Skip to main content
SpringerLink
Log in

Detection of mRNAs Anchored to the Nuclear Envelope During Export Inhibition in Living Cells

  • Protocol
  • First Online:
Imaging Gene Expression

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

Abstract

Export of mRNA transcripts from the cell nucleus is a complex and multistep process, regulated by various proteins and control mechanisms. Recent studies have demonstrated the rapid passage of mRNA–protein complexes (mRNPs) through the nuclear pore complex (NPC) as well as the ability to detect mRNPs stalled at the NPC during inhibition of the mRNA export process. In this chapter, we describe ways to block mRNA export and present an image analysis method to identify mRNPs stuck at the NPC during such blocks. Using the MS2 mRNA-tagging system to track single mRNPs in living cells we are able to examine their intracellular distribution and dynamics both in the nucleoplasm and at the nuclear periphery. We use this method to identify and count the number of static mRNPs anchored to the nuclear envelope under different conditions of mRNA export inhibition.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Fahrenkrog B, Aebi U (2003) The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat Rev Mol Cell Biol 4(10):757–766

    Article  CAS  Google Scholar 

  2. Fernandez-Martinez J, Rout MP (2012) A jumbo problem: mapping the structure and functions of the nuclear pore complex. Curr Opin Cell Biol 24(1):92–99. https://doi.org/10.1016/j.ceb.2011.12.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Adams RL, Wente SR (2013) Uncovering nuclear pore complexity with innovation. Cell 152(6):1218–1221. https://doi.org/10.1016/j.cell.2013.02.042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Maimon T, Elad N, Dahan I, Medalia O (2012) The human nuclear pore complex as revealed by cryo-electron tomography. Structure 20(6):998–1006. https://doi.org/10.1016/j.str.2012.03.025

    Article  CAS  PubMed  Google Scholar 

  5. Walde S, Kehlenbach RH (2010) The Part and the Whole: functions of nucleoporins in nucleocytoplasmic transport. Trends Cell Biol 20(8):461–469. https://doi.org/10.1016/j.tcb.2010.05.001

    Article  CAS  PubMed  Google Scholar 

  6. Lund MK, Guthrie C (2005) The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim. Mol Cell 20(4):645–651. https://doi.org/10.1016/j.molcel.2005.10.005

    Article  CAS  PubMed  Google Scholar 

  7. Ledoux S, Guthrie C (2011) Regulation of the Dbp5 ATPase cycle in mRNP remodeling at the nuclear pore: a lively new paradigm for DEAD-box proteins. Genes Dev 25(11):1109–1114. https://doi.org/10.1101/gad.2062611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hodge CA, Tran EJ, Noble KN, Alcazar-Roman AR, Ben-Yishay R, Scarcelli JJ, Folkmann AW, Shav-Tal Y, Wente SR, Cole CN (2011) The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1. Genes Dev 25(10):1052–1064. https://doi.org/10.1101/gad.2041611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Noble KN, Tran EJ, Alcazar-Roman AR, Hodge CA, Cole CN, Wente SR (2011) The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1. Genes Dev 25(10):1065–1077. https://doi.org/10.1101/gad.2040611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tran EJ, Zhou Y, Corbett AH, Wente SR (2007) The DEAD-box protein Dbp5 controls mRNA export by triggering specific RNA:protein remodeling events. Mol Cell 28(5):850–859. https://doi.org/10.1016/j.molcel.2007.09.019

    Article  CAS  PubMed  Google Scholar 

  11. Wente SR, Rout MP (2010) The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol 2(10):a000562. https://doi.org/10.1101/cshperspect.a000562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Daneholt B (1997) A look at messenger RNP moving through the nuclear pore. Cell 88(5):585–588

    Article  CAS  Google Scholar 

  13. Shav-Tal Y, Darzacq X, Shenoy SM, Fusco D, Janicki SM, Spector DL, Singer RH (2004) Dynamics of single mRNPs in nuclei of living cells. Science 304(5678):1797–1800

    Article  CAS  Google Scholar 

  14. Mor A, Suliman S, Ben-Yishay R, Yunger S, Brody Y, Shav-Tal Y (2010) Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nat Cell Biol 12(6):543–552. https://doi.org/10.1038/ncb2056

