Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

Regulation of mRNA export by nutritional status in fission yeast.

We have isolated a mutation in nup184(nup184-1) that is synthetically lethal with the mRNA export defective rae1-167 mutation in Schizosaccharomyces pombe. The consequence of the synthetic lethality is a defect in mRNA export. The predicted Nup184p is similar to Nup188p of Saccharomyces cerevisiae, and a Nup184p-GFP fusion localizes to the nuclear periphery in a punctate pattern. The Deltanup184 null mutant is viable and also is synthetically lethal with rae1-167. In a rae1(+) background, both the nup184-1 and Deltanup184 mutations confer sensitivity to growth in nutrient-rich medium (YES) that is accompanied by nuclear poly(A)+ RNA accumulation. Removal of the cAMP-dependent protein kinase, Pka1p, relieved the growth and mRNA export defects of nup184 mutants when grown in nutrient-rich medium. The activation of Pka1p is necessary, but not sufficient, to cause the severe poly(A)+ RNA export defects when nup184 mutant cells are incubated in YES, suggesting nutritional status can also regulate poly(A)+ RNA export. Our results suggest that the regulation of poly(A)+ RNA export by Pka1p kinase appears to be indirect, via a translation-dependent step, but post-translationally in response to YES.     

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.     

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.     

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.     

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 fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase

Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)+ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1+ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1+ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2+ gene isolated as a multi-copy suppressor of cdc7-PD1.     

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.     

Crp79p,like Mex67p,is an auxiliary mRNA export factor in Schizosaccharomyces pombe

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)(+) RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.     

The Prp1(+) Gene Required for Pre-mRNA Splicing in Schizosaccharomyces Pombe Encodes a Protein That Contains Tpr Motifs and Is Similar to Prp6p of Budding Yeast

The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)(+) RNA transport. The prp1(+) gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prp1p shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1(+) and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1(+) gene was found to be identical with the zer1(+) gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)(+) RNA nuclear export, in addition to pre-mRNA splicing.     

Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast

The yeast RAD27 gene encodes a functional homolog of the mammalian FEN1 protein, a structure-specific endo/exonuclease which plays an important role in DNA replication and repair. Previous genetic interaction studies, including a synthetic genetic array (SGA) analysis, showed that the survival of rad27Delta cells requires several DNA metabolic processes, in particular those mediated by all members of the Rad52-dependent recombinational repair pathway. Here, we report the results of our SGA analysis of the collection of non-essential yeast genes against the rad27Delta mutation, which resulted in the identification of a novel synthetic lethal interaction conferred by mutations affecting the Nup84 nuclear pore subcomplex (nup133Delta, nup120Delta and nup84Delta). Additional screens showed that all Rad52 group genes are required for the survival of the nup133Delta and nup120Delta mutants, which are defective in nuclear pore distribution and mRNA export, but not of the nup133DeltaN mutant, which is solely defective in pore distribution. This requirement for the DNA double-strand break (DSB) repair pathway is consistent with the observation that, like rad27Delta, the nup133Delta, nup120Delta and nup84Delta mutants are sensitive to methyl methanesulfonate (MMS). Furthermore, nup133Delta cells exhibit an increased number of spontaneous DNA repair foci containing Rad52. Altogether, these data suggest that the pathological interactions between the rad27Delta and specific nupDelta mutations result from the accumulation of unrepaired DNA damages.     

Isolation of synthetic lethal mutations in combination with spnab2 of fission yeast

spNab2 is a fission yeast, Schizosaccharomyces pombe, homologue of the budding yeast Nab2 protein that is an essential poly(A)⁺ RNA-binding protein required for both nuclear export of mRNA to cytoplasm and poly(A)⁺ tail length control. Here we performed a synthetic lethal genetic screen in the fission yeast to isolate mutants that are genetically linked to spnab2. We isolated three mutants that showed synthetic lethality under the repressed condition of the spnab2 expression. These mutants defined in different complementation groups. All the mutants exhibited the accumulation of poly(A)⁺ RNA in the nucleus under the restricted condition. In addition, the growth defects of one mutant (SLnab2) were complemented partially by some genes (mlo3 and rae1) required for mRNA export, while those of the rest (SLnab1 and SLnab3) were not complemented by any S. pombe genes we tested, which were known to be involved in mRNA export. These results suggest that the isolated mutants might harbor mutations in novel genes functionally linked to the spnab2 gene.     

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)⁺ RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.     

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.     

Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.

Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex. Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N-Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance.     

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37°?C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation.     

The fission yeast homologue of Glel is essential for growth and involved in mRNA export

We have isolated Glel homologue (named as spglel) as a partial multicopy suppressor of the synthetic lethality of rael-167 elfl-21 in fission yeast Schizosaccharomyces pombe. The spglel is also able to complement partially temperature-sensitive phenotype of rael-167 only at a lower restrictive temperature. The spglel gene contains one intron and encodes a 480 amino-acid protein with predicted molecular weight of 56.2 kDa. We showed that spglel gene is essential for vegetative growth and functional Glel-GFP protein is localized mainly in NPC. The accumulation of poly(A)(+) RNA in the nucleus is exhibited when expression of spglel is repressed or over-expressed. These results suggest that the spGle1 protein is also involved in mRNA export in fission yeast.     

Isolation and molecular characterization of mRNA transport mutants in Schizosaccharomyces pombe.

Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.