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

  • Rakefet Ben-Yishay
  • Yaron Shav-TalEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2038)


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.

Key words

mRNP mRNA export Nuclear pore complex (NPC) Gene expression Live cell imaging Single particle tracking Image processing and analysis 



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


  1. 1.
    Fahrenkrog B, Aebi U (2003) The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat Rev Mol Cell Biol 4(10):757–766CrossRefGoogle Scholar
  2. 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. Scholar
  3. 3.
    Adams RL, Wente SR (2013) Uncovering nuclear pore complexity with innovation. Cell 152(6):1218–1221. Scholar
  4. 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. Scholar
  5. 5.
    Walde S, Kehlenbach RH (2010) The Part and the Whole: functions of nucleoporins in nucleocytoplasmic transport. Trends Cell Biol 20(8):461–469. Scholar
  6. 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. Scholar
  7. 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. Scholar
  8. 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. Scholar
  9. 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. Scholar
  10. 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. Scholar
  11. 11.
    Wente SR, Rout MP (2010) The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol 2(10):a000562. Scholar
  12. 12.
    Daneholt B (1997) A look at messenger RNP moving through the nuclear pore. Cell 88(5):585–588CrossRefGoogle Scholar
  13. 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):1797–1800CrossRefGoogle Scholar
  14. 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. Scholar
  15. 15.
    Grunwald D, Singer RH (2010) In vivo imaging of labelled endogenous beta-actin mRNA during nucleocytoplasmic transport. Nature 467(7315):604–607. Scholar
  16. 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. Scholar
  17. 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. Scholar
  18. 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. Scholar
  19. 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. Scholar
  20. 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. Scholar
  21. 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. Scholar
  22. 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. Scholar
  23. 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. Scholar
  24. 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. Scholar
  25. 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. Scholar
  26. 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. Scholar
  27. 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. Scholar
  28. 28.
    Davis LI, Blobel G (1986) Identification and characterization of a nuclear pore complex protein. Cell 45(5):699–709CrossRefGoogle Scholar
  29. 29.
    Kalo A, Kafri P, Shav-Tal Y (2013) Single mRNP tracking in living mammalian cells. Methods Mol Biol 1042:87–99. Scholar
  30. 30.
    Yunger S, Shav-Tal Y (2011) Imaging mRNAs in living mammalian cells. Methods Mol Biol 714:249–263. Scholar

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

Authors and Affiliations

  1. 1.The Mina & Everard Goodman Faculty of Life Sciences and the Institute of Nanotechnology and Advanced Materials, Bar-Ilan UniversityRamat GanIsrael

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