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An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

Kendirgi F, Barry DM, Griffis ER, Powers MA, Wente SR - J. Cell Biol. (2003)

Bottom Line: An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA.Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1.These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-8240, USA.

ABSTRACT
Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

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Nuclear protein import and export are not perturbed in HeLa cells treated with AP–peptides. (A) HeLa cells transfected with a GR–GFP expression vector were treated with 5 μM of AP–peptide. Dexamethasone (Dex+) or an equivalent volume of ethanol (Dex−) was added in the last 30 min before fixation and DAPI staining. Dexamethasone addition specifically induces nuclear import of the cytoplasmic GR–GFP pool in the presence of either peptide. (B) Karyopherin/importin β1 (Kapβ1) intracellular localization in AP–hGle1-SD– and control peptide (scrSD)–treated cells is not perturbed. Cells were treated with peptides as described above, fixed, and processed for IIF using anti-Kapβ1 antibodies. Bars, 10 μm.
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fig5: Nuclear protein import and export are not perturbed in HeLa cells treated with AP–peptides. (A) HeLa cells transfected with a GR–GFP expression vector were treated with 5 μM of AP–peptide. Dexamethasone (Dex+) or an equivalent volume of ethanol (Dex−) was added in the last 30 min before fixation and DAPI staining. Dexamethasone addition specifically induces nuclear import of the cytoplasmic GR–GFP pool in the presence of either peptide. (B) Karyopherin/importin β1 (Kapβ1) intracellular localization in AP–hGle1-SD– and control peptide (scrSD)–treated cells is not perturbed. Cells were treated with peptides as described above, fixed, and processed for IIF using anti-Kapβ1 antibodies. Bars, 10 μm.

Mentions: To test whether the decrease in mRNA export resulted from a general perturbation of transport through NPCs, protein import and export activity was analyzed. For nuclear import, cells transiently expressing a glucocorticoid receptor (GR) GFP fusion protein were treated with AP–peptides for 4 h (Fig. 5 A). In the absence of the agonist dexamethasone, a substantial GR–GFP cytoplasmic pool was observed. When dexamethasone was added to induce import, nuclear accumulation of GR–GFP was observed in the presence of either peptide. To determine if protein export from the nucleus was perturbed, the intracellular distribution of karyopherin/importin β1 was monitored by IIF. We hypothesized that if the general nuclear protein export pathways were affected by AP–hGle1-SD peptide, karyopherin/importin β1 would accumulate in the nucleus of treated cells. However, the cellular distribution of this shuttling karyopherin was not affected (Fig. 5 B). Thus, protein import and export are not markedly inhibited under conditions where AP–hGle1-SD decreases poly(A)+ RNA export. These data suggest that hGle1-SD function is specifically essential for the mRNP export process.


An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

Kendirgi F, Barry DM, Griffis ER, Powers MA, Wente SR - J. Cell Biol. (2003)

Nuclear protein import and export are not perturbed in HeLa cells treated with AP–peptides. (A) HeLa cells transfected with a GR–GFP expression vector were treated with 5 μM of AP–peptide. Dexamethasone (Dex+) or an equivalent volume of ethanol (Dex−) was added in the last 30 min before fixation and DAPI staining. Dexamethasone addition specifically induces nuclear import of the cytoplasmic GR–GFP pool in the presence of either peptide. (B) Karyopherin/importin β1 (Kapβ1) intracellular localization in AP–hGle1-SD– and control peptide (scrSD)–treated cells is not perturbed. Cells were treated with peptides as described above, fixed, and processed for IIF using anti-Kapβ1 antibodies. Bars, 10 μm.
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Related In: Results  -  Collection

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fig5: Nuclear protein import and export are not perturbed in HeLa cells treated with AP–peptides. (A) HeLa cells transfected with a GR–GFP expression vector were treated with 5 μM of AP–peptide. Dexamethasone (Dex+) or an equivalent volume of ethanol (Dex−) was added in the last 30 min before fixation and DAPI staining. Dexamethasone addition specifically induces nuclear import of the cytoplasmic GR–GFP pool in the presence of either peptide. (B) Karyopherin/importin β1 (Kapβ1) intracellular localization in AP–hGle1-SD– and control peptide (scrSD)–treated cells is not perturbed. Cells were treated with peptides as described above, fixed, and processed for IIF using anti-Kapβ1 antibodies. Bars, 10 μm.
Mentions: To test whether the decrease in mRNA export resulted from a general perturbation of transport through NPCs, protein import and export activity was analyzed. For nuclear import, cells transiently expressing a glucocorticoid receptor (GR) GFP fusion protein were treated with AP–peptides for 4 h (Fig. 5 A). In the absence of the agonist dexamethasone, a substantial GR–GFP cytoplasmic pool was observed. When dexamethasone was added to induce import, nuclear accumulation of GR–GFP was observed in the presence of either peptide. To determine if protein export from the nucleus was perturbed, the intracellular distribution of karyopherin/importin β1 was monitored by IIF. We hypothesized that if the general nuclear protein export pathways were affected by AP–hGle1-SD peptide, karyopherin/importin β1 would accumulate in the nucleus of treated cells. However, the cellular distribution of this shuttling karyopherin was not affected (Fig. 5 B). Thus, protein import and export are not markedly inhibited under conditions where AP–hGle1-SD decreases poly(A)+ RNA export. These data suggest that hGle1-SD function is specifically essential for the mRNP export process.

Bottom Line: An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA.Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1.These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-8240, USA.

ABSTRACT
Gle1 is required for mRNA export in yeast and human cells. Here, we report that two human Gle1 (hGle1) isoforms are expressed in HeLa cells (hGle1A and B). The two encoded proteins are identical except for their COOH-terminal regions. hGle1A ends with a unique four-amino acid segment, whereas hGle1B has a COOH-terminal 43-amino acid span. Only hGle1B, the more abundant isoform, localizes to the nuclear envelope (NE) and pore complex. To test whether hGle1 is a dynamic shuttling transport factor, we microinjected HeLa cells with recombinant hGle1 and conducted photobleaching studies of live HeLa cells expressing EGFP-hGle1. Both strategies show that hGle1 shuttles between the nucleus and cytoplasm. An internal 39-amino acid domain is necessary and sufficient for mediating nucleocytoplasmic transport. Using a cell-permeable peptide strategy, we document a role for hGle1 shuttling in mRNA export. An hGle1 shuttling domain (SD) peptide impairs the export of both total poly(A)+ RNA and the specific dihydrofolate reductase mRNA. Coincidentally, SD peptide-treated cells show decreased endogenous hGle1 localization at the NE and reduced nucleocytoplasmic shuttling of microinjected, recombinant hGle1. These findings pinpoint the first functional motif in hGle1 and link hGle1 to the dynamic mRNA export mechanism.

Show MeSH