Limits...
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.

Show MeSH
hGle1 encodes two mRNA isoforms in HeLa cells. (A) Intracellular localization of endogenous hGle1 (top; using anti-hGle1 antibodies; Watkins et al., 1998), ectopically expressed EGFP–hGle1A (middle), and EGFP–hGle1B (bottom) in fixed HeLa cells (Fix-Triton; see Materials and methods). Double IIF with mAb414 was used to detect NPC/NE localization. Bar, 10 μm. (B) Schematic representation of hGle1. Exons are boxed, numbered 5′ to 3′, with nos. 15 and 16 specific to hGle1B (gray boxes). Primers used to specifically amplify hGle1A and hGle1B in RT-PCR experiments are F (common forward primer), A (hGle1A 3′ UTR–specific reverse primer), and B (hGle1B-specific reverse primer). (C) Semiquantitative RT-PCR analysis of hGle1A and hGleB mRNA levels in HeLa cells. Serial dilutions of total HeLa cell cDNA were tested using primer combinations F/A and F/B for hGle1A and hGle1B, respectively. Amplification products (202 bp and 180 bp, respectively) were separated on agarose gel and stained with ethidium bromide. Similar intensities at cDNA dilutions of 10−2 for hGle1A and 10−5 for hGle1B (arrowheads) suggest that hGle1B mRNA is 103-fold more abundant. (D) Sequence comparison (ClustalW) of the COOH-terminal regions of scGle1, hGle1A, and hGle1B (Pearson and Lipman, 1988). Boxed region 444–483 of hGle1A/B harbors shuttling activity. The putative LR-NES in scGle1 is double underlined. The unique four amino acids of hGle1A are underlined. *, identical residues; : or ., conserved residues.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172758&req=5

fig1: hGle1 encodes two mRNA isoforms in HeLa cells. (A) Intracellular localization of endogenous hGle1 (top; using anti-hGle1 antibodies; Watkins et al., 1998), ectopically expressed EGFP–hGle1A (middle), and EGFP–hGle1B (bottom) in fixed HeLa cells (Fix-Triton; see Materials and methods). Double IIF with mAb414 was used to detect NPC/NE localization. Bar, 10 μm. (B) Schematic representation of hGle1. Exons are boxed, numbered 5′ to 3′, with nos. 15 and 16 specific to hGle1B (gray boxes). Primers used to specifically amplify hGle1A and hGle1B in RT-PCR experiments are F (common forward primer), A (hGle1A 3′ UTR–specific reverse primer), and B (hGle1B-specific reverse primer). (C) Semiquantitative RT-PCR analysis of hGle1A and hGleB mRNA levels in HeLa cells. Serial dilutions of total HeLa cell cDNA were tested using primer combinations F/A and F/B for hGle1A and hGle1B, respectively. Amplification products (202 bp and 180 bp, respectively) were separated on agarose gel and stained with ethidium bromide. Similar intensities at cDNA dilutions of 10−2 for hGle1A and 10−5 for hGle1B (arrowheads) suggest that hGle1B mRNA is 103-fold more abundant. (D) Sequence comparison (ClustalW) of the COOH-terminal regions of scGle1, hGle1A, and hGle1B (Pearson and Lipman, 1988). Boxed region 444–483 of hGle1A/B harbors shuttling activity. The putative LR-NES in scGle1 is double underlined. The unique four amino acids of hGle1A are underlined. *, identical residues; : or ., conserved residues.

Mentions: Previous studies have shown endogenous hGle1 localization at the NPCs/NE and in the cytoplasm and nucleus (Watkins et al., 1998; Fig. 1 A). Here we show that ectopic expression of the hGle1 cDNA previously reported by Watkins et al. (1998), in fusion with EGFP, did not produce a protein that localized predominately to the NE of transfected cells (Fig. 1 A). We speculated that this isoform (hereafter designated hGle1A, a 659–amino acid polypeptide; accession no. AAC25561) lacked a critical region required for NPC/NE association. Recently, a human testis cDNA clone was deposited in the human EST database (accession no. AAH30012). The predicted protein product is a 698–amino acid polypeptide (designated hGle1B) identical to hGle1A except at the COOH terminus (Fig. 1, B and D). Based on genomic sequence information (Locus ID 2733), the hGle1 gene is comprised of 16 exons. Within exon 14, a 5′ cryptic splice donor site could account for the expression of two transcript variants, with two corresponding different 3′ untranslated regions (Nissim-Rafinia and Kerem, 2002). The predicted products result in the hGle1A version with a unique four–amino acid segment and hGle1B with a different 43–amino acid span at the COOH terminus (Fig. 1 D).


