

    Marlene Oeffinger

  6. Department of Viticulture and Enology, University of California, Davis, Davis, CA, USA

    Ben Montpetit

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  1. Azra Lari
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  4. Marlene Oeffinger
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Correspondence to Ben Montpetit .

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Editors and Affiliations

  1. The Mina & Everard Goodman, Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan, Israel

    Yaron Shav-Tal

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Lari, A. et al. (2019). Live-Cell Imaging of mRNP–NPC Interactions in Budding Yeast. In: Shav-Tal, Y. (eds) Imaging Gene Expression. Methods in Molecular Biology, vol 2038. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9674-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9674-2_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9673-5

  • Online ISBN: 978-1-4939-9674-2

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