The mechanism of mRNA export is a complex issue central to cellular physiology. We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A) ؉ RNA export in Saccharomyces cerevisiae. To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells. hGLE1 encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs. hGLE1 can complement a yeast gle1 temperature-sensitive export mutant only if a LR-NES is inserted into it. To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells. In situ hybridization of injected cells showed that poly(A) ؉ RNA export was inhibited. In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter. We conclude that hGle1p functions in poly(A) ؉ RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES. With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm.The nuclear export of proteins and ribonucleoprotein (RNP) particles through the nuclear pore complex (NPC) is a facilitated and signal-dependent process (1-3). Moreover, RNA processing and transport events are tightly coupled, such that splicing, polyadenylation, and capping all affect the export process (4-9). Throughout the processing and exit pathway, RNA is bound by distinct proteins and the critical signals for export are predicted to reside on these proteins (1, 2, 10). This has been demonstrated clearly in studies of the HIV-1 Rev protein, which specifically binds unspliced viral RNA (11-13). The RNA-binding domain of Rev is distinct from a region containing a leucine-rich (LR) nuclear export sequence (NES), which is both necessary and sufficient for mediating nuclear export (14, 15). The LR-NES is recognized in the nucleoplasm by a nuclear export receptor, Crm1p͞exportin (16), that is a member of a family of  nuclear transport factors (17,18). Thus, the NES of Rev directs export of the protein and bound viral RNA coincidentally through the NPC by interaction with an exporting .In vertebrate cells, different RNA classes are exported by independent pathways with each RNA type (mRNA, U snRNA, tRNA, or rRNA) potentially requiring at least a subset of distinct factors (reviewed in refs.
Studies of the essential nucleoporin Nup145p have shown that its depletion is coincident with a block in RNA export and that deletion of its amino-terminal domain results in clustering of nuclear pore complexes. To further define the functional domains of Nup145p, we have characterized a panel of nup145 mutants. Deletions from both the amino terminus and the carboxy terminus resulted in temperature sensitive mutants that accumulated polyadenylated RNA in the nucleus at the non-permissive temperature. In addition, these mutants also displayed constitutive clustering of nuclear pore complexes in localized patches of the nuclear envelope. These results suggested that an internal region of Nup145p consisting of amino acids 593–893 is essential for function. Accordingly, when this region was deleted, growth was not supported at any temperature, whereas the region alone was able to complement a null mutation when expressed on a high copy plasmid. Previous studies have suggested that Nup145p is cleaved into two polypeptides of approximately 65 and 80 kDa. Interestingly, our experiments suggest that cleavage occurs in vivo. However, a small internal deletion of 17 amino acid residues that abolished cleavage had no effect on cell growth. Therefore, cleavage is not necessary for Nup145p function. When a sequence harboring the Nup145p cleavage site required for Nup145p cleavage was inserted in a chimeric protein, it was not sufficient for mediating cleavage. Cleavage likely requires a second region from amino acid residues 247–524 in addition to the cleavage site.
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