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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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Delineating the hGle1 domain with nuclear export activity. (Top) Schematic representation and summary of the intracellular localizations for FLAG–hGle1A–NLS deletion constructs. C, cytoplasmic localization; N, nuclear localization; *, representative localization below. (Bottom) IIF localization of representative FLAG–hGle1–NLS proteins after transient expression in HeLa cells. Anti-FLAG monoclonal antibodies (top row) and staining for nuclear DNA with DAPI (bottom row) are shown. The cells in each figure are representative of the localization seen in the majority (∼70%; n = 500) of the cells across the total population of transfectants. Bar, 10 μm.
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fig2: Delineating the hGle1 domain with nuclear export activity. (Top) Schematic representation and summary of the intracellular localizations for FLAG–hGle1A–NLS deletion constructs. C, cytoplasmic localization; N, nuclear localization; *, representative localization below. (Bottom) IIF localization of representative FLAG–hGle1–NLS proteins after transient expression in HeLa cells. Anti-FLAG monoclonal antibodies (top row) and staining for nuclear DNA with DAPI (bottom row) are shown. The cells in each figure are representative of the localization seen in the majority (∼70%; n = 500) of the cells across the total population of transfectants. Bar, 10 μm.

Mentions: We speculated that if hGle1 is dynamic and transiently associates with NPCs while entering and exiting the nucleus, it should contain domains/sequences that harbor nucleocytoplasmic transport activities. The region of hGle1 spanning amino acid residues 444–606 was specifically targeted for internal in-frame deletion based on our previous studies of scGle1 (Murphy and Wente, 1996; Watkins et al., 1998). The COOH-terminal regions of scGle1 and hGle1A/B are highly similar, and the corresponding region in scGle1 contains a putative leucine-rich (LR) NES-like motif that is essential for scGle1 function in mRNA export (Fig. 1 D; see Discussion). As a tool to delineate the region(s) of hGle1 that may harbor nuclear export activity, we generated a panel of ectopically expressed FLAG-tagged hGle1A deletion proteins and analyzed their respective intracellular distributions independent of the endogenous hGle1 by indirect immunofluorescence (IIF) using anti-FLAG antibodies. To assay for export activity, we placed the SV40 large T antigen NLS in frame at the COOH terminus of FLAG–hGle1A. Based on the hypothesis that hGle1 harbors nuclear export activity, it should balance the activity of the heterologous NLS and result in cytoplasmic FLAG–hGle1A–NLS localization. Results from these experiments are summarized in Fig. 2. Although the majority of the FLAG–hGle1A–NLS was detected in the nucleus, FLAG–hGle1A–NLS was also clearly present in the cytoplasm (Fig. 2, lower left). Strikingly, the FLAG–hGle1AΔ444–606–NLS protein was localized exclusively in the nucleus. Various NH2- and COOH-terminal deletions of the 444–606 region were tested to map the export activity. Deletions from the NH2-terminal side eliminated cytoplasmic localization (Δ444–511), whereas deletions from the COOH-terminal side up to residue 483 retained the export activity (Δ484–606) (Fig. 2). Thus, a 39–amino acid region from residues 444–483 was necessary to confer cytoplasmic localization of FLAG–hGle1A–NLS.


An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

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

Delineating the hGle1 domain with nuclear export activity. (Top) Schematic representation and summary of the intracellular localizations for FLAG–hGle1A–NLS deletion constructs. C, cytoplasmic localization; N, nuclear localization; *, representative localization below. (Bottom) IIF localization of representative FLAG–hGle1–NLS proteins after transient expression in HeLa cells. Anti-FLAG monoclonal antibodies (top row) and staining for nuclear DNA with DAPI (bottom row) are shown. The cells in each figure are representative of the localization seen in the majority (∼70%; n = 500) of the cells across the total population of transfectants. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172758&req=5

fig2: Delineating the hGle1 domain with nuclear export activity. (Top) Schematic representation and summary of the intracellular localizations for FLAG–hGle1A–NLS deletion constructs. C, cytoplasmic localization; N, nuclear localization; *, representative localization below. (Bottom) IIF localization of representative FLAG–hGle1–NLS proteins after transient expression in HeLa cells. Anti-FLAG monoclonal antibodies (top row) and staining for nuclear DNA with DAPI (bottom row) are shown. The cells in each figure are representative of the localization seen in the majority (∼70%; n = 500) of the cells across the total population of transfectants. Bar, 10 μm.
Mentions: We speculated that if hGle1 is dynamic and transiently associates with NPCs while entering and exiting the nucleus, it should contain domains/sequences that harbor nucleocytoplasmic transport activities. The region of hGle1 spanning amino acid residues 444–606 was specifically targeted for internal in-frame deletion based on our previous studies of scGle1 (Murphy and Wente, 1996; Watkins et al., 1998). The COOH-terminal regions of scGle1 and hGle1A/B are highly similar, and the corresponding region in scGle1 contains a putative leucine-rich (LR) NES-like motif that is essential for scGle1 function in mRNA export (Fig. 1 D; see Discussion). As a tool to delineate the region(s) of hGle1 that may harbor nuclear export activity, we generated a panel of ectopically expressed FLAG-tagged hGle1A deletion proteins and analyzed their respective intracellular distributions independent of the endogenous hGle1 by indirect immunofluorescence (IIF) using anti-FLAG antibodies. To assay for export activity, we placed the SV40 large T antigen NLS in frame at the COOH terminus of FLAG–hGle1A. Based on the hypothesis that hGle1 harbors nuclear export activity, it should balance the activity of the heterologous NLS and result in cytoplasmic FLAG–hGle1A–NLS localization. Results from these experiments are summarized in Fig. 2. Although the majority of the FLAG–hGle1A–NLS was detected in the nucleus, FLAG–hGle1A–NLS was also clearly present in the cytoplasm (Fig. 2, lower left). Strikingly, the FLAG–hGle1AΔ444–606–NLS protein was localized exclusively in the nucleus. Various NH2- and COOH-terminal deletions of the 444–606 region were tested to map the export activity. Deletions from the NH2-terminal side eliminated cytoplasmic localization (Δ444–511), whereas deletions from the COOH-terminal side up to residue 483 retained the export activity (Δ484–606) (Fig. 2). Thus, a 39–amino acid region from residues 444–483 was necessary to confer cytoplasmic localization of FLAG–hGle1A–NLS.

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