Abstract
The flow of genetic information in eukaryotic cells occurs through the nucleocytoplasmic translocation of mRNAs. Knowledge of in vivo messenger RNA export kinetics remains poor in comparison with that of protein transport. We have established a mammalian system that allowed the real-time visualization and quantification of large single mRNA–protein complexes (mRNPs) during export. The in vivo dynamics of bulk mRNP transport and export, from transcription to the nuclear pore complex (NPC), occurred within a 5–40 minute time frame, with no NPC pile-up. mRNP export was rapid (about 0.5 s) and kinetically faster than nucleoplasmic diffusion. Export inhibition demonstrated that mRNA–NPC interactions were independent of ongoing export. Nucleoplasmic transport dynamics of intron-containing and intronless mRNAs were similar, yet an intron did increase export efficiency. Here we provide visualization and analysis at the single mRNP level of the various steps in nuclear gene expression and the inter-chromatin tracks through which mRNPs diffuse, and demonstrate the kinetics of mRNP–NPC interactions and translocation.
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References
Kohler, A. & Hurt, E. Exporting RNA from the nucleus to the cytoplasm. Nature Rev. Mol. Cell Biol. 8, 761–773 (2007).
Fahrenkrog, B. & Aebi, U. The nuclear pore complex: nucleocytoplasmic transport and beyond. Nature Rev. Mol. Cell Biol. 4, 757–766 (2003).
Terry, L. J., Shows, E. B. & Wente, S. R. Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science 318, 1412–1416 (2007).
Moore, M. J. From birth to death: the complex lives of eukaryotic mRNAs. Science 309, 1514–1518 (2005).
Iglesias, N. & Stutz, F. Regulation of mRNP dynamics along the export pathway. FEBS Lett. 582, 1987–1996 (2008).
Mehlin, H., Daneholt, B. & Skoglund, U. Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography. Cell 69, 605–613 (1992).
Daneholt, B. Assembly and transport of a premessenger RNP particle. Proc. Natl Acad. Sci. USA 98, 7012–7017 (2001).
Franke, W. W. & Scheer, U. in The Nucleus Vol. 1. (ed. Busch, H.) 219–347 (Academic, 1974).
Iborra, F. J., Jackson, D. A. & Cook, P. R. The path of transcripts from extra-nucleolar synthetic sites to nuclear pores: transcripts in transit are concentrated in discrete structures containing SR proteins. J. Cell Sci. 111, 2269–2282 (1998).
Iborra, F. J., Jackson, D. A. & Cook, P. R. The path of RNA through nuclear pores: apparent entry from the sides into specialized pores. J. Cell Sci. 113, 291–302 (2000).
Pante, N. et al. Visualizing nuclear export of different classes of RNA by electron microscopy. RNA 3, 498–513 (1997).
Huang, S., Deerinck, T. J., Ellisman, M. H. & Spector, D. L. In vivo analysis of the stability and transport of nuclear poly(A)+ RNA. J. Cell Biol. 126, 877–899 (1994).
Palazzo, A. F. et al. The signal sequence coding region promotes nuclear export of mRNA. PLoS Biol. 5, e322 (2007).
Luo, M. J. & Reed, R. Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl Acad. Sci. USA 96, 14937–14942 (1999).
Jarmolowski, A., Boelens, W. C., Izaurralde, E. & Mattaj, I. W. Nuclear export of different classes of RNA is mediated by specific factors. J. Cell Biol. 124, 627–635 (1994).
Dargemont, C. & Kuhn, L. C. Export of mRNA from microinjected nuclei of Xenopus laevis oocytes. J. Cell Biol. 118, 1–9 (1992).
Bastos, R. N. & Aviv, H. Globin RNA precursor molecules: biosynthesis and process in erythroid cells. Cell 11, 641–650 (1977).
Jelinek, W. et al. Further evidence on the nuclear origin and transfer to the cytoplasm of polyadenylic acid sequences in mammalian cell RNA. J. Mol. Biol. 75, 515–532 (1973).
Riedel, N., Bachmann, M., Prochnow, D., Richter, H. P. & Fasold, H. Permeability measurements with closed vesicles from rat liver nuclear envelopes. Proc. Natl Acad. Sci. USA 84, 3540–3544 (1987).
Schroder, H. C. et al. Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport. Biochem. J. 252, 777–790 (1988).
Mariman, E., Hagebols, A. M. & van Venrooij, W. On the localization and transport of specific adenoviral mRNA-sequences in the late infected HeLa cell. Nucleic Acids Res. 10, 6131–6145 (1982).
Braga, J., McNally, J. G. & Carmo-Fonseca, M. A reaction-diffusion model to study RNA motion by quantitative fluorescence recovery after photobleaching. Biophys. J. 92, 2694–2703 (2007).
Audibert, A., Weil, D. & Dautry, F. In vivo kinetics of mRNA splicing and transport in mammalian cells. Mol. Cell. Biol. 22, 6706–6718 (2002).
Shav-Tal, Y. et al. Dynamics of single mRNPs in nuclei of living cells. Science 304, 1797–1800 (2004).
Janicki, S. M. et al. From silencing to gene expression; real-time analysis in single cells. Cell 116, 683–698 (2004).
Chapdelaine, P. et al. Functional EGFP–dystrophin fusion proteins for gene therapy vector development. Protein Eng. 13, 611–615 (2000).
