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Genes to Cells (2008) 13, 105-116. doi:10.1111/j.1365-2443.2007.01154.x
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Rsp5 is required for the nuclear export of mRNA of HSF1 and MSN2/4 under stress conditions in Saccharomyces cerevisiae

 Yutaka Haitani and Hiroshi Takagi*

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Rsp5 is an essential and multi-functional E3 ubiquitin ligase in Saccharomyces cerevisiae. We previously isolated the Ala401Glu rsp5 mutant that is hypersensitive to various stresses. In rsp5A401E cells, the transcription of the stress protein genes was defective. To understand the mechanism by which Rsp5 regulates the expression of stress proteins, we analyzed the expression and localization of two major transcription factors, Hsf1 and Msn2/4, required for stress protein gene expression in S. cerevisiae. The mRNA levels of HSF1 and MSN2/4 in rsp5A401E cells were slightly lower than those of wild-type cells. An interesting finding is that the protein levels of HSF1 and Msn2/4 were remarkably defective in rsp5A401E cells after exposure to temperature up-shift and ethanol, although these proteins are mainly localized in the nucleus under these stress conditions. We also showed that the mRNAs of HSF1 and MSN2/4 were accumulated in the nucleus of rsp5A401E cells after exposure to temperature up-shift and ethanol, and even under non-stress conditions, suggesting that Rsp5 is required for the nuclear export of these mRNAs. These results indicate that, in response to environmental stresses, Rsp5 primarily regulates the expression of Hsf1 and Msn2/4 at the post-transcriptional level and is involved in the repair system of stress-induced abnormal proteins.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
During fermentation processes, yeast Saccharomyces cerevisiae cells are exposed to various stresses, including a high concentration of ethanol, freezing, desiccation and high osmolarity. Such stresses induce protein denaturation, generate abnormal proteins and lead to growth inhibition or cell death. We analyzed the stress–tolerance mechanisms in S. cerevisisae with a focus on stress-induced abnormal proteins (Hoshikawa et al. 2003; Morita et al. 2003; Nomura & Takagi 2004). The addition of some amino acid analogues can induce a transient physiological stress response in cells comparable to that of heat shock stress (Ananthan et al. 1986). Azetidine-2-carboxylic acid (AZC) is a toxic four-membered ring analogue of proline, which causes misfolding of only those proteins into which it is incorporated competitively with proline (Trotter et al. 2001). Therefore, the accumulation of abnormal proteins causes growth inhibition and cell death in yeast (Hoshikawa et al. 2003).

We previously isolated the AZC-hypersensitive mutant of S. cerevisiae and identified the RSP5 gene required for the growth when yeast cells were exposed to AZC (Hoshikawa et al. 2003). The essential ubiquitin ligase Rsp5 is structured in three domains: an amino-terminal C2 domain, three WW domains, and a carboxyl-terminal homologous to E6-AP carboxyl terminus (HECT) catalytic domain. This enzyme ligates ubiquitin to the target protein through the WW domains, which are the protein interaction modules that bind proline-rich ligands. Rsp5 participates in many biological events through ubiquitination of the target proteins: endocytosis of plasma membrane permeases (Vandenbol et al. 1987; Galan et al. 1996; Springael & Andre 1998), multivesicular body sorting (Katzmann et al. 2004), degradation of the large subunit of RNA polymerase II (Huibregtse et al. 1997), biosynthesis of unsaturated fatty acids (Hoppe et al. 2000), ER-associated degradation (Haynes et al. 2002) and heat shock element (HSE)-mediated gene expression (Kaida et al. 2003). In the Rsp5 sequence of the mutant, we found a single amino acid substitution, Ala401Glu, in the third WW domain (Dunn & Hicke 2001; Shcherbik et al. 2002). Interestingly, when yeast cells were exposed to stresses that induce protein denaturation, such as toxic amino acid analogues, high growth temperature in a rich medium, ethanol and heat shock treatment, the rsp5A401E cells showed much more sensitivity to these stresses than the wild-type strain (Hoshikawa et al. 2003). The mutation might impair the specific recognition and ubiquitination with the protein involved in cell growth under stress conditions. These results suggest that Rsp5 is involved in the degradation of stress-induced abnormal proteins.

In general, the accumulation of stress-induced abnormal proteins or misfolded proteins is a serious problem to cells. To overcome it, two strategies can be considered: (i) degradation of the proteins through a ubiquitin–proteasome system or (ii) refolding by molecular chaperones, including stress proteins. We thus considered that it would be of interest to determine whether Rsp5 is involved in the refolding of abnormal proteins by stress proteins. In yeast, two major transcription factors, Hsf1 and Msn2/4, are responsible for stress-induced gene expression (Hashikawa & Sakurai 2004; Ferguson et al. 2005). Each factor binds to HSE or stress response element (STRE) found in the promoters of many stress protein genes, such as HSP42 and DDR2. Some genes, such as HSP12, contain both elements in their promoters. Recently, misfolded proteins have been reported to mediate heat shock activation of heat shock factors and their target genes (Amoros & Estruch 2001; Kandror et al. 2004). Furthermore, HSE-mediated gene expression was defective in the rsp5 mutant under high temperature conditions (Kaida et al. 2003).

