Identification and characterization of the mouse nuclear export factor (Nxf) family members

Abstract

TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm.

Figure 1

Mouse Nxf2 and Nxf7. (A) Exon–intron structure of mouse Nxf2 and Nxf7 genes. The cDNAs were isolated and the sequences and the genomic structure were determined. Exons are shown in black boxes. ATG, translational initiation codon; TAA and TGA, translation termination codons. Asterisks indicate location of in-frame ATG codons. (B) Dendrogram illustrating the NXF family of proteins. Abbrevations: rn, Rattus norvegicus; cj, Coturnix japonica; dm, Drosophila melanogaster; sp, Schizosaccharomyces pombe; sc, Saccharomyces cerevisiae. The mouse family members are shown in bold. (C) Multiple sequence alignment of human TAP/hNXF1 and mouse NXF proteins. The protein domains based on Herold et al. (7) are indicated. The identified domains mediating nuclear localization of human TAP/hNXF1 and mNXF2, localization to cytoplasmic granules of mNXF7 and the rim association of mNXF2 are underlined. RBD, RNA-binding domain; LRR, leucine-rich repeat; NTF2, nuclear transport factor (NTF2)-like domain; UBA-like, ubiquitin associated-like domain.

Figure 1

Mouse Nxf2 and Nxf7. (A) Exon–intron structure of mouse Nxf2 and Nxf7 genes. The cDNAs were isolated and the sequences and the genomic structure were determined. Exons are shown in black boxes. ATG, translational initiation codon; TAA and TGA, translation termination codons. Asterisks indicate location of in-frame ATG codons. (B) Dendrogram illustrating the NXF family of proteins. Abbrevations: rn, Rattus norvegicus; cj, Coturnix japonica; dm, Drosophila melanogaster; sp, Schizosaccharomyces pombe; sc, Saccharomyces cerevisiae. The mouse family members are shown in bold. (C) Multiple sequence alignment of human TAP/hNXF1 and mouse NXF proteins. The protein domains based on Herold et al. (7) are indicated. The identified domains mediating nuclear localization of human TAP/hNXF1 and mNXF2, localization to cytoplasmic granules of mNXF7 and the rim association of mNXF2 are underlined. RBD, RNA-binding domain; LRR, leucine-rich repeat; NTF2, nuclear transport factor (NTF2)-like domain; UBA-like, ubiquitin associated-like domain.

Figure 2

Specific expression of NXF-related proteins in brain tissue. (A) Western immunoblot analysis of 293 cells transfected with untagged and tagged (HIS, GFP and Gβgal) mNXF2 (left panel) and different NXF expression plasmids (right panel) using a rabbit anti-mNXF2 antiserum. (B) Western blot analysis of mouse brain tissue using monospecific rabbit anti-mNXF2 antiserum. Pre-made ‘mouse brain aging’ blots (RNAWAY) contained normalized amounts of whole brain lysates from fetus to 1-year-old as indicated. (C) Western blot analysis of human tissues using the monospecific antiserum to human TAPX2/hNXF2. Pre-made blots (GenoTech) contained normalized amounts of proteins from the indicated tissues.

Figure 3

Distinct subcellular localization of mNXF2 and mNXF7 proteins. HeLa cells were transfected with GFP-tagged mNXF2 and mNXF7 expression plasmids, as indicated and the proteins were visualized in living cells. The images were obtained by fluorescent microscopy (Axiovert135TV, Zeiss) and by the use of a CCD camera and processed using IPLab Spectrum software (13). Similar results were obtained in numerous experiments and are typical of the vast majority of expressing cells in each individual experiment. Representative cells are shown.

Figure 4

Identification of NLS of mNXF2. (A and B) HeLa cells were transfected with plasmids producing mNXF2 and N- and C-terminal deletions fused to GFP (A) or GFP-βgal (B) and the proteins were visualized as described in Figure 2. The localization of the proteins is indicated. N, nucleus; C, cytoplasm. (C) Fine mapping of the NLS. Alanine substitution in 40–80Gβgal generated a series of mutants and the mutant proteins are visualized. The protein sequence containing the NLS regions is shown and the critical residues are indicated in bold.

Figure 5

Association of mNXF2 and mNXF7 with nuclear envelope. HeLa cells were transfected with plasmids producing the indicated GFP-tagged mNXF2 and mNXF7 deletion mutants and the images were analyzed as described in Figure 2.

Figure 6

Identification of signal mediating granule localization of mNXF7. (A) A panel of N- and C-terminal deletion mutants of GFP-tagged mNXF7 deletion mutants was analyzed upon transfection of HeLa cells. N, nucleus; C, cytoplasm. Confocal images of GFP fluorescence in the mid-sections through the nuclei. (B) Three-dimensional rendering of GFP images from confocal Z-stacks.

Figure 7

mNXF2 is an active mRNA export factor. (A) Human 293 cells were transfected with the cat reporter pDM128 either alone, in the presence of HIV-1 rev expression plasmid or in the presence of the indicated untagged NXF producing plasmids. As indicated, the transfection mixtures contained NXF expression plasmids together with human p15/NXT1 or p15/NXT1 alone. Two days later, the cells were harvested and the CAT production, measured as percentage of chloramphenicol acetylation, is shown by filled bars. Expression of the cotransfected luciferase expression plasmid was analyzed for each plate and the relative luciferase values are shown in open bars. A typical experiment is shown. (B) mNXF2 binds to TAP/hNXF1 export cofactors. Bacterially produced GST-tagged NXF proteins were immobilized on glutathione–Sepharose beads and used in pull-down assays with reticulocyte-produced, metabolically labeled factors (shown to the left). The bound (B) and 1:100 aliquots of the unbound (U) fractions were separated on SDS–PAGE and visualized on Phosphoimager.