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Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells

Nature Cell Biology volume 12, pages 543552 (2010) | Download Citation

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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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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Affiliations

  1. The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan 52900, Israel.

    • Amir Mor
    • , Shimrit Suliman
    • , Rakefet Ben-Yishay
    • , Sharon Yunger
    • , Yehuda Brody
    •  & Yaron Shav-Tal

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yaron Shav-Tal.

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https://doi.org/10.1038/ncb2056

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