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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 results in nuclear poly(A)+ RNA accumulation. (A) Schematic representation of the cell-permeable peptides used. AP–hGle1-SD, antennapedia 16–amino acid leader peptide (AP) followed by hGle1 amino acids 444–483 (hGle1-SD) or followed by randomly scrambled sequence of identical SD composition (AP–hGle1-scrSD). (B) In situ hybridization experiments detect nuclear poly(A)+ RNA accumulation in AP–hGle1-SD–treated cells. HeLa cells (<10 passages) were incubated for 4 h with 5 μM of AP–hGle1-SD (SD) or the control AP–hGle1-scrSD peptide (scrSD) in normal growth medium. In situ hybridization using digoxigenin-labeled oligo(dT)30 detected total poly(A)+ RNA. Nuclear DNA was visualized by DAPI staining. *, cell undergoing apoptosis. (C) Poly(A)+ RNA distribution in untreated HeLa cells shows distribution similar to AP–hGle1-scrSD–treated cells. (D) AP–hGle1-SD impairs nuclear export of DHFR mRNA. Cells treated with AP–peptides were processed for in situ hybridization using a digoxigenin-labeled DNA probe against DHFR mRNA. Bar, 10 μm. (B–D) Respective hybridization signals represent equivalent exposure times. (E) Semiquantitative analysis of intracellular DHFR mRNA distribution in subcellular fractions of AP–peptide-treated cells. After treatment with peptides, total RNA from nuclear and cytoplasmic fractions was isolated and reverse transcribed with oligo (dT)18, and serial dilutions were made. Subsaturating PCR-based amplifications with DHFR-specific primers were performed (see Materials and methods), and products were separated on agarose gels and stained with ethidium bromide.
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fig4: The AP–hGle1-SD peptide results in nuclear poly(A)+ RNA accumulation. (A) Schematic representation of the cell-permeable peptides used. AP–hGle1-SD, antennapedia 16–amino acid leader peptide (AP) followed by hGle1 amino acids 444–483 (hGle1-SD) or followed by randomly scrambled sequence of identical SD composition (AP–hGle1-scrSD). (B) In situ hybridization experiments detect nuclear poly(A)+ RNA accumulation in AP–hGle1-SD–treated cells. HeLa cells (<10 passages) were incubated for 4 h with 5 μM of AP–hGle1-SD (SD) or the control AP–hGle1-scrSD peptide (scrSD) in normal growth medium. In situ hybridization using digoxigenin-labeled oligo(dT)30 detected total poly(A)+ RNA. Nuclear DNA was visualized by DAPI staining. *, cell undergoing apoptosis. (C) Poly(A)+ RNA distribution in untreated HeLa cells shows distribution similar to AP–hGle1-scrSD–treated cells. (D) AP–hGle1-SD impairs nuclear export of DHFR mRNA. Cells treated with AP–peptides were processed for in situ hybridization using a digoxigenin-labeled DNA probe against DHFR mRNA. Bar, 10 μm. (B–D) Respective hybridization signals represent equivalent exposure times. (E) Semiquantitative analysis of intracellular DHFR mRNA distribution in subcellular fractions of AP–peptide-treated cells. After treatment with peptides, total RNA from nuclear and cytoplasmic fractions was isolated and reverse transcribed with oligo (dT)18, and serial dilutions were made. Subsaturating PCR-based amplifications with DHFR-specific primers were performed (see Materials and methods), and products were separated on agarose gels and stained with ethidium bromide.

Mentions: To test this hypothesis, a peptide corresponding to the SD was synthesized as a COOH-terminal fusion to a 16–amino acid portion of helix 3 from the homeodomain of the Drosophila transcription factor antennapedia (Prochiantz, 1999), designated AP–hGle1-SD (Fig. 4 A). The antennapedia peptide (AP) sequence mediates the uptake of peptides into cells and confers stability (Derossi et al., 1996, 1998). As a control, an AP fusion peptide was also synthesized with a randomly scrambled sequence of the same amino acid composition as the SD (designated AP–hGle1-scrSD; Fig. 4 A). As seen in Fig. 4 B, incubating cultured HeLa cells (at <10 passages, see Materials and methods) with media containing 5 μM AP–hGle1-SD peptide had distinct effects. After 4 h in the presence of AP–hGle1-SD peptide, ∼5% of cells appeared to undergo apoptosis (asterisk). Interestingly, all the remaining interphase cells showed marked accumulation of poly(A)+ RNA in the nucleus. In contrast, no cytotoxicity was detected with the AP–hGle1-scrSD incubation, and all the cells showed normal poly(A)+ RNA distribution by in situ hybridization (untreated control cells in Fig. 4 C). In culture media with <10% FBS or with AP–hGle1-SD peptide concentrations >5 μM, the viability of the cells was drastically reduced. However, if the inhibitory peptide was removed after the 4-h incubation, the cells recovered and showed normal poly(A)+ RNA distribution after 12 h (not depicted).


