An essential role for hGle1 nucleocytoplasmic shuttling in mRNA export.
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.
Kendirgi
Frederic
F
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232-8240, USA.
Barry
Dianne M
DM
Griffis
Eric R
ER
Powers
Maureen A
MA
Wente
Susan R
SR
D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000737:chemistry / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins; D011061:Poly A / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; C474082:Gle1 protein, human; D028884:Karyopherins; D008164:Luminescent Proteins; D029543:Nucleocytoplasmic Transport Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D049452:Green Fluorescent Proteins; D011061:Poly A