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¹ Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan
² Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan

	Abstract

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To identify novel factors involved in nuclear mRNA export in Schizosaccharomyces pombe, we isolated and characterized the ptr8⁺ gene, mutation of which causes nuclear accumulation of poly (A)⁺ RNA. The ptr8⁺ gene encodes an S. pombe homologue of human XPB, a component of TFIIH involved in nucleotide excision repair (NER) and transcription. A temperature-sensitive mutant of ptr8⁺ (ptr8-1) was highly sensitive to UV irradiation, as are human XPB cells. Northern blot analysis demonstrated that the amount of total poly (A)⁺ mRNAs does not decrease significantly at the nonpermissive temperature in ptr8-1 cells, whereas a pulse-labeling assay using ³⁵S-methionine showed that protein synthesis decreases rapidly after incubation of cells at the nonpermissive temperature, suggesting that ptr8-1 cells have a defect in nuclear mRNA export. In Saccharomyces cerevisiae, a mutation in the SSL2 gene encoding a homologue of Ptr8p also causes a block of mRNA export at the nonpermissive temperature. In addition, expression of human XPB in ptr8-1 cells rescued the ts phenotype and the mRNA export defects, suggesting that human XPB may also play a role in mRNA export. Furthermore, we revealed a functional interaction between Ptr8p and Tho2p, a component of the TREX complex involved in mRNA export. These results suggest that XPB/Ptr8p plays roles not only in NER and transcription, but also plays a conserved role in mRNA export.

	Introduction

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In eukaryotic cells, transcription of genes occurs in the nucleus and translation of mRNA to proteins takes place in the cytoplasm. Consequently, export of mRNA from the nucleus to the cytoplasm is an essential step for gene expression in eukaryotic cells.

After transcription, mRNAs associate with multiple proteins to form messenger ribonucleoprotein particles (mRNPs). Some components of the mRNPs were shown to interact with the nuclear pore complex (NPC) (Dimaano & Ullman 2004). One such protein, Mex67p in Saccharomyces cerevisiae (TAP in humans) forms a heterodimer with Mtr2p and functions as an essential mRNA export receptor that interacts with NPC (Sträßer et al. 2000). Yra1p (ALY in humans) is also essential for mRNA export in S. cerevisiae (Sträßer & Hurt 2000; Stutz et al. 2000). Yra1p and Mex67p directly interact, as do ALY and TAP in metazoans (Sträßer & Hurt 2000; Stutz et al. 2000). In addition, Yra1p associates with Sub2p (UAP56 in humans), a DEAD-box RNA helicase, in the nucleus (Sträßer & Hurt 2001). Yra1p and Sub2p form a multi-subunit complex with the transcription elongation factor complex, THO, which consists of four proteins (Hpr1p, Mft1p, Tho2p and Thp1p) (Sträßer et al. 2002). This complex is called TREX (transcription/export). The TREX complex is recruited to transcriptionally active genes where it loads the export receptor Mex67p onto nascent transcripts (Sträßer et al. 2002). The discovery of the TREX complex suggested a link between transcription and mRNA export (Abruzzi et al. 2004).

Several lines of evidence also support the notion of a link between transcription and nuclear mRNA export. In S. cerevisiae, Npl3p is an abundant protein that serves as a prototype for hnRNP functions in mRNA export (Singleton et al. 1995; Lee et al. 1996). Npl3p has been shown to interact directly with RNA polymerase II (Lei et al. 2001). Cotranscriptional recruitment of Npl3p to mRNAs is thought to begin at an early stage of transcription, either at initiation or soon after elongation begins (Lei et al. 2001).

In addition, a SAGA histone acetylase complex, which is a multi-subunit co-factor for RNA polymerase II transcription, was shown to interact with Sus1p, which interacts with the Sac3-Thp1 complex involved in mRNA export (Rodriguez-Navarro et al. 2004). Thus, Sus1p is thought to link the machinery for mRNA export and gene transcription involving the SAGA complex (Rodriguez-Navarro et al. 2004). Furthermore, we reported that a temperature-sensitive mutation in Schizosaccharomyces pombe Ptr6p, a homologue of human TAFII55 (a subunit of the general transcription factor complex TFIID), causes defective mRNA export and transcription at the nonpermissive temperature, suggesting that TAF is involved in nucleocytoplasmic transport of mRNA, in addition to the transcription of the protein-coding genes (Shibuya et al. 1999).

TFIIH is a multisubunit complex that plays essential roles in nucleotide excision repair (NER) and transcription of protein-coding genes (for a review, Egly 2001). In S. cerevisiae, Dbp5p/Rat8p localized at the cytoplasmic fibrils of NPC is required for nuclear mRNA export (Snay-Hodge et al. 1998). Interestingly, a mutation in Dbp5p/Rat8p was suppressed by a mutation in Ssl1p, a component of TFIIH in S. cerevisiae (Estruch & Cole 2003). Dbp5p/Rat8p has been shown to directly interact with components of TFIIH, suggesting a functional relationship between Dbp5p/Rat8p and the transcription machinery (Estruch & Cole 2003). However, direct involvement of TFIIH or its components in nuclear mRNA export has not been demonstrated.

In this study, we have isolated and characterized the ptr8⁺ gene in S. pombe. A temperature-sensitive ptr8 mutant (ptr8-1) accumulates poly (A)⁺ RNA in the nucleus at the nonpermissive temperature. This gene encodes the S. pombe homologue of human XPB, a component of TFIIH. We demonstrated that ptr8-1 is defective in nuclear mRNA export by in situ hybridization of poly (A)⁺ mRNA and a pulse-labeling analysis of newly translated proteins. Our results suggest a new function for XPB in posttranscriptional RNA metabolism.

	Results

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ptr8-1 accumulates poly(A)⁺ RNA in the nuclei rapidly after shifting to the nonpermissive temperature

To identify factors involved in mRNA export from the nucleus to the cytoplasm, we have screened an S. pombe ts^– mutant bank using fluorescent in situ hybridization with an oligo dT probe, and isolated 11 mutants (ptr1 to ptr11) that accumulate poly (A)⁺ RNA in the nuclei at the nonpermissive temperature of 37 °C (Azad et al. 1997; Shibuya et al. 1999; Ideue et al. 2004). Of these, ptr8-1 cells rapidly accumulated poly (A)⁺ RNA in the nuclei after shifting to the nonpermissive temperature and ceased growing (Fig. 1A,B). After 15 min incubation at 37 °C, we clearly detected dot-like nuclear signals in ptr8-1. The intensities of the nuclear signals increased with incubation time at 37 °C. The rapid accumulation of poly (A)⁺ RNA in the nuclei suggested that Ptr8p is directly involved in mRNA export.

View larger version (46K): [in this window] [in a new window]   Figure 1  Phenotypes of ptr8-1. (A) ptr8-1 accumulates poly (A)+ RNA in the nuclei after incubation at the nonpermissive temperature of 37 °C. The wild-type 972 or ptr8-1 cells were grown in YEAL medium at 26 °C and shifted to 37 °C for the indicated times. Cells were then subjected to fluorescent in situ hybridization using the biotin labeled oligo dT50 probe. Before observation, cells were stained with DAPI. (B) ptr8-1 is temperature sensitive for growth. Wild-type (972) and ptr8-1 strains were streaked on a YEAL plate and incubated at 26 °C or 37 °C for 4 days. (C) Poly (A)+ RNA is accumulated predominantly in the chromatin region in ptr8-1 cells at the nonpermissive temperature. After shifting to 37 °C for 2 h, ptr8-1 cells were subjected to in situ hybridization using the oligo dT50 probe and the Cy3-labeled probe for U3 snRNA. Cells were also stained with DAPI. Arrowheads indicate foci enriched with poly (A)+ RNA. In a merged image, green denotes poly (A)+ RNA, red denotes U3 snRNA and blue denotes DAPI.

The ptr8⁺ gene encodes a homologue of human XPB

To clone the ptr8⁺ gene, we transformed ptr8-1 cells with an S. pombe genomic library constructed in pDB248 and isolated a transformant grown at 37 °C on a MM plate. A plasmid that rescues the ts^– phenotype of ptr8-1 was then recovered from the transformant. After several steps of subcloning, we identified a single ORF (SPAC17A5.06) essential for complementation of the ptr8-1 mutation. The ORF encodes an 804-amino acid protein. Comparison of the sequence of Ptr8p with the sequences in the GENBANK database revealed a high degree of homology with human XPB/ERCC3 (53% amino acid sequence identity) and S. cerevisiae Ssl2p/Rad25p (66% identity) (Fig. 2). XPB and Ssl2p/Rad25p are known as DNA helicases and components of the TFIIH transcription factor complex, which is involved in gene transcription, NER and regulation of the cell cycle (Egly 2001).

View larger version (84K): [in this window] [in a new window]   Figure 2  Amino acid sequence alignment of Ptr8p and its homologues. The alignment was created by Multalin version 5.4.1 <http://prodes.toulouse.inra.fr/multalin/>. Lines under the sequence indicate conserved helicase motifs. The asterisk denotes the site of the mutation in the ptr8-1 gene.

We then examined localization of Ptr8p in S. pombe cells. We first constructed a plasmid that expresses Ptr8p tagged with GFP (Ptr8p-GFP) from its own promoter, which we subsequently introduced into ptr8-1 cells. Ptr8p-GFP was able to complement the ts^– phenotype of ptr8-1 (data not shown), demonstrating that the tagged protein is functional in S. pombe. Ptr8p-GFP was found to localize predominantly to the chromatin region, as has been shown for human XPB (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Figure 3  Localization of Ptr8p-GFP in the nucleus. Ptr8p-GFP was expressed in ptr8-1 cells and examined under a fluorescent microscope after staining with Hoechst 33342.

View larger version (14K): [in this window] [in a new window]   Figure 4  (A) ptr8-1 cells are highly sensitive to UV irradiation. Wild-type 972 (black) and ptr8-1 cells (grey) were cultured at 26 °C to mid-log phase and plated onto YPD plates. Cells were irradiated under a germicidal lamp for various times. After incubation for 24 h at 26 °C in the dark, plates were incubated at 26 °C (circle) or 30 °C (triangle) for 3 days, and the number of colonies grown was counted. (B) The ptr8+ gene is essential for viability in S. pombe. After incubation of ptr8+/ptr8::KanMX6 diploid cells on a SPA plate, the tetrad spores were dissected and incubated on a YPD plates at 26 °C for 7 days. None of the asci showed more than two viable spores.

To determine whether spXPB/Ptr8p is essential for viability in S. pombe, we disrupted one of the ptr8⁺ alleles in a diploid S. pombe cell by replacing the entire region of one of the ptr8⁺ genes with a KanMX6 selectable marker (see Experimental procedures section). Tetrad analysis of sporulated ptr8⁺/ptr8::KanMX6 diploid cells revealed no asci with three or four viable spores. Most asci showed 2:2 segregation of viable and nonviable spores (Fig. 4B). None of the spores were viable on a YEAL plate with 1 mg/mL geneticin disulfate (data not shown), indicating that the viable spores contain the wild type ptr8⁺ gene and that the ptr8-disrupted spores were nonviable. These results demonstrate that the ptr8⁺ gene is essential for viability in S. pombe.

The amount of total poly (A)⁺ mRNA does not decrease in ptr8-1 at the nonpermissive temperature

TFIIH plays an important role in transcription initiation (Guzder et al. 1994). Recent work has shown that the XPB helicase in TFIIH acts as an ATP-driven motor to recognize the preinitiation complex (promoter opening) (Lin et al. 2005). To investigate whether the ptr8-1 mutation causes a defect in gene transcription, Northern blot analysis was performed using the oligo dT probe (50mer) for total poly (A)⁺ mRNA, or oligonucleotide probes for pyk1, eno1, gpd1, pfk1, act1 and rpb1 mRNAs. As a control, we used a temperature sensitive mutant of the tafII55/ptr6⁺ gene, which encodes a subunit of the general transcription factor complex TFIID (Shibuya et al. 1999). The intensities of the hybridization signals associated with poly (A)⁺ mRNA were not reduced after shifting of the wild-type and ptr8-1 cells to the nonpermissive temperature, suggesting no drastic reduction of gene transcription has occurred in ptr8-1 at the nonpermissive temperature (Fig. 5A, a left panel). Contrary, the amount of total poly (A)⁺ mRNA in the tafII55/ptr6 mutant decreased due to the defective transcription after shifting to the nonpermissive temperature as previously reported (Shibuya et al. 1999). As for the individual mRNAs analyzed, four (pyk1, eno1, gpd1 and pfk1) of them showed no reduction in transcription after shifting of ptr8-1 cells to the nonpermissive temperature, whereas the amounts of act1 and rpb1 mRNAs decreased slightly at the nonpermissive temperature (Fig. 5A, a right panel). These results suggest that the ptr8-1 mutation does not decrease overall transcription significantly at the nonpermissive temperature, although it induces gene-specific transcriptional defects in some genes.

View larger version (36K): [in this window] [in a new window]   Figure 5  (A) Northern blot analyses. Wild-type, tafII55/ptr6 and ptr8-1 cells were shifted to 37 °C for 2 h and subjected to Northern blot analyses using the oligo dT probe for total poly (A)+ RNA (a left panel) or the oligonucleotide probe for rpb1, pyk1, eno1, gpd1, pfk1 or act1 mRNA (right panels). In the case act1 mRNA, we detected two hybridized bands derived from mRNAs containing different length of 3'-UTR. The bottom panel on the right shows 25s rRNA stained with ethidium bromide to indicate the quantity of total RNA in each lane. (B) In vivo pulse labeling assay. Wild-type and ptr8-1 cells were grown to mid-log phase and labeled with 35S-methionine for 30 min after shifting to 37 °C for the indicated times. After disruption of cells with glass beads, total proteins were fractionated on a 12% SDS-polyacrylamide gel and analyzed with BAS-1500. Protein synthesis rapidly decreases in ptr8-1 cells after shifting to the nonpermissive temperature.

It has been reported that major spliceosomal snRNAs are aberrantly polyadenylated in some yeast exosome mutants (Abou Elela & Ares 1998; van Hoof et al. 2000), which could potentially give rise to nuclear signals unrelated to mRNAs following in situ hybridization with the oligo dT probe. To demonstrate further defective nuclear mRNA export in ptr8-1 using a different assay, we measured the amount of cytoplasmic mRNAs in ptr8-1 cells at the nonpermissive temperature (Neville & Rosbash 1999). The amount of cytoplasmic mRNAs can be indirectly visualized by incorporation of ³⁵S-methionine into newly translated proteins. If the export of mature mRNAs to the cytoplasm is blocked, then the amount of cytoplasmic mRNAs and newly translated proteins will decrease. To test this, we carried out an in vivo pulse-labeling assay with ³⁵S-methionine. As shown in Fig. 5B, we observed no significant changes in protein translation in wild-type cells after shifting to 37 °C. In contrast, there was a strong decrease in protein synthesis in the ptr8-1 mutant cells at the nonpermissive temperature (Fig. 5B, a right panel), suggesting that the cytoplasmic pool of mature mRNAs was decreased. Taken together with the results of the Northern blot analysis, which indicated that there was no drastic decrease in gene transcription in ptr8-1 cells after shifting to the nonpermissive temperature, and the in situ hybridization that showed accumulation of poly (A)⁺ RNA in the nuclei, we conclude that ptr8-1 cells have a defect in mRNA export at the nonpermissive temperature.

ptr8-1 has no defect in transport of a protein with the NLS and the NES

To examine if protein transport is also arrested in ptr8-1 cells at the nonpermissive temperature, we subjected ptr8-1 cells to a protein transport assay using a GST-NLS-GFP-NES fusion protein (Kudo et al. 1999). The NLS and NES in the fusion protein are derived from SV40 large T antigen and Pap1, respectively (Kudo et al. 1999). The GFP fusion protein was expressed in 972 (a wild-type strain) and ptr8-1 cells, and its localization was observed under a fluorescence microscope. The fusion protein was distributed throughout wild-type cells at both 26 °C and 37 °C (Fig. 6A,B). However, after addition of leptomycin B (LMB), which inhibits Crm1p-dependent protein export, the fusion protein accumulated in the nuclei (Fig. 6C), suggesting that the fusion protein shuttles between the cytoplasm and the nucleus. The same phenomenon was observed in ptr8-1 cells. In the absence of LMB, the fusion protein was distributed throughout the cells at 26 °C and 37 °C (Fig. 6D,E), whereas after treatment of cells with LMB at 37 °C, the protein rapidly accumulated in the nuclei (Fig. 6F). These results demonstrate that there are no defects in transport of a protein with the NLS and the NES in ptr8-1 cells at the nonpermissive temperature, suggesting that the block in nuclear export is specific for mRNA in ptr8-1 cells.

View larger version (30K): [in this window] [in a new window]   Figure 6  ptr8-1 cells exhibit no defects in protein transport. The GST-NLS (SV40)-GFP-NES (pap1) fusion protein was expressed in wild type and ptr8-1 cells. Cells were incubated at 26 °C (A and D) and shifted to 37 °C for 2 h (B and E). After shifting to 37 °C for 2 h, cells were treated with 100 ng/mL leptomycin B for 1 h (C and F).

We next performed electron microscopic analysis to examine the nuclear structure of ptr8-1 cells at the ultrastructural level (Fig. 7). ptr8-1 cells were grown at 26 °C, followed by incubation at 37 °C for 2 h and subjected to cryofixation and freeze substitution. Thin sections were cut and then examined under the electron microscope. We observed structural alteration of the nucleolus in ptr8-1 cells at the nonpermissive temperature. Electron dense materials localized to the nucleolar region appeared to be aggregated and fragmented at the nonpermissive temperature (Fig. 7). We did not observe any abnormalities in the structure of the nuclear membrane in ptr8-1 cells, indicating that the defect in mRNA export is not due to modification of the structure of the nuclear membrane.

View larger version (41K): [in this window] [in a new window]   Figure 7  Electron microscopic analysis of ptr8-1 and wild-type cells. ptr8-1 and wild-type 972 cells grown at 26 °C were shifted to 37 °C for 2 h, fixed and embedded in Quetol-651. Thin sections were cut and examined under an electron microscope. Alteration of the nucleolar structure is observed in ptr8-1 cells at the nonpermissive temperature. N and No indicate the nucleus and the nucleolus, respectively. Bar, 1 µm.

To investigate whether the function of spXPB/Ptr8p in mRNA export is conserved between S. pombe and S. cerevisiae, we next analyzed four mutants (ssl2-1, ssl2-dead, ssl2-xp and ssl2-rtt) of the S. cerevisiae homologue of spXPB/Ptr8p, Ssl2p/Rad25p, which is also known to function in NER and transcription (kindly provided by Dr Garfinkel) (Gulyas & Donahue 1992; Lee et al. 1998). The ssl2-1 mutation was mapped between motifs I and II of the nucleotide binding fold of the Ssl2p DNA helicase (Gulyas & Donahue 1992). ssl2-xp contains a 3' truncation of the gene that eliminates 94 C-terminal amino acids residues. This mutation was designed to resemble the truncated protein predicted to be present in a XP patient (Lee et al. 1998). ssl2-dead mutation contains a mutation in the nucleotide binding motif II. In addition, a mutation of ssl2-rtt is located between the helicase motifs III and IV (Lee et al. 1998). Of these ssl2 mutants, only ssl2-rtt showed temperature-sensitive growth at 37 °C (data not shown, (Lee et al. 2000).

In situ hybridization analysis using the oligo dT probe revealed that ssl2-rtt accumulated poly (A)⁺ RNA in the nuclei at the nonpermissive temperature of 37 °C (Fig. 8). Other ssl2 mutants that lack the clear ts^- phenotype did not accumulate poly (A)⁺ RNA in the nuclei, suggesting that defective mRNA export at the nonpermissive temperature depends on the site of mutation in the SSL2 gene. From these results, we conclude that S. cerevisiae Ssl2p/Rad25p also plays a role in mRNA export, in addition to roles in NER and transcription.

View larger version (43K): [in this window] [in a new window]   Figure 8  A temperature-sensitive mutant (ssl2-rtt) of the S. cerevisiae homologue of spXPB/Ptr8p is also defective in mRNA export at the nonpermissive temperature. The wild-type and four mutant strains of ssl2 were grown at 26 °C and then either maintained at 26 °C (upper two panels) or shifted to 37 °C for 2 h (lower two panels), followed by in situ hybridization with the oligo dT probe. The ssl2-rtt mutant, which is temperature sensitive for growth, accumulated poly (A)+ RNA in the nuclei at the nonpermissive temperature.

We next examined whether human XPB could function in mRNA export. We first constructed pREP3-XPB expressing human XPB, which we subsequently introduced into ptr8-1 cells. Expression of human XPB suppressed partially the ts^- phenotype of ptr8-1 at 34 °C (Fig. 9A). Transformants could not grow at temperatures above 34 °C (data not shown). We also performed in situ hybridization of the transformants incubated at 34 °C using the oligo dT probe. As shown in Fig. 9B, defective mRNA export was restored in most of ptr8-1 cells transformed with pREP3-XPB, although we could still detect faint foci enriched with poly (A)⁺ mRNA in some cells. In contrast, all transformants harboring the pREP3 vector accumulated poly (A)⁺ RNA significantly in the nuclei at 34 °C. These results suggest that human XPB is able to substitute for the function of spXPB/Ptr8p in mRNA export, at least in part, in S. pombe.

View larger version (24K): [in this window] [in a new window]   Figure 9  Expression of human XPB suppresses the ptr8 phenotypes. (A) ptr8-1 cells were transformed with pREP41-ptr8+ (+Ptr8), pREP3 (+vec) or pREP3-XPB (+XPB). Transformants were spotted on a MM plate containing thiamine and incubated at 34 °C for 3 days. Thiamine was added to avoid over-expression of human XPB from the thiamine-repressible nmt1 promoter. pREP3-XPB transformants (+XPB) grew at 34 °C, whereas pREP3 transformants (+vec) did not. (B) ptr8-1 cells transformed with the indicated plasmid were grown at 26 °C in MM medium with thiamine and incubated at 34 °C for 2 h. Cells were then subjected to in situ hybridization with the oligo dT50 probe and counterstained with DAPI before observation.

To investigate if spXPB/Ptr8p interacts functionally with the TREX complex, which is involved in both nuclear mRNA export and transcription (Sträßer et al. 2002), we constructed a double mutant that has the ptr8-1 mutation and deletion of the tho2 gene (tho2). Tho2p is a component of the THO complex, a subcomplex of the TREX complex. Interestingly, the double mutant could grow at 34 °C, the temperature that the ptr8-1 single mutant could not survive at (Fig. 10A). This result suggests the functional interaction between spXPB/Ptr8p and Tho2p.

View larger version (34K): [in this window] [in a new window]   Figure 10  (A) Deletion of the tho2+ gene suppressed the ts- phenotype of ptr8-1. Wild-type (972), ptr8-1, tho2 and the double mutant with ptr8-1 and tho2 were streaked on YEAL plates and incubated at 26 °C or 34 °C. The ptr8-1 single mutant cells did not grow at 34 °C, whereas the ptr8-1 tho2 double mutant grew at the same temperature. tho2 cells showed no defects in growth, like wild-type cells, at 34 °C. (B) Wild-type (972), ptr8-1, tho2 and the double mutant grown at 26 °C were incubated at 35 °C for 6 h and subjected to in situ hybridization with the oligo dT probe.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
In this study, we cloned and characterized the S. pombe gene responsible for the ptr8-1 mutation, which causes accumulation of poly(A)⁺ RNA in the nuclei after incubation of cells at the nonpermissive temperature. The ptr8⁺ gene was found to encode an S. pombe homologue of human XPB and S. cerevisiae Ssl2p/Rad25p, components of the transcription factor complex TFIIH, thereby suggesting that spXPB/Ptr8p plays an essential role in nucleocytoplasmic transport of mRNA in addition to the roles in transcription and NER.

To demonstrate that ptr8-1 is actually defective in nuclear mRNA export, we labeled proteins in vivo to monitor the level of cytoplasmic mRNA, in addition to the evaluation of the nuclear accumulation of poly (A)⁺ RNA by in situ hybridization with the oligo dT probe (Figs 1A and 5B). Northern blot analysis using probes for total poly (A)⁺ RNA and several mRNAs revealed that the ptr8-1 mutation does not cause drastic reduction of transcription at the nonpermissive temperature (Fig. 5A). On the other hand, protein synthesis in ptr8-1 was shown to decrease rapidly following incubation at the nonpermissive temperature (Fig. 5B). These results strongly suggest that spXPB/Ptr8p functions in nuclear mRNA export. As far as we know, this is the first report to show involvement of a TFIIH component, XPB, in mRNA export.

A role for spXPB/Ptr8p in mRNA export is conserved in S. cerevisiae

We revealed that a temperature-sensitive mutant of the S. cerevisiae homologue of spXPB/Ptr8p, ssl2-rtt, is defective in mRNA export at the nonpermissive temperature, suggesting the conservation of a role for spXPB/Ptr8p in mRNA export between two species of yeast.

Of ssl2 mutants analyzed in this study (ssl2-1, ssl2-xp, ssl2-dead and ssl2-rtt), ssl2-1 was originally isolated as a dominant suppressor of his4-316, a mutation caused by a stable stem-loop structure in the 5' untranslated region of the HIS4 mRNA that blocks translation initiation (Gulyas & Donahue 1992). Interestingly, it was shown that destabilization of the stem-loop structure in the HIS4 mRNA or transcriptional enhancement of the HIS4 gene is not responsible for ssl2 suppression (Gulyas & Donahue 1992). It was thus thought that suppression of his4-316 by the ssl2 mutation is related to a post-transcriptional process, such as translation or mRNA export. On the other hand, ssl2-rtt (regulator of Ty transposition) was isolated as a mutation that permits a high level of spontaneous Ty1 retrotransposition in S. cerevisiae (Lee et al. 1998). This mutation stimulates Ty1 retrotransposition without altering the level of Ty1 RNA or proteins, suggesting that Ssl2p antagonizes Ty1 transposition post-transcriptionally. It is possible that the ssl2-rtt mutation affects nuclear export of Ty1 RNA and thereby stimulates RNA-mediated transposition of Ty1. These reports on S. cerevisiae ssl2 mutants suggest that Ssl2p/Rad25p plays a role in post-transcriptional process, in addition to roles in gene transcription and DNA repair.

What is a role of spXPB/Ptr8p in the mRNA export pathway?

So far, diverse factors involved in mRNA export have been identified by genetic screening in yeast (Lei & Silver 2002; Cullen 2003). Among the factors involved in mRNA export, S. cerevisiae Mex67p has been shown to be a major mRNA export receptor that interacts with FG repeat-containing nucleoporins, which are components of NPC (Segref et al. 1997). In S. pombe, Rae1p was shown to interact with Mex67p and is essential for nuclear mRNA export (Yoon et al. 2000). To examine the relationship between spXPB/Ptr8p and Mex67p, we over-expressed S. pombe Mex67p in ptr8-1 cells using a strong nmt1 promoter. However, over-expression of Mex67p did not suppress the defect in mRNA export in ptr8-1 (data not shown). We also constructed a double mutant containing ptr8-1 and rae1-167 mutations, which did not result in synthetic lethality, suggesting that there is no functional interaction between Rae1p and spXPB/Ptr8p in S. pombe.

In contrast to the cases of Mex67p and Rae1p, we found that ts^– phenotype of ptr8-1 is suppressed by deletion of the tho2⁺ gene, suggesting that Ptr8p interacts functionally with Tho2p, a component of the TREX complex. Recent studies have revealed functional cross-talk between transcription and mRNA export, as well as cross-talk among pre-mRNA splicing, poly (A)⁺ addition and mRNA export (for a review, see Reed 2003). It has been shown that Sub2p and Yra1p, which are involved in nuclear mRNA export, associate with the multisubunit THO complex required for transcription elongation to form the TREX complex (Jimeno et al. 2002). The TREX complex is thought to play a role in co-transcriptional loading of export factors to mRNPs. Through the interaction with Tho2p in the THO complex, spXPB/Ptr8p might be involved in co-transcriptional recruitment of mRNA export factors to sites of active transcription.

Alternatively, spXPB/Ptr8p might be required for steps for the release of mRNPs from transcription sites. The vaccinia virus A18R protein is a DNA helicase that shares sequence similarity with spXPB/Ptr8p (Simpson & Condit 1995). Interestingly, a biochemical analysis revealed that A18R DNA helicase is involved in the release of nascent RNA from a vaccinia virus transcription elongation complex (Lackner & Condit 2000). Similarly, spXPB/Ptr8p might also function in the release of nascent mRNAs from the transcription complex. Further experiments, such as measurement of the helicase or the release factor activity in the product of the ptr8-1 gene, will be necessary to elucidate the role of spXPB/Ptr8p in mRNA export.

mRNA export defects and Cockayne syndrome

We showed that expression of human XPB in S. pombe ptr8-1 cells partially complements defective mRNA export at 34 °C (Fig. 9). Thus, it is likely that human XPB also plays a role in mRNA export, in addition to its roles in transcription and NER.

