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Nucleic Acids Research, 2002, Vol. 30, No. 5 1145-1153
© 2002 Oxford University Press

Glucose-inducible expression of rrg1+ in Schizosaccharomyces pombe: post-transcriptional regulation of mRNA stability mediated by the downstream region of the poly(A) site

Min Ji Kim, Jae Bum Kim, Dong Sun Kim1 and Sang Dai Park*

School of Biological Sciences, Seoul National University, Kwanak-Ku, Shilim-dong, Seoul 151-742, Republic of Korea and 1Department of Anatomy, School of Medicine, Kyungpook National University, Taegu 700-422, Republic of Korea

Received November 16, 2001; Revised and Accepted January 16, 2002.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
rrg1+ (rapid response to glucose) has been isolated previously as a UV-inducible gene in Schizosaccharomyces pombe, designated as uvi22+. However, it was revealed that the transcript level of this gene was regulated by glucose, not by DNA-damaging agents. Glucose depletion led to a rapid decrease in the level of rrg1+ mRNA, by ~50% within 30 min. This effect was readily reversed upon re-introduction of glucose within 1 h. High concentrations (4 and 8%) of glucose showed similar effects on increasing the rrg1+ mRNA level compared with 2% glucose, while a low concentration (0.1%) was not effective in raising the rrg1+ mRNA level. In addition, sucrose and fructose could increase rrg1+ mRNA level. Interestingly, the rapid decline in mRNA level seen upon glucose deprivation resulted from precipitous reduction of mRNA half-life. Serial and internal deletions within the 3'-flanking region of rrg1+ revealed that a 210-nt region downstream of the distal poly(A) site was critical for glucose-regulated expression. Moreover, this downstream region participated in 3'-end formation of mRNA. Taken together, this is the first report on glucose-inducible expression regulated post-transcriptionally by control of mRNA stability in S.pombe.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucose, the preferred carbon source for most eukaryotic cells, plays a central role in metabolism, both as an energy source and a precursor of essential structural carbohydrates and other biomolecules. Many eukaryotic cells have developed mechanisms to sense and respond to changes in glucose levels, along with other nutrients in the environment, for flexible growth. One of the major mechanisms by which cells adapt is regulation of gene expression, accomplished primarily by transcriptional control, although mRNA stability, translation and proteolysis are also involved.

For the budding yeast Saccharomyces cerevisiae, it is well known that the expression of a large set of genes required for the utilization of alternative carbon sources is inhibited in the presence of glucose for optimal and efficient use of sugar available in the environment (1,2). This phenomenon is referred to as ‘glucose repression’ and many elements involved in glucose signaling have been studied (1–4). Recently, analysis of genomic expression by DNA microarrays revealed that transcript levels of numerous genes are differentially regulated in the process of diauxic shift (5). During the shift from fermentation to respiration upon glucose exhaustion, cells are subjected to widespread changes in the expression of genes involved in fundamental cellular processes such as protein synthesis and carbohydrate storage, as well as carbon metabolism. Among these, the expression of genes encoding low-affinity glucose transporter, glycolytic enzymes, ribosomal proteins, tRNA synthetase, and translation elongation and initiation factors are induced by glucose. Expression of another large set of genes, which are involved in utilization of alternative carbon sources, gluconeogenesis, respiration and peroxisomal functions, is repressed by glucose. However, about half of the differentially transcribed genes currently have no identified function and have not yet been named (5).

Although the main effect of glucose is exerted at the transcriptional level, it is clear that control of mRNA stability is also an important component of glucose-regulated gene expression (1). Glucose has been reported to destabilize the mRNA of CYC1 (6), MAL6S (7), PCK1 (8,9) and subunits of succinate dehydrogenase (10,11). Phosphoenolpyruvate carboxykinase gene, PCK1, is subjected to both transcriptional and mRNA stability controls, which were recently revealed to be coordinated by common signaling pathways (8,9). In the case of SDH2, encoding the iron protein (Ip) subunit of succinate dehydrogenase, glucose exerts a very strong effect on its mRNA stability and post-transcriptional regulation is regarded as the major determinant for SDH2 expression (10,11). An attempt to investigate the molecular mechanism governing glucose-dependent mRNA decay is currently under way in S.cerevisiae. However, the identities of glucose-regulated genes and the molecular mechanisms underlying glucose-regulated expression in the fission yeast Schizosaccharomyces pombe, a closer sibling to higher eukaryotic cells than S.cerevisiae, remain mostly elusive.

