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Importin-7 mediates nuclear trafficking of DNA in mammalian cells.

Dhanoya A, Wang T, Keshavarz-Moore E, Fassati A, Chain BM - Traffic (2012)

Bottom Line: Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls.Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response.Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

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Selective competition of plasmid DNA nuclear importNuclear import assays in permeabilized-HeLa cells in the presence of fluorescently labelled plasmid DNA (12.5 ng), 1 µm imp7, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Experiments were performed in parallel in the presence of 1 µm fluorescent MBP-M9 (M9) or GFP-NLS, 1 µm transportin or impα/β heterodimer, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Nuclear import was quantified using the Metamorph software. M9 import in the absence of competitor DNA is given an arbitrary value of 100. Values represent the average fluorescent intensity inside the nucleus relative to control (no DNA) ± SD of three independent experiments.
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fig02: Selective competition of plasmid DNA nuclear importNuclear import assays in permeabilized-HeLa cells in the presence of fluorescently labelled plasmid DNA (12.5 ng), 1 µm imp7, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Experiments were performed in parallel in the presence of 1 µm fluorescent MBP-M9 (M9) or GFP-NLS, 1 µm transportin or impα/β heterodimer, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Nuclear import was quantified using the Metamorph software. M9 import in the absence of competitor DNA is given an arbitrary value of 100. Values represent the average fluorescent intensity inside the nucleus relative to control (no DNA) ± SD of three independent experiments.

Mentions: If imp7 is part of an intrinsic pathway for DNA nuclear import, then this pathway should be inhibited specifically by competitor DNA. To test this prediction, nuclear import assays were performed in the presence of imp7, Ran and energy mix, a fixed amount of labelled plasmid DNA and increasing amounts of unlabelled competitor DNA (Figure 2). To test for the specificity of the assay, parallel experiments were carried out in the presence of Ran and energy mix, transportin and a constant amount of labelled maltose-binding protein (MBP) fused to the M9 peptide of hRNP A1, which binds to and is imported by transportin 27 or a green fluorescent protein (GFP) fused to the classical nuclear localizing signal (NLS) in the presence of the impα/β heterodimer, which binds directly to the NLS 17. Quantification of nuclear fluorescence revealed that competitor DNA inhibited nuclear import of fluorescently labelled DNA in a dose-dependent way but had no effect on the import of MBP-M9 or GFP-NLS (Figure 2).


Importin-7 mediates nuclear trafficking of DNA in mammalian cells.

Dhanoya A, Wang T, Keshavarz-Moore E, Fassati A, Chain BM - Traffic (2012)

Selective competition of plasmid DNA nuclear importNuclear import assays in permeabilized-HeLa cells in the presence of fluorescently labelled plasmid DNA (12.5 ng), 1 µm imp7, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Experiments were performed in parallel in the presence of 1 µm fluorescent MBP-M9 (M9) or GFP-NLS, 1 µm transportin or impα/β heterodimer, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Nuclear import was quantified using the Metamorph software. M9 import in the absence of competitor DNA is given an arbitrary value of 100. Values represent the average fluorescent intensity inside the nucleus relative to control (no DNA) ± SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672689&req=5

fig02: Selective competition of plasmid DNA nuclear importNuclear import assays in permeabilized-HeLa cells in the presence of fluorescently labelled plasmid DNA (12.5 ng), 1 µm imp7, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Experiments were performed in parallel in the presence of 1 µm fluorescent MBP-M9 (M9) or GFP-NLS, 1 µm transportin or impα/β heterodimer, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Nuclear import was quantified using the Metamorph software. M9 import in the absence of competitor DNA is given an arbitrary value of 100. Values represent the average fluorescent intensity inside the nucleus relative to control (no DNA) ± SD of three independent experiments.
Mentions: If imp7 is part of an intrinsic pathway for DNA nuclear import, then this pathway should be inhibited specifically by competitor DNA. To test this prediction, nuclear import assays were performed in the presence of imp7, Ran and energy mix, a fixed amount of labelled plasmid DNA and increasing amounts of unlabelled competitor DNA (Figure 2). To test for the specificity of the assay, parallel experiments were carried out in the presence of Ran and energy mix, transportin and a constant amount of labelled maltose-binding protein (MBP) fused to the M9 peptide of hRNP A1, which binds to and is imported by transportin 27 or a green fluorescent protein (GFP) fused to the classical nuclear localizing signal (NLS) in the presence of the impα/β heterodimer, which binds directly to the NLS 17. Quantification of nuclear fluorescence revealed that competitor DNA inhibited nuclear import of fluorescently labelled DNA in a dose-dependent way but had no effect on the import of MBP-M9 or GFP-NLS (Figure 2).

Bottom Line: Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls.Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response.Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

Show MeSH
Related in: MedlinePlus