We previously analyzed the ability of stress proteins to refold abnormal proteins in rsp5A401E cells (Haitani et al. 2006). The transcription of stress protein genes in rsp5A401E cells was significantly lower than that in the wild-type strain when exposed to temperature up-shift, ethanol or sorbitol. Interestingly, the amounts of transcription factors Hsf1 and Msn4 were remarkably low in rsp5A401E cells. Thus, the reduced induction of stress protein genes may cause the increased stress sensitivity of rsp5A401E cells. Here, to understand the mechanism by which Rsp5 regulates the expression of stress proteins, we analyzed the expression and localization of Hsf1 and Msn2/4 and showed that Rsp5 primarily regulates the expression of these transcription factors at the post-transcriptional level, e.g., the nuclear export of mRNA and tRNA.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
The ubiquitin ligase Rsp5 is involved in the transcription of HSF1 and MSN2/4

In yeast, two major transcription factors, Hsf1 and Msn2/4, are responsible for stress-induced gene expression (Sorger & Pelham 1987; Martinez-Pastor et al. 1996). Hsf1, which is best known for its involvement in heat shock response, regulates the transcription of hundreds of targets, including genes involved in protein folding, such as HSP42, HSP82 and HSP12 (Gasch et al. 2000). In response to several stresses, including heat shock, osmotic shock, oxidative stress, low pH, glucose starvation and high ethanol concentrations, Msn2 and Msn4 regulate the expression of 200 genes, such as DDR2, CTT1 and TPS2 (Causton et al. 2001).

We recently showed that the expression of stress protein genes was defective in rsp5A401E mutant cells when exposed to temperature up-shift, ethanol or sorbitol (Haitani et al. 2006). Accordingly, we consider that the reduced induction of stress proteins is the primary cause of the increased stress sensitivity of rsp5A401E cells. Rsp5 may play a new role in the expression of Hsf1- or Msn2/4-dependent genes and may regulate it by controlling the transcription of HSF1 and MSN2/4. To analyze the effect of Rsp5 on the expression of transcription factors, we examined the level of HSF1 and MSN2/4 transcription in wild-type and rsp5A401E mutant cells under stress conditions by real-time quantitative PCR. Strains CKY8 (wild-type) and CHT81 (rsp5A401E mutant) grown at 25 °C were exposed to elevated temperatures up to 37 °C or 10% ethanol. After the shift to 37 °C, the mRNA levels of HSF1 and MSN4 in wild-type cells were higher by 50% than those of wild-type cells under a non-stress condition (Fig. 1). The transcription of MSN4 was also slightly induced by addition of ethanol. On the other hand, under a non-stress condition, the mRNA levels of these genes in rsp5A401E cells grown at 25 °C were significantly (50%) lower than those of wild-type cells. It appears that the expression of these genes is induced by ethanol treatment in rsp5A401E cells. These results suggest that Rsp5 is involved in the transcription of HSF1 and MSN2/4.


Figure 1
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  		Figure 1  Rsp5 is involved in the transcription of HSF1 and MSN2/4. Strains CKY8 (wild-type) and CHT81 (rsp5A401E mutant) were cultured to the logarithmic growth phase in YPD medium and incubated for 10 min under the following conditions: at 25 °C (opened bar) at 37 °C (shaded bar) with 10% ethanol at 25 °C (hatched bar). Total RNA from each strain was prepared from samples collected before and after stress, and the transcription of HSF1 and MSN2/4 was assessed by real-time quantitative PCR. The mRNA level was normalized to that of ACT1 in the same sample. Values represent the means and error bars indicate the standard deviations of three independent experiments.

 
Rsp5 regulates the expression of Hsf1 and Msn2/4 at the post-transcriptional level

We next investigated how Rsp5 regulates the expression of HSF1 and MSN2/4. Rsp5 may have unknown function of regulating the expression of stress proteins through ubiquitination of these transcription factors. According to a recent paper, two homologous transcription factors, Spt23 and Mga2, are ubiquitinated by Rsp5 and partially degraded by the cytosolic 26S proteasome, and the truncated forms are imported to the nucleus for the transcription of OLE1 required for the oleic acid synthesis (Hoope et al. 2000). In fact, Hsf1 contains sequences such as the LPKY-motif that mediate an interaction with the substrate-binding domain of Rsp5 (Shcherbik et al. 2004). Therefore, we expected the same mechanism that Rsp5 would regulate the activation of Hsf1 and Msn2/4 through the same mechanism, i.e., through ubiquitination. However, our in vivo yeast two-hybrid assay and by in vitro immunoprecipitation assay using an ubiquitin-antibody provided no evidence that Rsp5 bound and ubiquitinated these proteins (data not shown).