rrg1+ was previously isolated as a UV-inducible gene in S.pombe and designated as uvi22+ (EMBL accession no. Z34299). However, this gene was found to be regulated by glucose, not by UV light or other DNA-damaging agents, after intensive investigations. Thus, we renamed this gene rrg1+ (rapid response to glucose), based on its prompt changes in expression in response to glucose. Here we examine the response of rrg1+ expression to glucose along with various other carbon sources and demonstrate that control of rrg1+ mRNA stability mediated by a downstream region of the poly(A) site was involved in the glucose-dependent expression of this gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, culture media, and glucose deprivation and re-addition
Schizosaccharomyces pombe strain JY1 (h– wild-type) obtained from Dr M. Yamamoto (University of Tokyo, Tokyo, Japan) was used for northern blot analysis. MJK1 (h– rrg1::ura4+ ade6-M210 ura4-D18 leu1-32) was used for integration of various rrg1+ constructs. The culture medium was Edinburgh minimal medium (EMM) supplemented with appropriate amino acids. For glucose deprivation, cells were incubated in water for 90 min. The cells were then transferred to EMM with 2% glucose and cultured for 60 min for glucose re-addition.

Construction of plasmids and deletions
The 2.3 kb HindIII fragment containing the rrg1+ open reading frame (ORF) and its 5'- and 3'-flanking regions was cloned into plasmid pJK148 (purchased from American Type Culture Collection, Rockville, MD) and integrated at the leu1-32 locus of the S.pombe chromosome as described by Keeney and Boeke (12). For swapping the promoter and the 5'-untranslated regions (UTRs) of rrg1+ for those of nmt1+ (nmt1-rrg1), a BamHI site was created in front of the translation start site (ATG) of rrg1+. The 1.2 kb PstI/NdeI fragment containing the 5' upstream region of nmt1+, which was obtained from pREP1 plasmid (13), was inserted at this BamHI site after deletion of the upstream region of rrg1+. For deletion in the 3'-flanking region of rrg1+, restriction endonuclease ClaI was used to generate a deletion to +1155. Deletions to +1374, +1070, +1043 and +911 were created by PCR with primers 3'-2100 (5'-TCGAAAGTACGAAGGGTAAG-3'), 3'-poly(A) (5'-AGTGATTCCCTTTGATACTG-3'), 3'-UTR (5'-ATACAAAAGAATCTATTGCTTTGC-3') and 3'-TAA (5'-TTATCCATGGTATTTCCATCTACTCC-3'), respectively. Internal deletions were produced by the overlap extension technique (14). The primers were int-poly(A) 5' (5'-CTTTTGTATTTTGGTTTTAAAGCTGGCTTTT-3') and int-poly(A) 3' (5'-AAAACCAAAATACAAAAGAATCTATTGCTTT-3') for {Delta}1043~1070; and int-210 5' (5'-ACAGGACATTCTTTAATCAGCATGCGAAGCTA-3') and int-210 3' (5'-GATTAAAGAATGTCCTGTTGATCTAAACCAA-3') for {Delta}1155~1374.

Chemical treatments and northern blot analysis
1,10-Phenanthroline (Sigma, P-9375) was dissolved in 100% ethanol and added to cells to a final concentration of 100 µg/ml. Cycloheximide (Sigma, C-6255) was dissolved in water and added to the cell culture to a final concentration of 200 µg/ml. For northern blot analysis, total RNA was isolated after extraction with phenol/chloroform/SDS as described by Jang et al. (15). About 15 µg of total RNA was separated in 1.5% agarose gel containing 0.67 M formaldehyde, transferred onto nylon membrane and hybridized with radiolabeled probes. Radiolabeled probes were made using double-stranded DNA fragments within the ORF of each gene, and random oligonucleotide primers (16). In the case of rrg1+, a 1.2 kb HincII/ClaI fragment containing the entire coding region of rrg1+ was utilized to generate the probe. After stringent washings, the blot was exposed to X-ray film or a phosphorimager (BAS1500; Fuji, Tokyo, Japan).

RT–PCR
About 5 µg of total RNA was reverse transcribed with 200 U Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) at 42°C for 50 min in the presence of 10 pmol of the following antisense primers: primer 1 (identical to 3'-TAA), primer 2 (identical to 3'-UTR), primer 3 [identical to 3'-poly(A)], primer 4 (5'-GATTTTCTACTAACCGTAATTC-3'), primer 5 (5'-ATAGAGACGAAACGATACTAAT-3'), primer 6 (identical to 3'-2100) and primer 7 (5'-AAAAGATCGAATTATAGAGAGA-3'). One-tenth of the cDNA products was amplified by PCR (30 amplification cycles) with 10 pmol of a specific 5' primer (5'-GTTGTATGTACAGATCTACC-3') and the corresponding antisense 3' primers using 2.5 U ExTaq (TaKaRa, Shiga, Japan). Various RT–PCR products were electrophoresed in 1.4% agarose gel and visualized by ethidium bromide staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of rrg1+ is regulated by glucose
Previously, it was found that rrg1+ was expressed in exponentially growing cells and that its transcripts were hardly detected in northern blot analysis when cells entered the stationary phase (data not shown). To determine the identity of regulating factors for rrg1+ expression, cells were resuspended in water to deprive them of all nutrients and each nutritional component was re-added, as shown in Figure 1A. Northern blot analysis of RNA isolated from the individual treatments revealed that the transcript level of rrg1+ decreased during nutrient starvation and was restored only in the presence of glucose. Different concentrations of sorbitol (solute) in culture media did not change the mRNA level, which ruled out the possibility that changes in osmolarity by glucose were involved in the regulation of rrg1+ expression (data not shown). Thus, it could be concluded that the changes in rrg1+ mRNA level were dependent on glucose as a carbon source.



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  		Figure 1. Glucose-dependent expression of rrg1+. The mRNA levels of rrg1+ were examined by northern blot analyses with the HincII/ClaI fragment of rrg1+ as probe. Ethidium bromide staining of rRNA showed an equal amount of RNA in each lane. (A) Wild-type S.pombe cells (JY1) pre-grown in EMM were transferred to water. After a 1 h incubation, glucose, salt mixture, NH4Cl, Na2HPO4 or various amino acids (a.a.) were added to the cells to the same final concentrations as EMM. At each time point, total RNA was isolated and northern blot analysis was performed. (B) The rapid changes in rrg1+ mRNA level. JY1 cells were transferred to EMM without glucose and harvested at each time point. After 3 h of glucose depletion, the cells were cultivated for another 6 h in the presence of 2% glucose and collected for RNA preparation. Northern blot analysis was carried out with probes for rps6-1, rpl32-1 and rrg1+. (C) Effect of glucose concentration on rrg1+ expression. After a 3 h incubation in EMM without glucose (–G), JY1 cells were provided with various concentrations of glucose (0.1–8%) and cultured for 2 h (left). JY1 cells were grown in EMM containing the indicated concentrations of glucose overnight (right). The cells in 0.1% glucose were subjected to 2% glucose upshift by adding an appropriate amount of glucose and incubation for 1 h. (D) Effect of other carbon sources on rrg1+ expression. JY1 cells were deprived of glucose by incubation in EMM without glucose for 3 h (–G). Then various carbon sources were added to the final concentration of 2%, followed by a 2 h incubation. Glc, glucose; Fru, fructose; Suc, sucrose; Mal, maltose; Raf, raffinose; Gly, glycerol; EtOH, ethanol.

 
Next we examined the time course of the changes in rrg1+ mRNA level in the presence or absence of glucose (Fig. 1B). The amount of rrg1+ mRNA was reduced dramatically by ~50% within 30 min and was hardly detectable in the northern blot after 3 h of glucose deprivation. This effect was reversed within 1 h by addition of glucose. This rapid response of rrg1+ mRNA to glucose is comparable with that of ribosomal protein (RP) mRNAs in budding yeast (17). In contrast, such rapid changes in mRNA level did not appear in rps6-1 and rpl32-1, S.pombe RP genes whose counterparts in S.cerevisiae are regulated rapidly by glucose at the transcriptional level (Fig. 1B). In addition, the eno1+ gene encoding enolase did not show such a prompt change in mRNA level by glucose (data not shown), although its steady-state mRNA level has been reported to be maximized when cells are grown in the presence of glucose (18).

The effects of glucose concentration have been investigated. After glucose starvation, the rrg1+ mRNA level was not raised by a low level (0.1%) of glucose, whereas the moderate (2%) and high (4 and 8%) levels were similarly effective in inducing rrg1+ expression (Fig. 1C, left). The steady-state level of rrg1+ mRNA was also similarly high when cells were grown in glucose above a concentration of 2%, while it was significantly reduced when cells were grown in 0.1% glucose (Fig. 1C, right). rrg1+ expression at 0.1% glucose increased by 6-fold after a 2% glucose up-shift. These results demonstrate that rrg1+ expression is regulated in a glucose-dependent manner, where its mRNA level is rapidly decreased and restored by the absence and the presence of glucose, respectively.

To investigate whether rrg1+ expression is specific to glucose, various other carbon sources were tested for the ability to raise the rrg1+ mRNA level after glucose starvation. Only fructose and sucrose, fermentable sugars, could restore the mRNA level, although the induced levels were slightly lower than the glucose effect (Fig. 1D). Sucrose is a disaccharide that can be readily converted to glucose and fructose by invertase, encoded by the inv1+ gene (19). Thus, it is probable that glucose and fructose, the favored sugars of yeast that trigger catabolite repression (1), are responsible for the regulation of rrg1+ gene expression, even though glucose seems to be the main regulatory factor.

The glucose-regulated expression of rrg1+ results from changes in mRNA stability
Transcript levels can be regulated by transcription initiation and mRNA stability. To distinguish between these two possibilities in the case of rrg1+, the contribution of transcription initiation was examined first. For this purpose, a plasmid was constructed in which the 5' upstream region of rrg1+ was substituted with that of nmt1+ (Fig. 2A). It has been determined that the nmt1+ promoter does not confer glucose-dependent transcription (data not shown). The wild-type and chimeric constructs (nmt1-rrg1) were then integrated into the leu1-32 locus of the chromosome to generate a stable single-copy background as described by Keeney and Boeke (12). With these integrants and the wild-type cells (JY1), the glucose deprivation and re-addition experiment was performed to measure rrg1+ transcript level by northern blot analysis. JY1 cells were used for the endogenous mRNA of rrg1+ as a control. Since the cells used to integrate the constructs were rrg1 deletion mutants, signals from the integrated constructs were not interfered with by those of the endogenous rrg1+. As shown in Figure 2B, the rrg1+ mRNA derived from the heterologous nmt1+ promoter still disappeared and accumulated under conditions of glucose withdrawal and re-addition, respectively. Although the relative level of mRNA in glucose deprivation was slightly increased in the chimeric construct, it can be said that the expression pattern of rrg1+ was not altered by promoter exchange. This result suggests that transcriptional control is not the major regulatory mechanism. This result is consistent with our previous observation that deletion of the whole rrg1+ promoter did not affect the glucose-dependent regulation of rrg1+ mRNA level (M.J.Kim and S.D.Park, unpublished data). Thus, these findings raised the possibility that the behavior of the rrg1+ transcript might result mainly from regulation of mRNA stability rather than transcription initiation control. The size difference between the transcripts driven from the rrg1+ and the nmt1+ promoters is due to different transcription start sites. In order to test our model, changes in the half-life of rrg1+ mRNA were examined during glucose depletion (Fig. 3). An inhibitor of RNA synthesis, 1,10-phenanthroline, was added to cell culture in the absence or presence of glucose and the amounts of remaining RNA were measured by northern blot analysis at the indicated time points. The result revealed that the rrg1+ mRNA in the glucose-deprived cells was dramatically degraded and hardly detected after 30 min, whereas the mRNA in the presence of glucose was very stable (Fig. 3A). Estimated half-lives of rrg1+ mRNAs were 6 and 58 min in the absence and the presence of glucose, respectively (Fig. 3B). In contrast, rpl32-1, the RP gene, did not show any changes in mRNA stability when glucose was withdrawn. Thus, these findings suggest that the primary cause of the rapid reduction in rrg1+ mRNA level under conditions of glucose deprivation may be accelerated RNA degradation rather than cessation of transcription initiation. Taken together, these results demonstrate that glucose regulation of rrg1+ expression is mainly mediated by control of mRNA turnover rather than transcriptional control.



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  		Figure 2. Exchange of a 5' upstream region does not alter the glucose-regulated expression of rrg1+. (A) Promoter and 5'-UTR regions of rrg1+ were substituted with those of the nmt1+ gene and the wild-type and the chimeric constructs were integrated into the leu1-32 locus in S.pombe chromosome (see Materials and Methods). (B) MJK1 cells harboring each construct and JY1 wild-type cells were subjected to glucose deprivation (D) and re-addition (R) by incubation in water for 1 h and post-incubation in EMM for 90 min. Total RNA was isolated and analyzed by northern blot analysis (top). The relative levels of rrg1+ mRNA, normalized to rRNA levels, was presented (bottom). The mRNA levels of JY1 cells were used as a control of endogenous rrg1+.

 


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  		Figure 3. Decrease in rrg1+ mRNA half-life upon glucose deprivation. Exponentially growing JY1 cells in EMM were divided into two, and either remained in EMM (+Glc) or were resuspended in water (–Glc), in the presence of 1,10-phenanthroline. Before drug addition, a portion of cells was harvested for control RNA (C). (A) Northern blot with RNA sample of each time point was hybridized with rrg1+ and rpl32-1 probes. (B) Plot representing decay kinetics of rrg1+ mRNA is shown. The values of the mRNA level were normalized to rRNA level. Each mRNA half-life (T1/2) is indicated in parentheses..

 
The glucose-dependent regulation of rrg1+ mRNA stability partially requires protein synthesis
It has been reported that the rapid glucose-inducible transcription of RP genes in S.cerevisiae and their inhibition by rapamycin do not require new protein synthesis (17,20). To address whether the changes in rrg1+ mRNA stability are dependent on de novo protein synthesis, the glucose deprivation and re-addition experiment was performed in the presence of cycloheximide. As shown in Figure 4, reduction in rrg1+ mRNA level by glucose withdrawal was partly alleviated in the cells treated with cycloheximide. In contrast, increase in the mRNA level by glucose re-addition was significantly affected by addition of cycloheximide. In the same northern membrane, actin (act1+) did not show any significant differences in its transcript level upon cycloheximide treatment. A control experiment demonstrated that cycloheximide was active under these experimental conditions (data not shown). Cycloheximide has been reported to increase the levels of various mRNAs by decreasing turnover rates of relatively unstable mRNAs (21). Considering the increased mRNA levels overall (compare lanes C of CON and CHX in Fig. 4A), it could be possible that the alleviation of reduction in rrg1+ mRNA stability during glucose deprivation might include the drug effect. However, the possible effect of defective protein synthesis cannot be ruled out, because the reduction rate of mRNA stability was altered (left panel in Fig. 4B). The increase in rrg1+ mRNA level upon glucose re-addition results from mRNA stability control, not by transcriptional induction (lane R of nmt1-rrg1 construct in Fig. 2B), and was almost abolished by cycloheximide. This result implied that the loss of protein synthesis might affect the restoration of rrg1+ mRNA stability in glucose re-addition more severely. Therefore, these results indicate that new protein synthesis is partially required for the glucose-dependent control of rrg1+ mRNA stability to enhance the decay rate of the mRNA in glucose deprivation and, more importantly, to recover mRNA stability to the glucose present state after glucose withdrawal.



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  		Figure 4. Changes in rrg1+ mRNA stability by glucose partially require de novo protein synthesis. (A) JY1 cells were grown in EMM, split into two, and either exposed to cycloheximide (CHX) or not (CON) for 30 min. After a fraction of each culture was harvested for control RNA (C), the cells were transferred to water and deprived of glucose with or without cycloheximide and incubated for indicated times. Then the cells were pelleted and resuspended in EMM containing 2% glucose with or without cycloheximide, followed by post-incubation. D and R denote deprivation and re-addition of glucose, respectively. Total RNA was prepared at the indicated intervals and analyzed by northern blot analysis with rrg1+ and act1+ probes. (B) Changes in relative levels of rrg1+ mRNA are presented, after normalization to ethidium bromide signals of rRNA.

 
A 210-nt region downstream of the poly(A) site is responsible for glucose-regulated mRNA turnover and mRNA 3'-end formation
In an attempt to identify regulatory elements involved in the glucose-regulation of rrg1+ mRNA stability, serial deletions were generated at the 3'-flanking region of rrg1+ (Fig. 5A). After the integration at the leu1-32 chromosome locus as described earlier, the mRNA behavior of rrg1+ derived from each deletion construct was investigated by northern blot analysis. When the deletion reached +1155 bp (construct II in Fig. 5), the reduction in mRNA level upon glucose deprivation was not observed. Instead, the mRNA level was raised by ~1.4-fold upon glucose deprivation, which was reinforced with further deletions (constructs IV and V in Fig. 5). Further evidence for the presence of a regulatory element between base pairs +1155 and +1374 was provided by the PCR-based internal deletion analysis of this region. Consistent with the results above, the construct devoid of this 210-nt region (construct i-II in Fig. 5) showed a markedly increased level of mRNA in the absence of glucose. These results suggested that the 210-nt region might encompass a major determinant to control mRNA decay in response to glucose.



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  		Figure 5. Deletion analyses of the 3'-flanking region of rrg1+. (A) A series of deletions was generated in the 3'-flanking region of rrg1+ by restriction enzyme digestion and by PCR (left). The intact 5' portion of each construct (up to –726 bp from ATG) is not shown and the coding region (filled boxes) is partly shown in schematic diagrams. The numbers indicate the deletion ends. The translation stop codon (UAA), the poly(A) tail addition site (arrow) and the 210-nt region (hatched box) are shown. After chromosomal integration of each construct into the leu1-32 locus of MJK1 cells (see Materials and Methods), glucose deprivation (D) and re-addition (R) were carried out with each integrant and the levels of rrg1+ mRNA were assessed by northern blot analysis. The relative amounts of mRNA upon glucose deprivation were calculated after normalization to rRNA signals (right). (B) The representative blot is shown. The roman numerals correspond to each construct illustrated in (A). JY1 denotes RNA from S.pombe wild-type cells, used for control of the endogenous rrg1+ mRNA band. The arrowhead on the right indicates the readthrough transcripts of rrg1+.

 
Another interesting observation was that the deletion of the 210-nt region led to a decrease in the basal level of rrg1+ mRNA and the appearance of larger transcripts (constructs II–V and i-II in Fig. 5B). Low steady-state levels of mRNA result from a weak poly(A) site and extended transcripts are frequently observed when a polyadenylation signal is inactivated (22). The cleavage of the rrg1+ transcripts with RNase H in the presence of oligo(dT) revealed that the larger RNA band was a readthrough transcript with a poly(A) tail (M.J.Kim and S.D.Park, unpublished data). Thus, this result suggested that deletion of the 210-nt region brought about loss of mRNA 3'-end formation ability at the major poly(A) site of rrg1+, which resulted in utilization of cryptic and weak poly(A) sites located downstream of the cloned rrg1+ gene. The decreased basal level of rrg1+ mRNA could be due to degradation of a large fraction of pre-mRNA that fails to be properly processed without this region.

To confirm that glucose-dependent mRNA stability of rrg1+ is regulated by the 210-nt region, the half-life of rrg1+ mRNA was measured in the absence of the 210-nt region as described earlier (Fig. 6). Upon glucose deprivation, the rrg1+ mRNA from the wild-type construct (W) was degraded as rapidly as the endogeneous rrg1+ mRNA (Fig. 3). However, the mRNAs from the serial and the internal deletion constructs of the 210-nt region (constructs II and i-II in Fig. 5, respectively) did not show significant changes in their half-lives. No changes in the half-life of rpl32-1 mRNA were detected, as expected. These results suggest that the 210-nt region indeed enhances the turnover rate of rrg1+ mRNA upon glucose starvation. The rrg1+ mRNA levels of the 210-nt-deficient constructs upon glucose depletion were lower than those detected in Figure 5B, which is considered as a consequence of no continuous transcription by the transcription inhibitor.