In humans, it has been shown that some mutations in the XPB gene give rise to Cockayne syndrome (CS), in addition to the well known autosomal recessive disorder xeroderma pigmentosum (XP). Patients with CS suffer from physical and mental retardation, UV sensitivity, progressive neurological and retinal degeneration and skeletal abnormalities (for a review, Rapin et al. 2000). Five genes, CSA, CSB, XPB, XPD and XPG, are known to cause CS in human. The precise mechanisms responsible for the failure of brain and somatic growth in CS patients are not known. It has been suggested, however, that CS might result at least partially from transcription defects, as mutations in XPB and XPD, subunits of the transcription complex TFIIH, can cause CS (Schaeffer et al. 1993; Drapkin et al. 1994). The recent discovery of a complex containing CSB, XPG, TFIIH and RNA polymerase I, which is involved in rRNA synthesis, led to an alternative hypothesis suggesting that destabilization of the CSB complex that reduces rRNA synthesis might cause the CS phenotype (Bradsher et al. 2002). In this study, we provide evidence that spXPB/Ptr8p is required for nucleocytoplasmic transport of mRNA and that human XPB might also function in nuclear mRNA export. Our results suggest that defective nuclear mRNA export could be another factor contributing to the CS phenotype.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Yeast strains, media, genetic methods and plasmids

The yeast strains used in this study are listed in Table 1. Complete (YPD or YEAL) or minimal (MM) media were used for standard cultures of S. pombe. The genetic methods for S. pombe used in this study have previously been described (Moreno et al. 1991; Alfa et al. 1993).

Fluorescent in situ hybridization

Fluorescent in situ hybridization was performed according to the previously described method (Azad et al. 1997). Yeast cells were grown at 26 °C and then shifted to 37 °C for the indicated periods of time, followed by fixation with freshly prepared 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 6.5) for 1 h at room temperature. After cells were washed 3 times with phosphate buffer, they were treated with Novozyme and Zymolyase 100T (ICN) in PEMS (100 mM PIPES, 0.1 mM MgCl₂, 1 mM EGTA and 1.2 mM sorbitol) for 10–15 min at 37 °C. Spheroplasts were applied to wells of Teflon-faced slides coated with poly-L-lysine. Cells were dehydrated by treatment for 5 min each with 70, 90, and 100% ethanol. Cells were then incubated with pre-hybridization buffer (4X SSC, 5X Denhardt's solution, and 1 mg/mL baker yeast tRNA) at 37 °C for 3 h. Hybridization was performed with the same buffer containing 1 µg/mL of an oligo dT (50mer) probe that had been end-labeled with biotin-16-dUTP (Boehringer Mannheim) using terminal transferase (Life Technologies), for 12–16 h at 42 °C in a humidified chamber. After hybridization, cells were washed 4 times in 4X SSC at 42 °C (10 min/wash). Cells were then briefly rinsed with 4X SSC, and then 0.1% Triton X-100. After washing, cells were incubated with FITC-conjugated avidin in 4X SSC and 1% bovine serum albumin for 1 h at room temperature. Unbound avidin was removed by washing 2 times in 4X SSC and twice in 4X SSC and 0.1% Triton X-100 at room temperature for 10 min each. Cells were counterstained with 0.1 µg/mL DAPI and mounted. Images of poly (A)⁺ RNA were obtained using an OLYMPUS AX70 fluorescence microscope equipped with a Photometrics Quantix cooled CCD camera. For triple staining, we used a mixture of a Cy3-labeled oligonucleotide probe that recognizes U3 snRNA and a biotin-labeled oligo dT probe for in situ hybridization, and stained cells with DAPI after hybridization.

Gene disruption

Disruption of the ptr8⁺ gene in the UDP6 diploid strain was carried out using a KanMX6 module, which confers G418 resistance to cells (Bahler et al. 1998). The fragments containing the 5' or 3' franking region of the ptr8⁺ gene (1173 and 316 bp in length) were inserted in pFA6a-KanMX6 using EcoRI-PmeI sites or BamHI-BglII sites. Transformation of UDP6 cells with the EcoRI-SalI fragment carrying the ptr8 gene disrupted with the KanMX6 module was performed as described (Okazaki et al. 1990). Cells were plated on YEAL plates and incubated at 30 °C for 18 h. After that, plates were replicated on YEAL plates containing 1 mg/mL geneticin disulfate to select for transformants. Disruption of the ptr8⁺ gene was confirmed by PCR analysis of the transformants.

To construct the tho2 deletion mutant, the open reading frame of the tho2⁺ gene was replaced with the KanMX6 module by homologous recombination using the KanMX6 fragment fused with the upstream region (741 bp) and the downstream region (660 bp) of the tho2⁺ gene.

UV survival experiments

Exponentially growing cells cultured in YPD liquid medium were diluted with distilled water. Then, 1000 or 10 000 cells were plated per YPD agar plate. UV irradiation (wavelength 254 nm) was performed using a CL-1000 UV crosslinker (UVP Inc.). We counted the number of colonies after incubation of plates for 24 h at 26 °C, and then for 3 days at 26 °C or 30 °C.