    Article  CAS  PubMed  Google Scholar 

  15. Grunwald D, Singer RH (2010) In vivo imaging of labelled endogenous beta-actin mRNA during nucleocytoplasmic transport. Nature 467(7315):604–607. https://doi.org/10.1038/nature09438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vargas DY, Raj A, Marras SA, Kramer FR, Tyagi S (2005) Mechanism of mRNA transport in the nucleus. Proc Natl Acad Sci U S A 102(47):17008–17013. https://doi.org/10.1073/pnas.0505580102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ben-Ari Y, Brody Y, Kinor N, Mor A, Tsukamoto T, Spector DL, Singer RH, Shav-Tal Y (2010) The life of an mRNA in space and time. J Cell Sci 123(Pt 10):1761–1774. https://doi.org/10.1242/jcs.062638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Halstead JM, Lionnet T, Wilbertz JH, Wippich F, Ephrussi A, Singer RH, Chao JA (2015) Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science 347(6228):1367–1671. https://doi.org/10.1126/science.aaa3380

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ma J, Liu Z, Michelotti N, Pitchiaya S, Veerapaneni R, Androsavich JR, Walter NG, Yang W (2013) High-resolution three-dimensional mapping of mRNA export through the nuclear pore. Nat Commun 4:2414. https://doi.org/10.1038/ncomms3414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Querido E, Gallardo F, Beaudoin M, Menard C, Chartrand P (2011) Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats. J Cell Sci 124. (Pt 10:1703–1714. https://doi.org/10.1242/jcs.073270

    Article  CAS  PubMed  Google Scholar 

  21. Thompson MA, Casolari JM, Badieirostami M, Brown PO, Moerner WE (2010) Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function. Proc Natl Acad Sci U S A 107(42):17864–17871. https://doi.org/10.1073/pnas.1012868107

    Article  PubMed  PubMed Central  Google Scholar 

  22. Veith R, Sorkalla T, Baumgart E, Anzt J, Haberlein H, Tyagi S, Siebrasse JP, Kubitscheck U (2010) Balbiani ring mRNPs diffuse through and bind to clusters of large intranuclear molecular structures. Biophys J 99(8):2676–2685. https://doi.org/10.1016/j.bpj.2010.08.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ben-Yishay R, Ashkenazy AJ, Shav-Tal Y (2016) Dynamic encounters of genes and transcripts with the nuclear pore. Trends Genet 32(7):419–431. https://doi.org/10.1016/j.tig.2016.04.003

    Article  CAS  PubMed  Google Scholar 

  24. 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(24):9426–9431. https://doi.org/10.1073/pnas.1201781109

    Article  PubMed  PubMed Central  Google Scholar 

  25. Smith C, Lari A, Derrer CP, Ouwehand A, Rossouw A, Huisman M, Dange T, Hopman M, Joseph A, Zenklusen D, Weis K, Grunwald D, Montpetit B (2015) In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p. J Cell Biol 211(6):1121–1130. https://doi.org/10.1083/jcb.201503135

    Article  PubMed  PubMed Central  Google Scholar 

  26. Kylberg K, Bjork P, Fomproix N, Ivarsson B, Wieslander L, Daneholt B (2010) Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport. Exp Cell Res 316(6):1028–1038. https://doi.org/10.1016/j.yexcr.2009.10.016

    Article  CAS  PubMed  Google Scholar 

  27. Mohr D, Frey S, Fischer T, Guttler T, Gorlich D (2009) Characterisation of the passive permeability barrier of nuclear pore complexes. EMBO J 28(17):2541–2553. https://doi.org/10.1038/emboj.2009.200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Davis LI, Blobel G (1986) Identification and characterization of a nuclear pore complex protein. Cell 45(5):699–709

    Article  CAS  Google Scholar 

  29. Kalo A, Kafri P, Shav-Tal Y (2013) Single mRNP tracking in living mammalian cells. Methods Mol Biol 1042:87–99. https://doi.org/10.1007/978-1-62703-526-2_7

    Article  CAS  PubMed  Google Scholar 

  30. Yunger S, Shav-Tal Y (2011) Imaging mRNAs in living mammalian cells. Methods Mol Biol 714:249–263. https://doi.org/10.1007/978-1-61779-005-8_16

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Y.S.T. is supported by the Israel Science Foundation and German–Israeli Foundation.

Author information

Authors 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

    Rakefet Ben-Yishay & Yaron Shav-Tal

Authors
  1. Rakefet Ben-Yishay
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yaron Shav-Tal
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yaron Shav-Tal .

Editor information

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

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ben-Yishay, R., Shav-Tal, Y. (2019). Detection of mRNAs Anchored to the Nuclear Envelope During Export Inhibition in Living Cells. 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_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9674-2_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9673-5

  • Online ISBN: 978-1-4939-9674-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation