Mex67p of Schizosaccharomyces pombe interacts with Rae1p in mediating mRNA export

We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 ts mutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast to scMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Deltamex67) is synthetically lethal with the rae1-167 mutation and accumulates poly(A)(+) RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of the rae1-167 Deltamex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149-505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149-505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149-505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.     

The nuclear export signal of splicing factor Uap56p interacts with nuclear pore-associated protein Rae1p for mRNA export in Schizosaccharomyces pombe

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.     

Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions

Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe. Here we show that S. pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential. We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67. These residues map within the known NTF2-like fold of TAP (amino acids 371-551). We show that the homologs of these two NESs are present and are functionally conserved in TAP. The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S. pombe Nup159 and Nup98 but not with human p62. Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S. pombe and in HeLa cells. Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S. pombe strain, suggesting that the NESs can function in the absence of p15. These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex.     

Elf1p,a member of the ABC class of ATPases,functions as a mRNA export factor in Schizosacchromyces pombe

Rae1p and Mex67p/Tap are conserved mRNA export factors. We have used synthetic lethal genetic screens in Schizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67 in mRNA export. From these screens, we have isolated mutations in a putative S. pombe homologue of the Candida albicans elf1 gene. The elf1 of S. pombe is not an essential gene. When elf1 mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants. This inhibition can be suppressed by the multicopy expression of mex67 suggesting that Mex67p can substitute for the loss of Elf1p function. Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm. Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP. A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro. This mutant protein no longer functions in mRNA export. Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S. pombe.     

Schizosaccharomyces pombe nup97, which Genetically Interacts with mex67, is essential for growth and involved in mRNA export

We have isolated previously three synthetic lethal mutants in Schizosaccharomyces pombe, which genetically interact with mex67, in order to identify the genes involved in mRNA export. A novel nup97 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex3. The nup97 gene contains one intron and encodes an 851 amino-acid protein that is similar to nucleoporins, Npp106p in S. pombe and Nic96p in Saccharomyces cerevisiae. The nup97 gene is essential for vegetative growth, and nup97 null mutant harboring pREP41X-Nup97 showed poly(A)+ RNA export defect when expression of nup97 is repressed in the presence of thiamine. These results suggest that nup97 is involved in mRNA export from the nucleus to cytoplasm.     

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.

An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.     

The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim

Eukaryotic mRNAs are exported from the nucleus to the cytoplasm as complex mRNA-protein particles (mRNPs), and translocation through the nuclear pore complex (NPC) is accompanied by extensive structural changes of the mRNP. We have tested the hypothesis that the DEAD-box ATPase Dbp5p is required for such an mRNP rearrangement. In dbp5 mutant cells, the mRNA export receptor Mex67p accumulates on mRNA. This aberrant accumulation of Mex67p with RNA and the cold-sensitive growth phenotype of a dbp5 allele are suppressed by a mex67 mutation. Moreover, Mex67 bound mRNA accumulates at the nuclear rim in a temperature-sensitive dbp5 mutant when the nuclear exosome is impaired. Importantly, although accumulation of Mex67p-containing mRNPs is also observed when a nuclear basket component is mutated, these mRNPs still contain the nuclear export factor Yra1p. In contrast, the dbp5-trapped mRNPs lack Yra1p. We propose that Dbp5p's function is specifically required to displace Mex67p from exported mRNPs, thus terminating export.     

Npp106p, a Schizosaccharomyces pombe nucleoporin similar to Saccharomyces cerevisiae Nic96p, functionally interacts with Rae1p in mRNA export.

To identify components of the mRNA export machinery in Schizosaccharomyces pombe, a screen was developed to identify mutations that were synthetically lethal with the conditional mRNA export allele rae1-167. Mutations defining three complementation groups were isolated, and here we report the characterization of npp106 (for nuclear pore protein of 106 kDa). This gene encodes a predicted protein that has significant similarity to the Nic96p nucleoporin of Saccharomyces cerevisiae. Consistent with Npp106p being a nucleoporin, a functional green fluorescent protein (GFP)-tagged Npp106p localized to the nuclear periphery. In contrast to NIC96, the npp106 gene is not essential. Moreover, a delta npp106 mutant did not show cytoplasmic mislocalization of a simian virus 40 nuclear localization signal-GFP-LacZ reporter protein, and a fraction of cells had accumulation of poly(A)+ RNA in the nucleus. A consequence of the synthetic lethality between rae1-167 and npp106-1 was the accumulation of poly(A)+ RNA in the nucleus when cells were grown under synthetic lethal conditions. In addition to npp106-1, which is a nonsense mutation that truncates the protein at amino acid 292, the delta npp106 mutation was synthetically lethal with rae1-167, suggesting that the synthetic lethality is a consequence of the loss of a function of npp106. We further demonstrate that a region between amino acids 74 and 348 of Npp106p is required for complementation of the synthetic lethality. These results uncover a potential direct or indirect involvement of Npp106p in mRNA export.     

Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells.

In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p. Both are essential for mRNA export. Here we report that Dbp5p and Rat7p interact through their Nterminal domains. Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores. Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1. Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p. Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner. It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors. Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect. In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1. Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim.     

Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.     

Homolog of BRCA2-interacting Dss1p and Uap56p link Mlo3p and Rae1p for mRNA export in fission yeast

The breast cancer tumor suppressor BRCA2-interacting protein, DSS1, and its homologs are critical for DNA recombination in eukaryotic cells. We found that Dss1p, along with Mlo3p and Uap56p, Schizosaccharomyces pombe homologs of two messenger RNA (mRNA) export factors of the NXF-NXT pathway, is required for mRNA export in S. pombe. Previously, we showed that the nuclear pore-associated Rae1p is an essential mRNA export factor in S. pombe. Here, we show that Dss1p and Uap56p function by linking mRNA adapter Mlo3p to Rae1p for targeting mRNA-protein complex (mRNP) to the proteins of the nuclear pore complex (NPC). Dss1p preferentially recruits to genes in vivo and interacts with -FG (phenylalanine glycine) nucleoporins in vivo and in vitro. Thus, Dss1p may function at multiple steps of mRNA export, from mRNP biogenesis to their targeting and translocation through the NPC.     

Schizosaccharomyces pombe rsm1 genetically interacts with spmex67, which is involved in mRNA export

We have previously isolated three synthetic lethal mutants from Schizosaccharomyces pombe in order to identify mutations in the genes that are functionally linked to spmex67 with respect to mRNA export. A novel rsm1 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMexl. The rsm1 gene contains no introns and encodes a 296 amino-acid-long protein with the RING finger domain, a C3HC4 in the N-terminal half. The deltarsm1 null mutant is viable, but it showed a slight poly(A)+ RNA accumulation in the nucleus. Also, the combination of deltarsm1 and deltaspmex67 mutations confers synthetic lethality that is accompanied by the severe poly(A)+ RNA export defect. These results suggest that rsm1 is involved in mRNA export from the nucleus.     

The mitochondrial protein targeting suppressor (mts1) mutation maps to the mRNA-binding domain of Npl3p and affects translation on cytoplasmic polysomes

In all eukaryotic organisms, messenger RNA (mRNA) is synthesized in the nucleus and then exported to the cytoplasm for translation. The export reaction requires the concerted action of a large number of protein components, including a set of shuttle proteins that can exit and re-enter the nucleus through the nuclear pore complex. Here, we show that, in Saccharomyces cerevisiae, the shuttle protein Npl3p leaves the nuclear pore complex entirely and continues to function in the cytoplasm. A mutation at position 219 in its RNA-binding domain leaves Npl3p lingering in the cytoplasm associated with polysomes. Yeast cells expressing the mutant Npl3(L-219S) protein show alterations in mRNA stability that can affect protein synthesis. As a result, defects in nascent polypeptide targeting to subcellular compartments such as the mitochondria are also suppressed.     

An anti-apoptotic protein, Hax-1, inhibits the HIV-1 rev function by altering its sub-cellular localization

The human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the nuclear export of viral mRNA containing the Rev response element (RRE). Although several host proteins co-operating with Rev in viral RNA export have been reported, little is known about the innate host defense factors that Rev overcomes to mediate the nuclear export of unspliced viral mRNAs. We report here that an anti-apoptotic protein, HS1-associated protein X-1 (Hax-1), a target of HIV-1 Vpr, interacts with Rev and inhibits its activity in RRE-mediated gene expression. Co-expression of Sam68 emancipates Rev activity from Hax-1-mediated inhibition. Hax-1 does not bind to RRE RNA by itself, but inhibits Rev from binding to RRE RNA in vitro. The impact of Hax-1 on Rev/RRE interactions in vitro correlates well with the reduced level of RRE-containing mRNA in vivo. Immunofluorescence studies further reveal that Hax-1 and Rev are cytoplasmic and nuclear proteins, respectively, when expressed independently. However, in Hax-1 co-expressing cells, Rev is translocated from the nucleus to the cytoplasm, where it is co-localized with Hax-1 in the cytoplasm. We propose that over-expression of Hax-1, possibly through binding to Rev, may interfere with the stability/export of RRE-containing mRNA and target the RNA for degradation.