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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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Analysis of GST–hGle1 dynamics in HeLa cells by microinjection. (A) The hGle1 39–amino acid region from 444–483 has intrinsic nucleocytoplasmic shuttling activity. HeLa cells were comicroinjected with purified proteins and TR-labeled 70-kD dextran. Cells were incubated for 5 h at 37°C or 4°C after injection and processed for IIF microscopy using anti-GST antibodies (top). Nuclear DNA was stained with DAPI (bottom). At 37°C (left), microinjection of GST–hGle1444–483 (SD) in one nucleus of a binucleate HeLa cell (top, arrow), as indicated by the TR-labeled dextran (middle), results in GST staining in the second nucleus (arrowhead). At 4°C (right), nuclear export and import activity of GST–hGle1444–483 (SD) is not detected. (arrow, nuclear injection; asterisk, cytoplasmic injection). (B and D) The SD is necessary for GST–hGle1B export. Full-length GST–hGle1B (B) and GST–hGle1ΔSD (D) were analyzed by microinjection. (B) After injection into one nucleus of a binucleate cell (arrowhead), GST–hGle1B is in both the cytoplasm and the uninjected nucleus after incubation at 37°C (right). Cytoplasmically microinjected GST–hGle1B (asterisk) shows strong nuclear staining. At 4°C (left), injected protein remains at the site of microinjection. (C) FLIP analysis reveals that EGFP–hGle1B shuttles between the nucleus and cytoplasm in HeLa cells. An area of the cytoplasm was repeatedly bleached, and the loss of nuclear fluorescence was monitored over time. The data points plotted for EGFP–hGle1B represent averages (n = 6). EGFP–coilin and EGFP–hNup98 data are representative of the loss of fluorescence detected in several time courses and are consistent with data previously reported (Griffis et al., 2002). (D) Microinjection of GST–hGle1BΔSD protein in the nucleus results in no staining outside the microinjection site after incubation at 37°C (both panels) (or 4°C; not depicted). In the right panel, partial nuclear localization is observed in two cytoplasmically injected cells (asterisks). Bars, 10 μm.
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fig3: Analysis of GST–hGle1 dynamics in HeLa cells by microinjection. (A) The hGle1 39–amino acid region from 444–483 has intrinsic nucleocytoplasmic shuttling activity. HeLa cells were comicroinjected with purified proteins and TR-labeled 70-kD dextran. Cells were incubated for 5 h at 37°C or 4°C after injection and processed for IIF microscopy using anti-GST antibodies (top). Nuclear DNA was stained with DAPI (bottom). At 37°C (left), microinjection of GST–hGle1444–483 (SD) in one nucleus of a binucleate HeLa cell (top, arrow), as indicated by the TR-labeled dextran (middle), results in GST staining in the second nucleus (arrowhead). At 4°C (right), nuclear export and import activity of GST–hGle1444–483 (SD) is not detected. (arrow, nuclear injection; asterisk, cytoplasmic injection). (B and D) The SD is necessary for GST–hGle1B export. Full-length GST–hGle1B (B) and GST–hGle1ΔSD (D) were analyzed by microinjection. (B) After injection into one nucleus of a binucleate cell (arrowhead), GST–hGle1B is in both the cytoplasm and the uninjected nucleus after incubation at 37°C (right). Cytoplasmically microinjected GST–hGle1B (asterisk) shows strong nuclear staining. At 4°C (left), injected protein remains at the site of microinjection. (C) FLIP analysis reveals that EGFP–hGle1B shuttles between the nucleus and cytoplasm in HeLa cells. An area of the cytoplasm was repeatedly bleached, and the loss of nuclear fluorescence was monitored over time. The data points plotted for EGFP–hGle1B represent averages (n = 6). EGFP–coilin and EGFP–hNup98 data are representative of the loss of fluorescence detected in several time courses and are consistent with data previously reported (Griffis et al., 2002). (D) Microinjection of GST–hGle1BΔSD protein in the nucleus results in no staining outside the microinjection site after incubation at 37°C (both panels) (or 4°C; not depicted). In the right panel, partial nuclear localization is observed in two cytoplasmically injected cells (asterisks). Bars, 10 μm.

Mentions: To further examine the transport properties of the 39–amino acid hGle1 region defined above, we conducted a series of microinjection experiments with bacterially expressed and purified GST fusion proteins (Fig. 3). Binucleate HeLa cells were used for nuclear microinjection such that the export and nucleocytoplasmic shuttling activities of GST fusion proteins could be coincidentally examined. First, a GST fusion with only the 39–amino acid putative domain (hGle1444–483 (SD)) was tested by coinjection with Texas red (TR)–labeled 70-kD dextran into one nucleus of a binucleate HeLa cell (Fig. 3 A). The TR–dextran identified injected cells and the subcellular injection site (arrows). The cells were subsequently incubated at either 37°C (Fig. 3 A, left) or 4°C (right), fixed, and processed for IIF microscopy with anti-GST antibodies. At 37°C, 86.5% (±2.8%; n =112) of the microinjected cells showed GST–hGle1444–483 (SD) at the injection site, in the cytoplasm, and in the second nucleus (Fig. 3 A, arrowhead). In contrast, at 4°C, GST–hGle1444–483 (SD) remained at the site of injection in 87% (±4.3%; n = 81) of the injected cells. These results indicated that GST–hGle1444–483 (SD) was actively exported from the injected nucleus. In time course experiments, export was detected at full levels within 2 h after injection (unpublished data). Thus, residues 444–483 of hGle1 were sufficient to mediate nuclear export. In addition, the presence of GST–hGle1444–483 (SD) in the second nucleus of binucleate cells suggested that this region also had nuclear import activity. These experiments support the hypothesis that hGle1 harbors a nucleocytoplasmic transport domain, for both import and export. Hence, we have designated this 39–amino acid span the hGle1 nucleocytoplasmic shuttling domain (SD).


