Advertisement

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

  • Azra Lari
  • Farzin Farzam
  • Pierre Bensidoun
  • Marlene Oeffinger
  • Daniel Zenklusen
  • David Grunwald
  • Ben MontpetitEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

mRNP export Nuclear pore complex NPC Live-cell imaging Single molecule Budding yeast S. cerevisiae Fluorescent imaging PP7 Superregistration 

Notes

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.

References

  1. 1.
    Kim SJ, Fernandez-Martinez J, Nudelman I et al (2018) Integrative structure and functional anatomy of a nuclear pore complex. Nature 555:475CrossRefGoogle Scholar
  2. 2.
    Folkmann A, Noble K, Cole C (2011) Dbp5, Gle1-IP6, and Nup159: a working model for mRNP export. Nucleus 2(6):540–548CrossRefGoogle Scholar
  3. 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.0336594100CrossRefPubMedPubMedCentralGoogle Scholar
  4. 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.201503070CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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/nature09438CrossRefPubMedPubMedCentralGoogle Scholar
  6. 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.201503135CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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.1201781109CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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/ncb2056CrossRefGoogle Scholar
  9. 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/cr400002gCrossRefPubMedGoogle Scholar
  10. 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/B978-0-12-417160-2.00001-1CrossRefGoogle Scholar
  11. 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.2012.02.011CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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.201600124CrossRefGoogle Scholar
  13. 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.2018.07.022CrossRefPubMedGoogle Scholar
  14. 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.2305CrossRefPubMedGoogle Scholar
  15. 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)80143-4CrossRefGoogle Scholar
  16. 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.1202142CrossRefPubMedPubMedCentralGoogle Scholar
  17. 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.4502CrossRefPubMedGoogle Scholar
  18. 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 24:2519–2524CrossRefGoogle Scholar
  19. 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. 20.
    Sherman BF, Sherman MF, Enzymol M (2003) Getting started with yeast. Contents 41:3–41Google Scholar
  21. 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)50957-5CrossRefPubMedGoogle Scholar
  22. 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-UCrossRefPubMedGoogle Scholar
  23. 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/s41596-018-0037-2CrossRefPubMedGoogle Scholar
  24. 24.
    Amberg DC, Burke DJ, Strathern JN (2006) Tetrad dissection. Cold Spring Harb Protoc 2006:pdb.prot4181.  https://doi.org/10.1101/pdb.prot4181CrossRefGoogle Scholar
  25. 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.2019CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefGoogle Scholar
  27. 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/btp184CrossRefPubMedPubMedCentralGoogle Scholar
  28. 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 300:1–59CrossRefGoogle Scholar
  29. 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.200411005CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Shaner NC, Lambert GG, Chammas A et al (2013) A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods 10:407CrossRefGoogle Scholar
  31. 31.
    Shcherbo D, Merzlyak EM, Chepurnykh TV et al (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4:741CrossRefGoogle Scholar
  32. 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.200209116CrossRefPubMedPubMedCentralGoogle Scholar
  33. 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.2016.01.006CrossRefPubMedGoogle Scholar
  34. 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_4CrossRefPubMedGoogle Scholar
  35. 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.051797.115CrossRefPubMedPubMedCentralGoogle Scholar
  36. 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.057786.116CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Sengupta SK, Kay SM (1995) Fundamentals of statistical signal processing: estimation theory. Technometrics 37:465.  https://doi.org/10.2307/1269750CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Azra Lari
    • 1
  • Farzin Farzam
    • 2
  • Pierre Bensidoun
    • 3
    • 4
  • Marlene Oeffinger
    • 3
    • 4
    • 5
  • Daniel Zenklusen
    • 3
  • David Grunwald
    • 2
  • Ben Montpetit
    • 1
    • 6
    Email author
  1. 1.Department of Cell BiologyUniversity of AlbertaEdmontonCanada
  2. 2.RNA Therapeutics InstituteUniversity of Massachusetts Medical SchoolWorcesterUSA
  3. 3.Département de Biochimie et Médecine MoléculaireUniversité de MontréalMontréalCanada
  4. 4.Institut de Recherches Cliniques de MontréalMontréalCanada
  5. 5.Faculty of Medicine, Division of Experimental MedicineMcGill UniversityMontréalCanada
  6. 6.Department of Viticulture and EnologyUniversity of California, DavisDavisUSA

Personalised recommendations