Subsequently, to examine the effect of Rsp5 on the protein amounts of transcriptional factors, we constructed three high-copy-number plasmids, pAD-HSF1, pAD-MSN2 and pAD-MSN4, which contained the carboxyl-termini of the hemagglutinin (HA)-tagged fusion genes of HSF1, MSN2 and MSN4, respectively. These plasmids were introduced into wild-type and rsp5A401E cells. We found that rsp5A401E cells were sensitive to 37 °C on SC-Leu plates (Fig. 2A). It should be noted that these proteins were undetectable in rsp5A401E cells when these genes were introduced in the centromeric plasmid (data not shown). The transformant cells were grown in SC-Leu medium at 25 °C and exposed to temperature up-shift and ethanol stress. Real-time PCR analysis showed that the mRNA levels of HSF1 and MSN2/4 were almost the same between the two strains, and were not significantly affected by stress (Fig. 2B). This result seems reasonable, since these genes were placed under the constitutive ADH1 promoter in the plasmids. In Western blot analysis using HA antibody, we found that the protein levels of Hsf1 and Msn2/4 were unchanged or increased after exposure to temperature up-shift and ethanol stress in both strains, but the amounts of fusion proteins dramatically decreased in the rsp5A401E mutant even under a non-stress condition (Fig. 2C). It is noteworthy that Hsf1 and Msn2 were abundant in wild-type cells after exposure to temperature up-shift and ethanol. It is unlikely that plasmid-based ADH1 expression is regulated by Rsp5, because similar results were obtained from pYES2-based high-copy-number plasmids pYES-HSF1 and pYES-MSN4 under the control of their original promoters (data not shown).


Figure 2
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  		Figure 2  The protein levels of Hsf1 and Msn2/4 were defective in rsp5A401E cells. (A) Strains CKY8 (wild-type) harboring empty vector (pAD4) and CHT81 (rsp5A401E mutant) harboring empty vector (pAD4) or RSP5 (pAD-RSP5). Approximately 106 cells of each strain and serial dilutions of 10–1 to 10–5 (from left to right) were spotted and incubated onto SC-Leu for 3 days. (B) Recombinant strains CKY8 (wild-type) harboring pAD-HSF1, pAD4-MSN2, and pAD-MSN4, and CHT81 (rsp5A401E mutant) harboring pAD-HSF1, pAD4-MSN2 and pAD-MSN4 were cultured to the logarithmic growth phase in SC-Leu medium at 25 °C and incubated for 10 min under the following conditions: at 25 °C (opened bar) at 37 °C (shaded bar) with 10% ethanol at 25 °C (hatched bar). Total RNAs from each strain were prepared, and the transcription of HSF1 and MSN2/4 was assessed by real-time quantitative PCR, as shown in Fig. 1. Values represent the means and error bars indicate the standard deviations of three independent experiments. (C) The above strains were cultured to the logarithmic growth phase in SC-Leu medium at 25 °C and incubated in the absence (–) or presence (+) of 100 µg/mL CX for 10 min under the following conditions: at 25 °C (–) at 37 °C (Temp.) with 10% ethanol at 25 °C (EtOH). Whole cell extracts from each strain were analyzed by Western blot analysis using anti-HA monoclonal antibody. The 3-phosphoglycerate kinase Pgk1 was used as a protein-loading control.

 
We also examined the stability of Hsf1 and Msn2/4 using the protein synthesis inhibitor cycloheximide (CX) in rsp5A401E cells. In the presence of CX, Western blot analysis showed that the protein level of Hsf1 was unchanged in rsp5A401E cells under the stress conditions (Fig. 2C). This result suggests that HSF1-mRNA is hardly exported from the nucleus and is little translated in rsp5A401E cells. On the other hand, the amounts of Msn2 and Msn4 were decreased in the presence or absence of stresses after the addition of CX in wild-type and rsp5A401E cells (Fig. 2C). It might be possible that the mRNAs of MSN2 and MSN4 are slightly exported from the nucleus, and then the translated products Msn2 and Msn4 are degraded via some proteolytic pathway(s). These results indicate that Rsp5 is involved in the post-transcriptional regulation of Hsf1 and Msn2/4 and raise the possibility that Rsp5 may directly influence the expressions of Hsf1 and Msn2/4.

Rsp5 has little effect on the intracellular localization of Hsf1 and Msn2/4

Hsf1 binds to the conserved HSE motif found in the promoters of its target genes as a homotrimeric complex in both a constitutive and an inducible manner. In unstressed cells, Hsf1 is constitutively phosphorylated in the nucleus, but under certain stresses, such as alkaline pH, oxidative stress, heat stress or glucose starvation, it becomes hyperphosphorylated and adopts an activated conformation resulting in the transcription of target genes (Hashikawa et al. 2006). The gene expression of MSN4 itself is induced by stress in an Msn2/4-dependent manner, while MSN2 is constitutively expressed. The two proteins share 41% identity and are similar in size and amino acid composition (Estruch & Carlson 1993). While the single deletion mutants of msn2 and msn4 have no obvious phenotype, msn2msn4 double null mutants are hypersensitive to carbon source starvation, heat shock, and osmotic and oxidative stresses. Msn2 and Msn4, which are located in the cytoplasm under non-stress conditions (Gorner et al. 1998), are hyperphosphorylated and relocalized to the nucleus upon stress.

To determine whether Rsp5 influences the nuclear transport of Hsf1 and Msn2/4, the localization of these proteins was examined under stress conditions (Fig. 3). We constructed GFP-fusion genes of HSF1, MSN2, and MSN4 and introduced them into wild-type and rsp5A401E cells to construct six recombinant strains (CYH01-CYH06) (Table 1). In wild-type cells (CYH01, CYH02 and CYH03), Hsf1 was mainly observed in the nucleus. Msn2/4 were usually distributed in the whole cell, but were partly localized in the nucleus after exposure to elevated temperatures up to 37 °C or 10% ethanol. In particular, Msn2 was observed in both the nucleus and cytoplasm at high temperatures, while Msn4 was clearly detected after ethanol treatment in wild-type cells. On the other hand, we found similar patterns of intracellular localization in rsp5A401E cells (CYH04, CYH05 and CYH06), but the amounts of fusion proteins were significantly decreased. In good agreement with the previous results (Fig. 2C), the stress-induced increases in these transcription factors in rsp5A401E cells were considerably lower than those in wild-type cells. These results showed that Rsp5 has little direct effect on the nuclear transport of Hsf1 and Msn2/4 under stress conditions.


Figure 3
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  		Figure 3  Hsf1 and Msn2/4 are mainly localized in the nucleus under stress conditions. Strains CYH01, CYH02, CYH03, CYH04, CYH05 and CYH06 were observed in the fluorescence microscope for the localization of Hsf1- or Msn2/4-GFP after shifting to 37 °C (Temp.) or adding 10% ethanol (EtOH) for 10 min. Cell morphology was observed through differential interference contrast (DIC) and was also stained with DAPI to visualize nuclei. Scale bars: 5 µm.

 

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  		Table 1  Yeast strains used in this study
 
The rsp5A401E cells are defective in nuclear export of HSF1- and MSN2/4-mRNA

Rsp5 has been shown to play a pivotal role in the nuclear export and modification of mRNA, rRNA and tRNA (Rodriguez et al. 2003; Kwapisz et al. 2005). Ubiquitination of some mRNA nuclear transport factors contributes to the regulation of this transport pathway. The mRNA export requires that newly synthesized precursor mRNAs undergo several processing steps, which include 5' capping, splicing, 3'-end cleavage and polyadenylation. The different steps leading to the ribonucleo-protein complex formation are linked to each other and often mediated by interactions with the RNA polymerase II transcription machinery. Recently, Neumann et al. (2003) reported that the rsp5-3 mutant having the mutations Thr104Ala, Glu673Gly and Gln716Pro was strongly impaired in the nuclear export of mRNA and ribosomal 60S subunits after a shift from 25 °C to 37 °C. In addition, tRNA and rRNA export defects in the rsp5-3 mutant are preceded by a severe inhibition of pre-tRNA and pre-rRNA processing (Neumann et al. 2003).

Based on the results described above, we predicted that the rsp5A401E mutant would fail to export mRNA because the protein amounts of transcription factors were defective in this mutant (Fig. 2C). To analyze the intracellular localization of HSF1- and MSN2/4-mRNA, we performed fluorescence in situ hybridization (FISH) as described in the Experimental procedures. Strains CKY8 and CHT81 were cultured at 25 °C to the exponential growth phase in YPD and were exposed to elevated temperatures up to 37 °C or 10% ethanol. As shown in Fig. 4, RNA Poly(A)+, intron-containing tRNAIle and intron-less tRNAMet were normally exported to the cytoplasm in wild-type and rsp5A401E cells under non-stress conditions. Interestingly, we observed that the mRNAs of HSF1 and MSN2/4 were accumulated in the nucleus of rsp5A401E cells after exposure to temperature up-shift and ethanol, and even under a non-stress condition. This result suggests that Rsp5 is involved in the nuclear export of HSF1- and MSN2/4-mRNA. It also appears that rsp5A401E cells were impaired in the nuclear export of many mRNA and tRNA molecules after temperature up-shift and ethanol treatment, although we do not exclude the possibility that impairment of overall protein synthesis occurred in rsp5A401E cells. Ethanol can disrupt protein folding by a mechanism distinct from that of high temperature (Piper 1995). Therefore, it is probable that the rsp5A401E mutant cannot adequately respond to ethanol stress through the transcription of stress proteins. These results suggest that Rsp5 probably fails to ubiquitinate the target protein(s) required for the nuclear export of HSF1- and MSN2/4 mRNA.


Figure 4
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  		Figure 4  The nuclear export of HSF1- and MSN2/4-mRNA is inhibited in rsp5A401E cells. Strains CKY8 (wild-type) and CHT81 (rsp5A401E mutant) cells were shifted to 37 °C (Temp.) or added 10% ethanol (EtOH) for 10 min. FISH was performed using specific digoxigenin-labeled probes recognizing intron-containing tRNAIle and intron-less tRNAMet, and probes specific for poly(A)+, HSF1- and MSN2/4-mRNA. DNA was stained with DAPI. Scale bars: 1 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We previously showed that HSE- or STRE-mediated gene expression was defective in the rsp5A401E mutant under various stresses, suggesting that Rsp5 participates in the refolding of denatured proteins by regulating the expression of stress proteins through Hsf1 and Msn4 (Haitani et al. 2006). The expressions of HSF1 and MSN4 were increased in wild-type cells at the shift to 37 °C, and the transcription of MSN4 was also induced by addition of ethanol (Fig. 1). Thus, it appears that these gene expressions are necessary for the adaptive stress response. However, the expression of MSN2 in response to temperature up-shift and ethanol treatment was decreased in wild-type cells. It is probable that the activation of Msn4 primarily affects the expression of stress protein genes, even though Msn2/4 itself regulates the MSN4 expression under various stresses. In S. cerevisiae, it is well understood that Hsf1 and Msn2/4 regulate the expression of many stress protein genes in response to high temperature and high ethanol concentrations, respectively (Hashikawa & Sakurai 2004; Ferguson et al. 2005). Although we do not know the mechanistic basis for this induction, such differences in the induction of HSF1 and MSN2/4 may reflect the different kind of stresses or different target genes, which were regulated by these factors. As shown in Fig. 1, the mRNA levels of HSF1 and MSN2/4 in rsp5A401E cells were significantly lower than that of wild-type cells even at the permissive temperature (a non-stress condition). Therefore, it appears that the sensitivity of rsp5A401E mutant to the restricted temperature does not cause the reduction of transcription of HSF1 at high temperature. In addition, we think that the over-expression of HSP12 does not restore the transcription level of HSF1, because Hsp12 is located downstream of Hsf1 and Msn2/4 in the stress response. Although Rsp5 may participate in many biological events, rsp5A401E cells shows hypersensitivity to various stresses mainly due to the impaired export of mRNA.

Despite the fact that the mRNA levels of HSF1 and MSN2/4 were almost the same between wild-type and rsp5A401E cells, the protein levels of Hsf1 and Msn2/4 dramatically decreased in rsp5A401E cells in the presence or absence of stresses (Fig. 2). In S. cerevisiae, it is well known that the binding of Hsf1 and Msn2/4 to HSEs and STREs, respectively, triggers HSE- and STRE-mediated gene expression in response to both heat and oxidative stresses. However, it remains unclear how Hsf1 and Msn2/4 are transported into the nucleus. In this process, Rsp5 may play a significant role in regulation of the expression and the transport of Hsf1 and Msn2/4 through ubiquitination. We previously considered a possible mechanism in which three transcription factors in rsp5A401E cells would be degraded by unknown protease(s), probably because of their failure to transport into the nucleus. It is also possible that the rsp5A401E mutant, which has a single amino acid change of Ala401 by Glu in the third WW domain, cannot recognize and bind to the substrate(s) via its WW domains to conjugate ubiquitin. We have attempted to confirm that there is a physical interaction between Rsp5 and these transcription factors using various methods, but no evidence has been obtained so far (data not shown). Therefore, we examined the localization of Hsf1 and Msn2/4 in rsp5A401E cells, and found that these transcription factors are usually distributed in the whole cell, but in part are localized in the nucleus after exposure to stresses, although the amounts of GFP-fusion proteins were remarkably decreased (Fig. 3).

Another interesting point is that the protein levels of Hsf1 and Msn2/4 increased in the rsp5A401E mutant after exposure to high temperature and ethanol (Fig. 2C), although the levels of mRNAs were not changed by these stresses (Fig. 2B). It has been suggested that Hsf1 and Msn2/4 are activated and stabilized probably due to phosphorylation or structural change under stress conditions, and then induce transcription of stress proteins (Hashikawa & Sakurai, 2004). Therefore, two possibilities can be considered: (i) to avoid the rapid turnover of Hsf1 and Msn2/4 through stabilization or (ii) to stop the degradation of Hsf1 and Msn2/4 by ubiquitin-mediated proteolysis.