England, S. B. et al. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature 343, 180–182 (1990).
Michael, W. M., Choi, M. & Dreyfuss, G. A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell 83, 415–422 (1995).
Valencia, P., Dias, A. P. & Reed, R. Splicing promotes rapid and efficient mRNA export in mammalian cells. Proc. Natl Acad. Sci. USA 105, 3386–3391 (2008).
Saxton, M. J. & Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).
Braga, J., Rino, J. & Carmo-Fonseca, M. Photobleaching microscopy reveals the dynamics of mRNA-binding proteins inside live cell nuclei. Prog. Mol. Subcell. Biol. 35, 119–134 (2004).
Siebrasse, J. P. et al. Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin. Proc. Natl Acad. Sci. USA 105, 20291–20296 (2008).
Lim, R. Y., Aebi, U. & Fahrenkrog, B. Towards reconciling structure and function in the nuclear pore complex. Histochem. Cell Biol. 129, 105–116 (2008).
Davis, L. I. & Blobel, G. Identification and characterization of a nuclear pore complex protein. Cell 45, 699–709 (1986).
Blobel, G. Gene gating: a hypothesis. Proc. Natl Acad. Sci. USA 82, 8527–8529 (1985).
Vargas, D. Y., Raj, A., Marras, S. A., Kramer, F. R. & Tyagi, S. Mechanism of mRNA transport in the nucleus. Proc. Natl Acad. Sci. USA 102, 17008–17013 (2005).
Politz, J. C., Tuft, R. A., Pederson, T. & Singer, R. H. Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells. Curr. Biol. 9, 285–291 (1999).
Albiez, H. et al. Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks. Chromosome Res. 14, 707–733 (2006).
Carmo-Fonseca, M., Platani, M. & Swedlow, J. R. Macromolecular mobility inside the cell nucleus. Trends Cell Biol. 12, 491–495 (2002).
Yang, W., Gelles, J. & Musser, S. M. Imaging of single-molecule translocation through nuclear pore complexes. Proc. Natl Acad. Sci. USA 101, 12887–12892 (2004).
Yang, W. & Musser, S. M. Nuclear import time and transport efficiency depend on importin beta concentration. J. Cell Biol. 174, 951–961 (2006).
Dange, T., Grunwald, D., Grunwald, A., Peters, R. & Kubitscheck, U. Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. J. Cell Biol. 183, 77–86 (2008).
Kubitscheck, U. et al. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J. Cell Biol. 168, 233–243 (2005).
Siebrasse, J. P. & Peters, R. Rapid translocation of NTF2 through the nuclear pore of isolated nuclei and nuclear envelopes. EMBO Rep. 3, 887–892 (2002).
Ribbeck, K. & Gorlich, D. Kinetic analysis of translocation through nuclear pore complexes. EMBO J. 20, 1320–1330 (2001).
Kustanovich, T. & Rabin, Y. Metastable network model of protein transport through nuclear pores. Biophys. J. 86, 2008–2016 (2004).
Mohr, D., Frey, S., Fischer, T., Guttler, T. & Gorlich, D. Characterisation of the passive permeability barrier of nuclear pore complexes. EMBO J. 28, 2541–2553 (2009).
Visser, A. E., Jaunin, F., Fakan, S. & Aten, J. A. High resolution analysis of interphase chromosome domains. J. Cell Sci. 113, 2585–2593 (2000).
Fakan, S. The functional architecture of the nucleus as analysed by ultrastructural cytochemistry. Histochem. Cell Biol. 122, 83–93 (2004).
Darzacq, X. et al. Stepwise RNP assembly at the site of H/ACA RNA transcription in human cells. J. Cell Biol. 173, 207–218 (2006).
Tsukamoto, T. et al. Visualization of gene activity in living cells. Nature Cell Biol. 2, 871–878 (2000).
Dultz, E. et al. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J. Cell Biol. 180, 857–865 (2008).
Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).
Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nature Struct. Mol. Biol. 14, 796–806 (2007).
Acknowledgements
We thank J. Tremblay (Laval University, Canada) for GFP–Dys and GFP–mini-Dys, J. Ellenberg (EMBL Heidelberg, Germany) for the POM121-mCherry plasmid, and R. Drummer (Bar-Ilan University (BIU), Israel) for statistical analysis. This work was supported by grants to Y.S.T. from the Israel Science Foundation (grant 250/06) and the Israel Ministries of Health and Science. Y.S.T. thanks the Israel Science Foundation for the fluorescence live-cell imaging microscope. Y.B. is an Azrielli fellow. Y.S.T. is the Jane Stern Lebell Family Fellow in Life Sciences at BIU.
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A.M. designed, performed and analysed most of the experiments in this study. S.S. generated the Dys plasmids and stable cell lines. R.B.Y. generated the full-Dys cell line and performed the immunofluorescence experiments. S.Y. made the three-dimensional rendering of the mRNPs and chromatin. Y.B. generated the constructs E1–E6. Y.S.T. conceived the project, designed and supervised the study, and wrote the paper.
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Mor, A., Suliman, S., Ben-Yishay, R. et al. Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nat Cell Biol 12, 543–552 (2010). https://doi.org/10.1038/ncb2056
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DOI: https://doi.org/10.1038/ncb2056
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