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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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The AP–hGle1-SD peptide inhibits the shuttling of GST–hGle1. (A) HeLa cells were pretreated with AP–peptides (3 h) and microinjected with GST–hGle1A in the cytoplasm (left) or nucleus (right) of the cells. After recovery (2 h), the intracellular distribution of GST–hGle1A was analyzed by IIF using anti-GST antibodies. Compared with cells treated with the scrSD control peptide, less GST–hGle1 is detected outside the microinjection site in the majority of cells analyzed. (B) NPC/NE association of nucleoporins recognized by mAb414 is not perturbed by AP–hGle1-SD treatment. HeLa cells incubated with AP–hGle1-SD (SD) or control peptide (scrSD) were processed for in situ hybridization with oligo(dT)30 and coincident IIF with mAb414. The mAb414 staining is similar in AP–hGle1-SD and scrSD cells despite the poly(A)+ RNA export defect in the AP–hGle1-SD cells. Bars, 10 μm.
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fig7: The AP–hGle1-SD peptide inhibits the shuttling of GST–hGle1. (A) HeLa cells were pretreated with AP–peptides (3 h) and microinjected with GST–hGle1A in the cytoplasm (left) or nucleus (right) of the cells. After recovery (2 h), the intracellular distribution of GST–hGle1A was analyzed by IIF using anti-GST antibodies. Compared with cells treated with the scrSD control peptide, less GST–hGle1 is detected outside the microinjection site in the majority of cells analyzed. (B) NPC/NE association of nucleoporins recognized by mAb414 is not perturbed by AP–hGle1-SD treatment. HeLa cells incubated with AP–hGle1-SD (SD) or control peptide (scrSD) were processed for in situ hybridization with oligo(dT)30 and coincident IIF with mAb414. The mAb414 staining is similar in AP–hGle1-SD and scrSD cells despite the poly(A)+ RNA export defect in the AP–hGle1-SD cells. Bars, 10 μm.

Mentions: To more directly address the effect of AP–hGle1-SD on hGle1 nucleocytoplasmic shuttling, we analyzed the distribution of microinjected GST–hGle1A in cells treated with the AP–hGle1-SD peptide. Compared with the control AP–hGle1-scrSD cells, the presence of the AP–hGle1-SD peptide resulted in less efficient import of cytoplasmically injected GST–hGle1A into the nucleus (90 ± 2.5%; n = 49 microinjected HeLa cells) (Fig. 7 A, left). Similarly, the AP–hGle1-SD peptide impaired the export of nuclear-injected GST–hGle1A (88.5 ± 3%; n = 43 injected cells) (Fig. 7 A, right).


An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

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

The AP–hGle1-SD peptide inhibits the shuttling of GST–hGle1. (A) HeLa cells were pretreated with AP–peptides (3 h) and microinjected with GST–hGle1A in the cytoplasm (left) or nucleus (right) of the cells. After recovery (2 h), the intracellular distribution of GST–hGle1A was analyzed by IIF using anti-GST antibodies. Compared with cells treated with the scrSD control peptide, less GST–hGle1 is detected outside the microinjection site in the majority of cells analyzed. (B) NPC/NE association of nucleoporins recognized by mAb414 is not perturbed by AP–hGle1-SD treatment. HeLa cells incubated with AP–hGle1-SD (SD) or control peptide (scrSD) were processed for in situ hybridization with oligo(dT)30 and coincident IIF with mAb414. The mAb414 staining is similar in AP–hGle1-SD and scrSD cells despite the poly(A)+ RNA export defect in the AP–hGle1-SD cells. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172758&req=5

fig7: The AP–hGle1-SD peptide inhibits the shuttling of GST–hGle1. (A) HeLa cells were pretreated with AP–peptides (3 h) and microinjected with GST–hGle1A in the cytoplasm (left) or nucleus (right) of the cells. After recovery (2 h), the intracellular distribution of GST–hGle1A was analyzed by IIF using anti-GST antibodies. Compared with cells treated with the scrSD control peptide, less GST–hGle1 is detected outside the microinjection site in the majority of cells analyzed. (B) NPC/NE association of nucleoporins recognized by mAb414 is not perturbed by AP–hGle1-SD treatment. HeLa cells incubated with AP–hGle1-SD (SD) or control peptide (scrSD) were processed for in situ hybridization with oligo(dT)30 and coincident IIF with mAb414. The mAb414 staining is similar in AP–hGle1-SD and scrSD cells despite the poly(A)+ RNA export defect in the AP–hGle1-SD cells. Bars, 10 μm.
Mentions: To more directly address the effect of AP–hGle1-SD on hGle1 nucleocytoplasmic shuttling, we analyzed the distribution of microinjected GST–hGle1A in cells treated with the AP–hGle1-SD peptide. Compared with the control AP–hGle1-scrSD cells, the presence of the AP–hGle1-SD peptide resulted in less efficient import of cytoplasmically injected GST–hGle1A into the nucleus (90 ± 2.5%; n = 49 microinjected HeLa cells) (Fig. 7 A, left). Similarly, the AP–hGle1-SD peptide impaired the export of nuclear-injected GST–hGle1A (88.5 ± 3%; n = 43 injected cells) (Fig. 7 A, right).

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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