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  		Figure 6. Deletion effect of the 210-nt region on the reduction of rrg1+ mRNA half-life upon glucose starvation. Each MJK1 cell harboring the wild type construct (W), the serial (II) and the internal (i-II) deletion constructs of the 210-nt region (see Fig. 5) was treated with 1,10-phenanthroline and the rrg1+ mRNA half-life was measured upon glucose deprivation as described in Figure 3. (A) Northern blot with RNA sample of each time point was hybridized with rrg1+ and rpl32-1 probes. (B) Plot for decay kinetics of rrg1+ mRNA is presented. The values of the mRNA level were normalized to rRNA level.

 
The 210-nt region resides downstream of the distal poly(A) site and it is not included in the mature rrg1+ mRNA, in contrast to other regulatory elements for post-transcriptional control. However, some recent studies on S.pombe have shown that RNA pol II transcription proceeds beyond the poly(A) site and that the downstream sequences located in the 3' non-coding region are responsible for transcription termination and mRNA 3'-end formation, which are closely coupled to efficient gene expression (23–26). Moreover, we previously demonstrated that a 54-nt element downstream of the poly(A) site in the uvi15+ gene was involved in mRNA stabilization upon exposure to UV light (27). Therefore, it was necessary to examine whether this 210-nt region could be transcribed into a precursor RNA by RT–PCR. DNase-treated total RNA was hybridized to various antisense 3' primers spanning the downstream region of the rrg1+ gene and was subjected to reverse transcription (Fig. 7A). The synthesized cDNA products were subsequently amplified by PCR with the same 3' primers and a common 5' primer located within the rrg1+ ORF and the same amount of each PCR sample was separated electrophoretically (Fig. 7B). The transcripts were detected within the 210-nt region (primers 4–6), although the amounts were much less than those of transcripts amplified with primers upstream of the poly(A) site. The transcripts were detected even downstream of this region as far as at least 130 bases (primer 7) after 30 cycles of amplification.



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  		Figure 7. Detection of rrg1+ transcript downstream of the poly(A) site. (A) Strategy for RT–PCR analysis of the 3'-flanking region of rrg1+. The relative positions of 5' primer and each antisense 3' primer (1~7) are illustrated with the deduced sizes of their PCR products. The hatched box and the broken filled box indicate the 210-nt region and the coding region of rrg1+, respectively. (B) RT–PCR products were separated on 1.4% agarose gel. The lane numbers correspond to each antisense 3' primer shown in (A). Lane C shows a control reaction where the reverse transcriptase was omitted during cDNA synthesis, proving no DNA contamination occurred. M, 1 kb DNA ladder marker (Life Technologies).

 
Collectively, these results provide evidence that the 210-nt region of rrg1+ pre-mRNA is involved in glucose-dependent control of mRNA stability and mRNA 3'-end formation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Glucose-inducible expression of rrg1+ regulated by post-transcriptional control of mRNA stability
In S.pombe, a few reports are available regarding glucose-inducible gene expression, which only describe increased steady-state levels of transcripts when cells were cultured in the presence of glucose (18,28). In this study, rapid glucose-inducible expression of rrg1+ and its molecular regulatory mechanism were investigated, providing a hint towards understanding the glucose regulation of gene expression in the fission yeast. The regulation of rrg1+ expression is mainly accomplished at the transcript level because changes in Rrg1 protein level were no different to those of the rrg1+ mRNA level (M.J.Kim and S.D.Park, unpublished data). Although contributions to transcription initiation should be precisely determined by transcription run-on assay, control of mRNA stability appeared to be the major determinant for the glucose-dependent expression of rrg1+ for the following reasons. First, exchange or loss of the promoter region did not abolish the pattern of glucose-dependent rrg1+ expression (Fig. 2). Along the same lines, the yeast Ip subunit of succinate dehydrogenase was shown to be regulated by mRNA stability rather than transcription by the similar experiment with its truncated promoter (10). Secondly, rrg1+ mRNA under conditions of glucose deprivation disappeared faster in the presence of transcription inhibitor (Fig. 3A) than in its absence (Fig. 1B), indicating that the reduction rate of mRNA half-life surpassed that of the steady-state mRNA level. This result implies that the increase in mRNA turnover rate alone was rapid enough to generate the decline in rrg1+ transcript level upon glucose starvation, regardless of transcriptional repression. Therefore, it could be concluded that the glucose-dependent expression of rrg1+ might be achieved by rapid degradation of rather constitutively produced mRNA upon glucose depletion and its inactivation by glucose.