In contrast, the localization of RNA molecules was dramatically changed in rsp5A401E cells (Fig. 4). It is interesting that mRNAs of HSF1 and MSN2/4 were accumulated in the nucleus even under a non-stress condition in rsp5A401E cells, though the bulk mRNAs and tRNAs were normally exported. It is believed that high temperature as well as ethanol stress causes selective mRNA export in yeast (Tani et al. 1995; Saavedra et al. 1996; Krebber et al. 1999). Bulk poly(A)+ mRNA accumulates in the nucleus, whereas the mRNA of SSA4 encoding a heat shock protein is exported from the nucleus in ethanol-stressed cells. Thus, it is necessary that Hsf1 and Msn2/4 are constitutively expressed and present in the cytoplasm even under a non-stress condition. Therefore, Rsp5 may be directly or indirectly concerned with the processing of pre-mRNA of HSF1 and MSN2/4 and the selective export of their mRNAs from the nucleus. In addition, we found that HSF1- and MSN2/4-mRNAs were accumulated in the nucleus of rsp5A401E cells after exposure to temperature up-shift and ethanol and most of the bulk mRNAs and tRNAs were strictly prevented from the nuclear export in rsp5A401E cells under these stress conditions. It is probable that the mRNA nuclear export receptor Mex67 (Gwizdek et al. 2006) is not recruited to mRNAs through the RNA-binding adaptors-one of which includes the THO/TREX (transcription/export) complex component Hpr1 (Gwizdek et al. 2005) that couple transcription to mRNA export, and that this absence of Mex67 impairs the nuclear export. Rsp5 has been shown to take part in the regulation of mRNA export in S. cerevisiae through the ubiquitination and degradation of the nuclear export factor Hpr1, but this export mechanism remains unclear. In addition to the catalytic domain of Rsp5, proper nuclear export of RNAs is also regulated by its second and third WW repeats in Rsp5. These repeats are thought to interact with Rsp5 substrates through a PXY motif (Rodriguez et al. 2003). The Rsp5 activity is dispensable for mRNA transport at 25 °C, but essential at 37 °C. However, it is possible that mRNA of HSF1 and MSN2/4 could be differentially sorted into the extranucleus by different kinds of stresses. Furthermore, we should examine the localization of other genes, including stress protein genes, in the rsp5A401E mutant under stress or non-stress conditions.

Despite the fact that the amount of Pgk1 was almost the same between wild-type and rsp5A401E cells under a non-stress condition, the protein levels of Hsf1 and Msn2/4 were remarkably decreased in rsp5A401E cells in the presence or absence of stresses (Fig. 2C). These results suggest that PGK1-mRNA was non-selectively exported from the nucleus, but most of HSF1- and MSN2/4-mRNAs were not exported even under a non-stress condition. Moreover, it should be noted that yeast cells were exposed to temperature up-shift and ethanol for only 10 min, suggesting that pre-existed Pgk1 before stress treatment was only detected.

We previously demonstrated that over-expression of HSP12 can rescue the growth defect of rsp5A401E cells to some extent under stress conditions (Haitani et al. 2006). Therefore, HSF1, MSN2 and MSN4 were constitutively over-expressed under control of the ADH1 promoter in the rsp5A401E mutant. However, over-expression of these transcription factors did not rescue any growth defect of rsp5A401E cells under various stresses such as high growth temperature, ethanol, H2O2 and toxic amino acid analogues that induce protein misfolding in the cell (data not shown). These results suggest that rsp5A401E cells are defective in nuclear export of HSF1- and MSN2/4-mRNA. In contrast, we consider a possible mechanism in which HSP12 as well as SSA4 in rsp5A401E cells would be selectively exported and translated to repair stress-induced abnormal proteins (Tani et al. 1995; Saavedra et al. 1996; Krebber et al. 1999).

A simple but important conclusion is that Rsp5 would be involved in both repair and degradation of stress-induced abnormal proteins. In particular, based on the fact that Rsp5 participates in the proper export of RNA molecules from the nucleus, we think that Rsp5 primarily regulates the expression of transcription factors required for stress responses through the post-transcriptional event. The Rsp5 activity is required for proper mRNA export, indicating that the Rsp5-mediated ubiquitination acts directly or indirectly to affect the export of RNA molecules. The rsp5A401E mutant probably causes a more pronounced defect at high concentrations of ethanol and high temperatures, suggesting that ubiquitination of nuclear export factors by Rsp5 may be an essential event for cellular stress responses. To elucidate the mechanism, we are currently investigating the nuclear export of mRNA and tRNA in wild-type and rsp5A401E cells. It is believed that Rsp5 might control the major nuclear RNA biogenesis/export pathways in S. cerevisiae, and identification of the ubiquitin ligase substrates is an important challenge in clarifying how this modification may modulate the export of RNAs. This could be a useful method for studying stress responses and breeding novel stress-resistant yeast strains.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains, plasmids and media