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 results in nuclear poly(A)+ RNA accumulation. (A) Schematic representation of the cell-permeable peptides used. AP–hGle1-SD, antennapedia 16–amino acid leader peptide (AP) followed by hGle1 amino acids 444–483 (hGle1-SD) or followed by randomly scrambled sequence of identical SD composition (AP–hGle1-scrSD). (B) In situ hybridization experiments detect nuclear poly(A)+ RNA accumulation in AP–hGle1-SD–treated cells. HeLa cells (<10 passages) were incubated for 4 h with 5 μM of AP–hGle1-SD (SD) or the control AP–hGle1-scrSD peptide (scrSD) in normal growth medium. In situ hybridization using digoxigenin-labeled oligo(dT)30 detected total poly(A)+ RNA. Nuclear DNA was visualized by DAPI staining. *, cell undergoing apoptosis. (C) Poly(A)+ RNA distribution in untreated HeLa cells shows distribution similar to AP–hGle1-scrSD–treated cells. (D) AP–hGle1-SD impairs nuclear export of DHFR mRNA. Cells treated with AP–peptides were processed for in situ hybridization using a digoxigenin-labeled DNA probe against DHFR mRNA. Bar, 10 μm. (B–D) Respective hybridization signals represent equivalent exposure times. (E) Semiquantitative analysis of intracellular DHFR mRNA distribution in subcellular fractions of AP–peptide-treated cells. After treatment with peptides, total RNA from nuclear and cytoplasmic fractions was isolated and reverse transcribed with oligo (dT)18, and serial dilutions were made. Subsaturating PCR-based amplifications with DHFR-specific primers were performed (see Materials and methods), and products were separated on agarose gels and stained with ethidium bromide.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172758&req=5

fig4: The AP–hGle1-SD peptide results in nuclear poly(A)+ RNA accumulation. (A) Schematic representation of the cell-permeable peptides used. AP–hGle1-SD, antennapedia 16–amino acid leader peptide (AP) followed by hGle1 amino acids 444–483 (hGle1-SD) or followed by randomly scrambled sequence of identical SD composition (AP–hGle1-scrSD). (B) In situ hybridization experiments detect nuclear poly(A)+ RNA accumulation in AP–hGle1-SD–treated cells. HeLa cells (<10 passages) were incubated for 4 h with 5 μM of AP–hGle1-SD (SD) or the control AP–hGle1-scrSD peptide (scrSD) in normal growth medium. In situ hybridization using digoxigenin-labeled oligo(dT)30 detected total poly(A)+ RNA. Nuclear DNA was visualized by DAPI staining. *, cell undergoing apoptosis. (C) Poly(A)+ RNA distribution in untreated HeLa cells shows distribution similar to AP–hGle1-scrSD–treated cells. (D) AP–hGle1-SD impairs nuclear export of DHFR mRNA. Cells treated with AP–peptides were processed for in situ hybridization using a digoxigenin-labeled DNA probe against DHFR mRNA. Bar, 10 μm. (B–D) Respective hybridization signals represent equivalent exposure times. (E) Semiquantitative analysis of intracellular DHFR mRNA distribution in subcellular fractions of AP–peptide-treated cells. After treatment with peptides, total RNA from nuclear and cytoplasmic fractions was isolated and reverse transcribed with oligo (dT)18, and serial dilutions were made. Subsaturating PCR-based amplifications with DHFR-specific primers were performed (see Materials and methods), and products were separated on agarose gels and stained with ethidium bromide.
Mentions: To test this hypothesis, a peptide corresponding to the SD was synthesized as a COOH-terminal fusion to a 16–amino acid portion of helix 3 from the homeodomain of the Drosophila transcription factor antennapedia (Prochiantz, 1999), designated AP–hGle1-SD (Fig. 4 A). The antennapedia peptide (AP) sequence mediates the uptake of peptides into cells and confers stability (Derossi et al., 1996, 1998). As a control, an AP fusion peptide was also synthesized with a randomly scrambled sequence of the same amino acid composition as the SD (designated AP–hGle1-scrSD; Fig. 4 A). As seen in Fig. 4 B, incubating cultured HeLa cells (at <10 passages, see Materials and methods) with media containing 5 μM AP–hGle1-SD peptide had distinct effects. After 4 h in the presence of AP–hGle1-SD peptide, ∼5% of cells appeared to undergo apoptosis (asterisk). Interestingly, all the remaining interphase cells showed marked accumulation of poly(A)+ RNA in the nucleus. In contrast, no cytotoxicity was detected with the AP–hGle1-scrSD incubation, and all the cells showed normal poly(A)+ RNA distribution by in situ hybridization (untreated control cells in Fig. 4 C). In culture media with <10% FBS or with AP–hGle1-SD peptide concentrations >5 μM, the viability of the cells was drastically reduced. However, if the inhibitory peptide was removed after the 4-h incubation, the cells recovered and showed normal poly(A)+ RNA distribution after 12 h (not depicted).

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