Analysis of protein import and export

Cells transformed with pR1GLFPA6 (Kudo et al. 1999) expressing a GST-GFP protein tagged with NLS (SV40) and NES (Pap1) from the thiamine-repressible nmt1 promoter were grown at 26 °C in MM medium with 5 µg/mL thiamine. The expression of the GFP fusion protein was then induced by incubation of the cells at 26 °C in MM medium without thiamine for 20 h. After induction, cells were shifted to 37 °C for 2 h and treated with leptomycin B (LMB) at a concentration of 200 ng/mL for 30 min. Localization of the GFP fusion protein was examined using the OLYMPUS AX70 fluorescence microscope equipped with the Photometrics Quantix cooled CCD camera.

RNA preparation and Northern blot analysis

Wild-type and ptr8-1 cells were cultured in YEAL at 26 °C and shifted to 37 °C for 2 h. Cells were harvested and disrupted by mixing vigorously with 0.2 g of glassbeads in 200 µL of a TELS solution containing 10 mM Tris–HCl (pH 7.5), 10 mM EDTA, 100 mM LiCl and 1% SDS and 200 µL of phenol/chloroform/isoamylalcohol (PCI). After centrifugation, lysate was treated with the same volume of PCI. Ethanol was then added and the mixture was centrifuged at 14 000 rpm at 4 °C for 20 min to precipitate the RNA. RNA (15 µg) was dissolved in a solution containing 4% formaldehyde, 20% formamide, 20 mM MOPS (pH 7.0), 2.5 mM sodium acetate, 1 mM EDTA, 0.05 mg/mL ethidium bromide, XC and BPB, heated at 70 °C for 20 min and fractionated on a 1% agarose gel containing 6% formaldehyde. After electrophoresis, the gel was treated with 50 mM NaOH and washed with 200 mM sodium acetate (pH 4.5). RNA was then transferred to a Genescreen filter by a capillary action. The filter was irradiated with UV to fix RNA on the membrane. Pre-hybridization was done in a solution containing 6 X SSC, 50 mM Na-phosphate, 5 X Denhardt's solution, 0.1% SDS and 100 µg/mL boiled ssDNA. Hybridization was performed in the same buffer containing a ³²P-labelled probe at 42 °C. After hybridization, the filter was washed three times with 6 X SSC and then with 6 X SSC containing 0.1% SDS at 42 °C (5 min each). The filter was dried and analyzed with Bioimaging Analyzer BAS-1500 (Fuji Photo Film Co., Ltd). Nucleotide sequences of oligonucleotide probes used in this study are as follows.

pyk1: 5'-GAG GCC GAA CAT CTC TTG GTA AAG GCT GCC-3'

eno1: 5'-CTT GCC GTC AAC GTA GAA CTC GGA GGA GGC-3'

gpd1: 5'-TTT CCT GCA TTG CCT TTAAGC ACC CAC CCG ACG TTT CTT A -3'

pfk1: 5'-GTT AAG CTC GGA GTC AAT AGC ACC CTC TGC-3'

act1: 5'-CAG AGT CCA AGA CGA TAC CAG TGG TAC GAC-3'

rpb1: 5'-CAA CCA CCG TGT CCC ATA TTG GCG GAG GGA-3'

In vivo pulse labeling assay

Cells were grown to 0.3 OD₅₉₅ in 1 mL of YPD at 26 °C. Cultures were then maintained at 26 °C or shifted to 37 °C for the indicated times. ³⁵S-methionine was added to the cultures which were incubated further for 30 min at the same temperatures. After centrifugation at 4 °C, the cells were broken with glass beads in a 20 µL solution containing PBS, 1 mM PMSF, 1% Triton X-100 and 0.1% SDS. After centrifugation for 20 min at 4 °C, the supernatants were mixed with an equal volume of 2 X SDS sample buffer and boiled for 5 min. A 10–15 µL of sample per lane were electrophoresed through a 12% SDS-polyacrylamide gel. After electrophoresis, gels were dried, exposed to a Fuji imaging plate, and analyzed with BAS-1500.

Electron microscopic analysis

It was carried out using a cryofixation and freeze substitution method. ptr8-1 cells were grown in YEAL to a mid-log phase at 26 °C, shifted to 37 °C for 2 h and harvested by centrifugation. Cells were frozen in liquid propane at –190 °C, transferred to a 2% osmium/acetone solution at –80 °C and kept at –80 °C for 3 days. Samples were then kept at –35 °C, 4 °C, 4 °C and room temperature for 2 h each. Cells were washed 3 times with acetone and twice with propylene oxide for 10 min. The fixed samples were then embedded in Quetol-651. Thin sections were cut and stained with 2% uranyl acetate and Reynold's lead citrate. Sections were observed at 80 kV in a JOEL JEM 1210 electron microscope.

	Acknowledgements

 
We thank Dr Minoru Yoshida for providing leptomycin B and the reporter plasmid expressing GST-NES-GFP-NLS, and Dr Ravi Dhar for the rae1-167 strain. We also thank Dr Masaru Yamaizumi for providing pcDNA3.1-ERCC3. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T.T.

	Footnotes

 
Communicated by: Masayuki Yamamoto

^aPresent address: Department of Applied Life Science, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto 860-0082, Japan

^* Correspondence: E-mail: ttani{at}sci.kumamoto-u.ac.jp

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Received: 13 February 2006
Accepted: 26 September 2006

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