The S. cerevisiae strains used in this study are listed in Table 1. Strains CYH01, CYH02, CYH03, CYH04, CYH05 and CYH06 were isolated by homologous recombination (Petracek & Longtine 2002). The integration cassette from plasmid pFA6a-GFP-kan (supplied by S. Harashima) (Bahler et al. 1998) was amplified by PCR performed with oligonucleotide primers HSF1-GFP-F, HSF1-GFP-R, MSN2-GFP-F, MSN2-GFP-R, MSN4-GFP-F and MSN4-GFP-R (Table 2). These PCR fragments were introduced into strain CKY8 (wild-type) or CHT81 (the Ala401Glu rsp5 mutant) (Hoshikawa et al. 2003) and genetycin-resistant transformants integrated with Kan-GFP were selected to generate CYH01, CYH02, CYH03, CYH04, CYH05 and CYH06. The correct integration was confirmed by PCR. Escherichia coli strain DH5{alpha} [F–{lambda}–{Phi}80lacZ{Delta} M15 {Delta}(lacZYA argF) U169 deoR recA1 endA1 hsdR17(Formula ) supE44 thi-1 gyrA96] was used to subclone the yeast gene.


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  		Table 2  Oligonucleotides used in this study
 
The 2 µ-based high-copy-number plasmid pAD4 (supplied by J. Nikawa), which contains LEU2, was used for expressing the yeast gene. Plasmid pFA6a-GFP-kan, which contains the bacterial ampicillin-resistant gene and the kanMX6 cassette for selective marker for E. coli and S. cerevisiae, respectively, was used to generate GFP-fused HSF1 or MSN2/4 at its carboxyl-terminus. Plasmids pAD-HSF1 (Haitani et al. 2006), pAD-MSN4 (Haitani et al. 2006) and pAD-MSN2 (in this study) were used for expressing HA-tagged HSF1, MSN4 and MSN2 under the control of the ADH1 promoter, respectively. The DNA fragment of MSN2 was amplified by PCR performed with genomic DNA from CKY8 and oligonucleotide primers MSN2-PstI and MSN2-SmaI (Table 2). The unique amplified bands of 2.1 kb corresponding to MSN2 was digested with PstI and SmaI and then ligated into the PstI and SmaI sites of pAD4 to construct pAD-MSN2, which has HA-tagged MSN2. The nucleotide sequences of the cloned genes in this study were confirmed by DNA sequencing.

The media used for growth of S. cerevisiae were yeast extract/peptone/dextrose (YPD) (2% glucose, 1% yeast extract, 2% peptone) and synthetic complete (SC) (Rose et al. 1990) consisting of 2% glucose, 0.67% Bacto Yeast Nitrogen Base with ammonium sulfate (Difco Laboratories, Detroit, MI) and drop-out mix lacking leucine (SC-Leu).

RNA extraction, reverse transcription-PCR and real-time PCR

Strains CKY8 and CHT81 cells were grown to the exponential growth phase (OD600 of 1.0) in SC-Leu medium at 25 °C and subjected to temperature up-shift (to 37 °C) and ethanol (10%). The cells were harvested and washed, and the whole-cell extracts were prepared by vortexing the cells with glass beads. Total RNA from S. cerevisiae was isolated by RNeasy Mini Kit (Qiagen, Valencia, CA) and incubated with RNase-free DNase set (Qiagen). Using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA) 2 µg of total RNA was reverse transcribed following the supplier's guidelines. cDNA were amplified with the target gene-specific primers (Table 2) and analyzed by real-time quantitative PCR performed with a 7300 Real-Time PCR System (Applied Biosystems). The mRNA level of the target gene was normalized to that of ACT1, which encodes β-actin, in the same sample. The cycle threshold (CT) value for each reaction was determined using the 7300 Real-Time PCR System software package (Applied Biosystems). CT values were used to calculate the mean fold change of the reactions through the 2–{Delta}{Delta}CT method for each sample in triplicate, for which one indicates no change in abundance (Livak & Schmittgen 2001). For each gene tested, the non-stress transcription level measured in wild-type cells was arbitrarily set to 1.0 and all other values were represented relative to this standard.