An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

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

Analysis of GST–hGle1 dynamics in HeLa cells by microinjection. (A) The hGle1 39–amino acid region from 444–483 has intrinsic nucleocytoplasmic shuttling activity. HeLa cells were comicroinjected with purified proteins and TR-labeled 70-kD dextran. Cells were incubated for 5 h at 37°C or 4°C after injection and processed for IIF microscopy using anti-GST antibodies (top). Nuclear DNA was stained with DAPI (bottom). At 37°C (left), microinjection of GST–hGle1444–483 (SD) in one nucleus of a binucleate HeLa cell (top, arrow), as indicated by the TR-labeled dextran (middle), results in GST staining in the second nucleus (arrowhead). At 4°C (right), nuclear export and import activity of GST–hGle1444–483 (SD) is not detected. (arrow, nuclear injection; asterisk, cytoplasmic injection). (B and D) The SD is necessary for GST–hGle1B export. Full-length GST–hGle1B (B) and GST–hGle1ΔSD (D) were analyzed by microinjection. (B) After injection into one nucleus of a binucleate cell (arrowhead), GST–hGle1B is in both the cytoplasm and the uninjected nucleus after incubation at 37°C (right). Cytoplasmically microinjected GST–hGle1B (asterisk) shows strong nuclear staining. At 4°C (left), injected protein remains at the site of microinjection. (C) FLIP analysis reveals that EGFP–hGle1B shuttles between the nucleus and cytoplasm in HeLa cells. An area of the cytoplasm was repeatedly bleached, and the loss of nuclear fluorescence was monitored over time. The data points plotted for EGFP–hGle1B represent averages (n = 6). EGFP–coilin and EGFP–hNup98 data are representative of the loss of fluorescence detected in several time courses and are consistent with data previously reported (Griffis et al., 2002). (D) Microinjection of GST–hGle1BΔSD protein in the nucleus results in no staining outside the microinjection site after incubation at 37°C (both panels) (or 4°C; not depicted). In the right panel, partial nuclear localization is observed in two cytoplasmically injected cells (asterisks). Bars, 10 μm.
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fig3: Analysis of GST–hGle1 dynamics in HeLa cells by microinjection. (A) The hGle1 39–amino acid region from 444–483 has intrinsic nucleocytoplasmic shuttling activity. HeLa cells were comicroinjected with purified proteins and TR-labeled 70-kD dextran. Cells were incubated for 5 h at 37°C or 4°C after injection and processed for IIF microscopy using anti-GST antibodies (top). Nuclear DNA was stained with DAPI (bottom). At 37°C (left), microinjection of GST–hGle1444–483 (SD) in one nucleus of a binucleate HeLa cell (top, arrow), as indicated by the TR-labeled dextran (middle), results in GST staining in the second nucleus (arrowhead). At 4°C (right), nuclear export and import activity of GST–hGle1444–483 (SD) is not detected. (arrow, nuclear injection; asterisk, cytoplasmic injection). (B and D) The SD is necessary for GST–hGle1B export. Full-length GST–hGle1B (B) and GST–hGle1ΔSD (D) were analyzed by microinjection. (B) After injection into one nucleus of a binucleate cell (arrowhead), GST–hGle1B is in both the cytoplasm and the uninjected nucleus after incubation at 37°C (right). Cytoplasmically microinjected GST–hGle1B (asterisk) shows strong nuclear staining. At 4°C (left), injected protein remains at the site of microinjection. (C) FLIP analysis reveals that EGFP–hGle1B shuttles between the nucleus and cytoplasm in HeLa cells. An area of the cytoplasm was repeatedly bleached, and the loss of nuclear fluorescence was monitored over time. The data points plotted for EGFP–hGle1B represent averages (n = 6). EGFP–coilin and EGFP–hNup98 data are representative of the loss of fluorescence detected in several time courses and are consistent with data previously reported (Griffis et al., 2002). (D) Microinjection of GST–hGle1BΔSD protein in the nucleus results in no staining outside the microinjection site after incubation at 37°C (both panels) (or 4°C; not depicted). In the right panel, partial nuclear localization is observed in two cytoplasmically injected cells (asterisks). Bars, 10 μm.
Mentions: To further examine the transport properties of the 39–amino acid hGle1 region defined above, we conducted a series of microinjection experiments with bacterially expressed and purified GST fusion proteins (Fig. 3). Binucleate HeLa cells were used for nuclear microinjection such that the export and nucleocytoplasmic shuttling activities of GST fusion proteins could be coincidentally examined. First, a GST fusion with only the 39–amino acid putative domain (hGle1444–483 (SD)) was tested by coinjection with Texas red (TR)–labeled 70-kD dextran into one nucleus of a binucleate HeLa cell (Fig. 3 A). The TR–dextran identified injected cells and the subcellular injection site (arrows). The cells were subsequently incubated at either 37°C (Fig. 3 A, left) or 4°C (right), fixed, and processed for IIF microscopy with anti-GST antibodies. At 37°C, 86.5% (±2.8%; n =112) of the microinjected cells showed GST–hGle1444–483 (SD) at the injection site, in the cytoplasm, and in the second nucleus (Fig. 3 A, arrowhead). In contrast, at 4°C, GST–hGle1444–483 (SD) remained at the site of injection in 87% (±4.3%; n = 81) of the injected cells. These results indicated that GST–hGle1444–483 (SD) was actively exported from the injected nucleus. In time course experiments, export was detected at full levels within 2 h after injection (unpublished data). Thus, residues 444–483 of hGle1 were sufficient to mediate nuclear export. In addition, the presence of GST–hGle1444–483 (SD) in the second nucleus of binucleate cells suggested that this region also had nuclear import activity. These experiments support the hypothesis that hGle1 harbors a nucleocytoplasmic transport domain, for both import and export. Hence, we have designated this 39–amino acid span the hGle1 nucleocytoplasmic shuttling domain (SD).

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