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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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AP–hGle1-SD peptide results in mislocalization of endogenous hGle1 localization when poly(A)+ RNA export is inhibited. HeLa cells were incubated with AP–hGle1-SD (SD) or control peptide (scrSD), processed for IIF using affinity-purified anti-hGle1 antibodies (A–C), and in situ hybridized with oligo (dT)30 (A) or stained with DAPI (B and C). Hybridized probe and bound hGle1 antibodies were simultaneously detected using rhodamine-labeled anti–Dig Fab antibodies and FITC-labeled anti–rabbit antibodies, respectively. Images showing in situ hybridization results reflect equivalent exposure times. (A) Fix-Triton permeabilization detects total hGle1 pool and shows changes in anti-hGle1 localization in AP–hGle1-SD–treated cells. (B) Digitonin-Fix permeabilization detects only the cytoplasmically accessible pool. AP–hGle1-SD results in decreased levels of cytoplasmically accessible hGle1 (asterisk) compared with unaffected cells (arrowheads, at passage >150; see Materials and methods) or cells treated with control peptide. The absence of lamin B staining using anti–lamin B antibodies (far right) confirms NE integrity under these conditions. (C) Detection of nuclear hGle1 was accomplished by combined Digitonin-Fix-Triton permeabilization. Lamin B staining confirms access of antibodies to intranuclear proteins. Bars, 10 μm.
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fig6: AP–hGle1-SD peptide results in mislocalization of endogenous hGle1 localization when poly(A)+ RNA export is inhibited. HeLa cells were incubated with AP–hGle1-SD (SD) or control peptide (scrSD), processed for IIF using affinity-purified anti-hGle1 antibodies (A–C), and in situ hybridized with oligo (dT)30 (A) or stained with DAPI (B and C). Hybridized probe and bound hGle1 antibodies were simultaneously detected using rhodamine-labeled anti–Dig Fab antibodies and FITC-labeled anti–rabbit antibodies, respectively. Images showing in situ hybridization results reflect equivalent exposure times. (A) Fix-Triton permeabilization detects total hGle1 pool and shows changes in anti-hGle1 localization in AP–hGle1-SD–treated cells. (B) Digitonin-Fix permeabilization detects only the cytoplasmically accessible pool. AP–hGle1-SD results in decreased levels of cytoplasmically accessible hGle1 (asterisk) compared with unaffected cells (arrowheads, at passage >150; see Materials and methods) or cells treated with control peptide. The absence of lamin B staining using anti–lamin B antibodies (far right) confirms NE integrity under these conditions. (C) Detection of nuclear hGle1 was accomplished by combined Digitonin-Fix-Triton permeabilization. Lamin B staining confirms access of antibodies to intranuclear proteins. Bars, 10 μm.

Mentions: In parallel with the study of poly(A)+ RNA distribution, we analyzed the localization of endogenous hGle1 by IIF microscopy in cells treated with AP–peptides. Different permeabilization and fixation conditions were used to discriminate between cytoplasmically accessible hGle1 and nuclear hGle1 (Fig. 6). In untreated wild-type cells processed by fixation and complete Triton X-100 permeabilization (Fix-Triton), a pool of hGle1 is localized at the NPC/NE, and a fraction of the hGle1 is cytoplasmically accessible (Watkins et al., 1998; Fig. 1 A). In cells treated with the control AP–hGle1-scrSD peptide and processed by Fix-Triton, endogenous hGle1 was localized in a typical wild-type pattern, and cells exhibited normal poly(A)+ RNA distribution (Fig. 6 A, top). However, when cells were treated with the AP–hGle1-SD peptide, distinct hGle1 staining at the NPC/NE was less apparent in cells with impaired poly(A)+ RNA export (Fig. 6 A, bottom).


An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

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

AP–hGle1-SD peptide results in mislocalization of endogenous hGle1 localization when poly(A)+ RNA export is inhibited. HeLa cells were incubated with AP–hGle1-SD (SD) or control peptide (scrSD), processed for IIF using affinity-purified anti-hGle1 antibodies (A–C), and in situ hybridized with oligo (dT)30 (A) or stained with DAPI (B and C). Hybridized probe and bound hGle1 antibodies were simultaneously detected using rhodamine-labeled anti–Dig Fab antibodies and FITC-labeled anti–rabbit antibodies, respectively. Images showing in situ hybridization results reflect equivalent exposure times. (A) Fix-Triton permeabilization detects total hGle1 pool and shows changes in anti-hGle1 localization in AP–hGle1-SD–treated cells. (B) Digitonin-Fix permeabilization detects only the cytoplasmically accessible pool. AP–hGle1-SD results in decreased levels of cytoplasmically accessible hGle1 (asterisk) compared with unaffected cells (arrowheads, at passage >150; see Materials and methods) or cells treated with control peptide. The absence of lamin B staining using anti–lamin B antibodies (far right) confirms NE integrity under these conditions. (C) Detection of nuclear hGle1 was accomplished by combined Digitonin-Fix-Triton permeabilization. Lamin B staining confirms access of antibodies to intranuclear proteins. Bars, 10 μm.
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fig6: AP–hGle1-SD peptide results in mislocalization of endogenous hGle1 localization when poly(A)+ RNA export is inhibited. HeLa cells were incubated with AP–hGle1-SD (SD) or control peptide (scrSD), processed for IIF using affinity-purified anti-hGle1 antibodies (A–C), and in situ hybridized with oligo (dT)30 (A) or stained with DAPI (B and C). Hybridized probe and bound hGle1 antibodies were simultaneously detected using rhodamine-labeled anti–Dig Fab antibodies and FITC-labeled anti–rabbit antibodies, respectively. Images showing in situ hybridization results reflect equivalent exposure times. (A) Fix-Triton permeabilization detects total hGle1 pool and shows changes in anti-hGle1 localization in AP–hGle1-SD–treated cells. (B) Digitonin-Fix permeabilization detects only the cytoplasmically accessible pool. AP–hGle1-SD results in decreased levels of cytoplasmically accessible hGle1 (asterisk) compared with unaffected cells (arrowheads, at passage >150; see Materials and methods) or cells treated with control peptide. The absence of lamin B staining using anti–lamin B antibodies (far right) confirms NE integrity under these conditions. (C) Detection of nuclear hGle1 was accomplished by combined Digitonin-Fix-Triton permeabilization. Lamin B staining confirms access of antibodies to intranuclear proteins. Bars, 10 μm.
Mentions: In parallel with the study of poly(A)+ RNA distribution, we analyzed the localization of endogenous hGle1 by IIF microscopy in cells treated with AP–peptides. Different permeabilization and fixation conditions were used to discriminate between cytoplasmically accessible hGle1 and nuclear hGle1 (Fig. 6). In untreated wild-type cells processed by fixation and complete Triton X-100 permeabilization (Fix-Triton), a pool of hGle1 is localized at the NPC/NE, and a fraction of the hGle1 is cytoplasmically accessible (Watkins et al., 1998; Fig. 1 A). In cells treated with the control AP–hGle1-scrSD peptide and processed by Fix-Triton, endogenous hGle1 was localized in a typical wild-type pattern, and cells exhibited normal poly(A)+ RNA distribution (Fig. 6 A, top). However, when cells were treated with the AP–hGle1-SD peptide, distinct hGle1 staining at the NPC/NE was less apparent in cells with impaired poly(A)+ RNA export (Fig. 6 A, bottom).

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
Related in: MedlinePlus