Western blot analysis

Yeast cells were cultured to the exponential growth phase in SC-Leu medium at 25 °C and subjected to temperature up-shift (from 25 to 37 °C) and 10% ethanol in the absence or presence of 100 µg/mL CX for 10 min. The cells were harvested and washed, and the whole-cell extracts were prepared by vortexing the cells with glass beads in 1 M Tris–HCl buffer (pH 7.5) containing 0.5 M EDTA, 1 M MgCl2, 1 M KCl, 5% glycerol and 2% SDS (Sambrook & Russell 2001). The supernatant (50 µg of solubilized proteins) after centrifugation (30 min at 15 000 g) was boiled for 5 min and loaded on a 7.5% SDS-polyacrylamide gel. Hsf1-HA and Msn2/4-HA were detected by using an ECL plus Western blotting Detection System (Amersham Biosciences), anti-HA (12CA5) monoclonal antibody at 1 : 2000 dilutions (Roche, Basel, Switzerland) and anti-Pgk1 monoclonal antibody at 1 : 1000 dilutions (Molecular Probes, Eugene, OR). Protein concentrations were determined using a Bio-Rad dye staining kit (Hercules, CA).

Fluorescence in situ hybridization (FISH)

FISH was performed as described by Sarkar & Hopper (1998). Strain CKY8 and CHT81 were cultivated to the exponential growth phase in YPD medium at 25 °C and exposed to elevated temperatures up to 37 °C or 10% ethanol. Cells were prefixed in the culture by the addition of 0.1 volume of 37% formaldehyde. After 30 min, 5 mL cells were harvested by centrifugation and resuspended in 6 mL of 4% paraformaldehyde, 0.1 M KPO4 (pH 6.5) and 5 mM MgCl2. After 3 h, cells were washed with solution B (1.2 M sorbitol and 0.1 M KPO4, pH 6.5) and resuspended in 2.8 mL of solution B containing 0.05% β-mercaptoethanol and 50 µL of 2 mg/mL freshly prepared Zymolyase-100T (Seikagaku, Tokyo, Japan). Spheroplasting was conducted at 37 °C for 20 min. Spheroplasts were washed in solution B and resuspended in 0.3 mL of solution B. Cells were adhered to the wells of Teflon-faced slides that had been pre-treated with a 0.1% poly-L-lysine-containing solution. Nonadhered cells were removed by aspiration. Cells were treated with 70%, 90% and 100% ethanol successively for duration of 5 min each, and the slides were placed in a dessicator for 5 min. Cells were then incubated in pre-hybridization buffer containing 2 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M Na-citrate), 10% dextran sulfate, 0.2% BSA, 125 µg/mL E. coli tRNA (Sigma Chemical, St. Louis, MO) and 500 µg/mL denatured sonicated salmon sperm DNA (Roche) for 2 h at 37 °C in a humid chamber. Hybridization buffer had the same composition with 450 pg/mL digoxigenin-labeled probes. All probes were labeled at their 3' end using digoxigenin-11-UTP. Both the pre-hybridization and hybridization buffers contained RNase Inhibitor (Applied Biosystems) at a concentration of 1 U/µL, and hybridization was carried out at 37 °C overnight. Cells were washed with 2 x SSC at 45 °C for tRNA probes and at 37 °C for the oligo(dT)50 probe, and then washed for 10 min with 1 x SSC at room temperature. Cells were briefly washed with 4 x SSC containing 1% Triton X-100 and then blocked for 2 h using 1% BSA containing 4 x SSC. Anti-digoxigenin-fluorescein Fab fragment (Roche) was diluted at 1 : 200 in solution containing 1% BSA and 4 x SSC, and the cells were incubated with the diluted antibody for 2 h. Cells were washed twice with 4 x SSC followed by two more washes with 4 x SSC containing 1% Triton X-100, each wash lasting for 10 min. After two more rapid washes with 4 x SSC, cell nuclei were counterstained with 0.1 µg/mL 4',6-diamidino-2-phenylindole dihydrochloride (DAPI). After two rapid washes with water, the slides were mounted under 90% glycerol and 1 x PBS containing 1 mg/mL p-phenylenediamine and stored at –20 °C.

Fluorescence microscopy

Cells were viewed with Axiovert 200M (Carl Zeiss, Oberkochen, Germany) microscope equipped with appropriate fluorescence light filter sets. Images were captured with a HBO 100 Microscope Illuminating System (Carl Zeiss) digital camera and processed using Adobe Photoshop Elements 2.0 (Adobe Systems, San Jose, CA). Internalization experiments of mRNA, tRNA and GFP-fusion protein were performed at least 3 times.


   Acknowledgements
 
We thank Drs Chris A. Kaiser (Massachusetts Institute of Technology, USA), Kenji Kitamura (Hiroshima University, Japan), Jun-ichi Nikawa (Kyushu Institute of Technology, Japan), and Satoshi Harashima (Osaka University, Japan) for providing strains and plasmids, and Drs Hiroyuki Hiraishi, Hiroshi Sakurai (Kanazawa University, Japan), and Satoshi Kagiwada (Nara Women's University, Japan) for helpful discussion. This work was supported by a grant to H.T. from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).


   Footnotes
 
Communicated by: Yoshinori Ohsumi

* Correspondence: E-mail: hiro{at}bs.naist.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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