An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

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

hGle1 encodes two mRNA isoforms in HeLa cells. (A) Intracellular localization of endogenous hGle1 (top; using anti-hGle1 antibodies; Watkins et al., 1998), ectopically expressed EGFP–hGle1A (middle), and EGFP–hGle1B (bottom) in fixed HeLa cells (Fix-Triton; see Materials and methods). Double IIF with mAb414 was used to detect NPC/NE localization. Bar, 10 μm. (B) Schematic representation of hGle1. Exons are boxed, numbered 5′ to 3′, with nos. 15 and 16 specific to hGle1B (gray boxes). Primers used to specifically amplify hGle1A and hGle1B in RT-PCR experiments are F (common forward primer), A (hGle1A 3′ UTR–specific reverse primer), and B (hGle1B-specific reverse primer). (C) Semiquantitative RT-PCR analysis of hGle1A and hGleB mRNA levels in HeLa cells. Serial dilutions of total HeLa cell cDNA were tested using primer combinations F/A and F/B for hGle1A and hGle1B, respectively. Amplification products (202 bp and 180 bp, respectively) were separated on agarose gel and stained with ethidium bromide. Similar intensities at cDNA dilutions of 10−2 for hGle1A and 10−5 for hGle1B (arrowheads) suggest that hGle1B mRNA is 103-fold more abundant. (D) Sequence comparison (ClustalW) of the COOH-terminal regions of scGle1, hGle1A, and hGle1B (Pearson and Lipman, 1988). Boxed region 444–483 of hGle1A/B harbors shuttling activity. The putative LR-NES in scGle1 is double underlined. The unique four amino acids of hGle1A are underlined. *, identical residues; : or ., conserved residues.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: hGle1 encodes two mRNA isoforms in HeLa cells. (A) Intracellular localization of endogenous hGle1 (top; using anti-hGle1 antibodies; Watkins et al., 1998), ectopically expressed EGFP–hGle1A (middle), and EGFP–hGle1B (bottom) in fixed HeLa cells (Fix-Triton; see Materials and methods). Double IIF with mAb414 was used to detect NPC/NE localization. Bar, 10 μm. (B) Schematic representation of hGle1. Exons are boxed, numbered 5′ to 3′, with nos. 15 and 16 specific to hGle1B (gray boxes). Primers used to specifically amplify hGle1A and hGle1B in RT-PCR experiments are F (common forward primer), A (hGle1A 3′ UTR–specific reverse primer), and B (hGle1B-specific reverse primer). (C) Semiquantitative RT-PCR analysis of hGle1A and hGleB mRNA levels in HeLa cells. Serial dilutions of total HeLa cell cDNA were tested using primer combinations F/A and F/B for hGle1A and hGle1B, respectively. Amplification products (202 bp and 180 bp, respectively) were separated on agarose gel and stained with ethidium bromide. Similar intensities at cDNA dilutions of 10−2 for hGle1A and 10−5 for hGle1B (arrowheads) suggest that hGle1B mRNA is 103-fold more abundant. (D) Sequence comparison (ClustalW) of the COOH-terminal regions of scGle1, hGle1A, and hGle1B (Pearson and Lipman, 1988). Boxed region 444–483 of hGle1A/B harbors shuttling activity. The putative LR-NES in scGle1 is double underlined. The unique four amino acids of hGle1A are underlined. *, identical residues; : or ., conserved residues.
Mentions: Previous studies have shown endogenous hGle1 localization at the NPCs/NE and in the cytoplasm and nucleus (Watkins et al., 1998; Fig. 1 A). Here we show that ectopic expression of the hGle1 cDNA previously reported by Watkins et al. (1998), in fusion with EGFP, did not produce a protein that localized predominately to the NE of transfected cells (Fig. 1 A). We speculated that this isoform (hereafter designated hGle1A, a 659–amino acid polypeptide; accession no. AAC25561) lacked a critical region required for NPC/NE association. Recently, a human testis cDNA clone was deposited in the human EST database (accession no. AAH30012). The predicted protein product is a 698–amino acid polypeptide (designated hGle1B) identical to hGle1A except at the COOH terminus (Fig. 1, B and D). Based on genomic sequence information (Locus ID 2733), the hGle1 gene is comprised of 16 exons. Within exon 14, a 5′ cryptic splice donor site could account for the expression of two transcript variants, with two corresponding different 3′ untranslated regions (Nissim-Rafinia and Kerem, 2002). The predicted products result in the hGle1A version with a unique four–amino acid segment and hGle1B with a different 43–amino acid span at the COOH terminus (Fig. 1 D).

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