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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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Comparison of the hGle1 SD with nucleocytoplasmic shuttling sequences. The amino acid sequence of the hGle1 SD does not share significant similarities with other nucleocytoplasmic transport signals (Michael et al., 1995, 1997; Fan and Steitz, 1998). There are limited similarities with the hnRNP A1 M9 transport signal (boxed), but the hGle1 SD lacks the critical GPM triplet (asterisks) required for efficient transport activity of M9 (Bogerd et al., 1999).
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fig8: Comparison of the hGle1 SD with nucleocytoplasmic shuttling sequences. The amino acid sequence of the hGle1 SD does not share significant similarities with other nucleocytoplasmic transport signals (Michael et al., 1995, 1997; Fan and Steitz, 1998). There are limited similarities with the hnRNP A1 M9 transport signal (boxed), but the hGle1 SD lacks the critical GPM triplet (asterisks) required for efficient transport activity of M9 (Bogerd et al., 1999).

Mentions: Interestingly, the amino acid sequence of the hGle1 SD is not similar to any of the previously characterized nucleocytoplasmic SDs (Fig. 8). A direct alignment of the hGle1 SD with the M9 of hnRNP A1 suggests weak similarity, which could result in a potential interaction with the M9-binding karyopherin β2A/transportin-1 (Siomi et al., 1997). However, the key consensus residues implicated in transportin-1 binding are not present in hGle1 (Pollard et al., 1996). We tested for an hGle1-SD and transportin-1 interaction and found that bacterially expressed GST–transportin-1 does not directly bind immobilized hGle1-SD peptide (unpublished data). This suggests that transportin-1 is not directly involved in hGle1 shuttling. Further deletions within the hGle1 SD and selective point mutations based on interspecies homology analysis have not yet been successful in separating the nuclear import and export activities (unpublished data).


An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.

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

Comparison of the hGle1 SD with nucleocytoplasmic shuttling sequences. The amino acid sequence of the hGle1 SD does not share significant similarities with other nucleocytoplasmic transport signals (Michael et al., 1995, 1997; Fan and Steitz, 1998). There are limited similarities with the hnRNP A1 M9 transport signal (boxed), but the hGle1 SD lacks the critical GPM triplet (asterisks) required for efficient transport activity of M9 (Bogerd et al., 1999).
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Related In: Results  -  Collection

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

fig8: Comparison of the hGle1 SD with nucleocytoplasmic shuttling sequences. The amino acid sequence of the hGle1 SD does not share significant similarities with other nucleocytoplasmic transport signals (Michael et al., 1995, 1997; Fan and Steitz, 1998). There are limited similarities with the hnRNP A1 M9 transport signal (boxed), but the hGle1 SD lacks the critical GPM triplet (asterisks) required for efficient transport activity of M9 (Bogerd et al., 1999).
Mentions: Interestingly, the amino acid sequence of the hGle1 SD is not similar to any of the previously characterized nucleocytoplasmic SDs (Fig. 8). A direct alignment of the hGle1 SD with the M9 of hnRNP A1 suggests weak similarity, which could result in a potential interaction with the M9-binding karyopherin β2A/transportin-1 (Siomi et al., 1997). However, the key consensus residues implicated in transportin-1 binding are not present in hGle1 (Pollard et al., 1996). We tested for an hGle1-SD and transportin-1 interaction and found that bacterially expressed GST–transportin-1 does not directly bind immobilized hGle1-SD peptide (unpublished data). This suggests that transportin-1 is not directly involved in hGle1 shuttling. Further deletions within the hGle1 SD and selective point mutations based on interspecies homology analysis have not yet been successful in separating the nuclear import and export activities (unpublished data).

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