Post-transcriptional control of mRNA stability is involved in expression of several glucose-repressible genes in S.cerevisiae (1). Among these, Ip transcript stability seems to change most dramatically in response to glucose, decreasing the half-life from >60 to <5 min (10). Regulation of its mRNA turnover by glucose is mediated by 5'-UTR and 5'-exonuclease Xrn1. The nucleolytic degradation of the Ip mRNA is slowed down in the presence of cycloheximide (10). Recently, each step of the currently accepted mRNA turnover mechanism was examined for its involvement in the glucose-induced degradation of the Ip mRNA (11). rrg1+ mRNA displayed similar rapid changes in half-life in response to glucose (T1/2 = 58 and 6 min with or without glucose, respectively; Fig. 3B). Thus, it is likely that a mRNA degradation system sensitive to glucose level might also exist in the fission yeast, which controls the stability of rrg1+ mRNA. Future studies regarding the general mRNA turnover mechanism and its relationship with glucose signaling in S.pombe will reveal the exact mechanism of the glucose-dependent mRNA decay system governing rrg1+ expression.

As an example of rapid glucose-inducible expression, RP genes of S.cerevisiae show quite similar changes in their transcript levels as that of rrg1+ (29). However, at least two of their counterparts in S.pombe seem to be regulated by other processes (Fig. 1B), such as translational control of RP syntheses in eubacteria and vertebrates (30). The prompt regulation of RP mRNA levels in S.cerevisiae results from rapid transcriptional control of the RP genes, coupled to the naturally short half-life (5–7 min) of their transcripts (17,31). Thus, the RP genes produce massive numbers of very short-lived mRNAs and regulate the amount mainly by transcriptional turn-on and -off, which is totally opposite to the case of rrg1+. A possible explanation for the different regulating systems adopted by rrg1+ and RPs could be that efficient use of energy and tight regulation dependent on glucose might be more critical to expression of rrg1+ and RPs, respectively. The rapid change into a new steady-state level of mRNA in response to environmental signals can be achieved more easily by the transcriptional control of RP genes, considering that the short mRNA half-life and no requirement of new protein synthesis for transcriptional control (17,20). The prompt response is inevitable for RP expression because of the extremely large portion of their expression inside of the cell and plausible deleterious effects of their excess (31). However, Rrg1 protein was required for optimal cell growth but it did not have lethal effects on cell viability when it was deleted or overproduced (M.J.Kim and S.D.Park, unpublished data). Thus, the efficiency of regulation would be more beneficial to rrg1+ expression.

The 210-nt downstream region as a destabilizing determinantA 210-nt region located downstream of the poly(A) site was identified as playing a pivotal role in enhancing the turnover rate of rrg1+ mRNA upon glucose starvation. So far, most stability determinants (destabilizing sequences) have been identified within mRNA, such as 3'-coding or -UTRs (32,33). Thus, the 210-nt region of rrg1+ is peculiar in its ability to function by post-transcriptional mode from its location downstream of the poly(A) site in pre-mRNA. The notion that pre-mRNA sequences excluded from mRNA could be involved in post-transcriptional regulation of gene expression has been supported by recent studies on the human N-myc gene (34) and uvi15+ gene of S.pombe (27). The exact mechanisms, however, remain largely elusive.

Based on our observations, three possible models for the effect of the 210-nt region on mRNA stability have been considered: (i) inhibition of normal mRNA 3'-end formation to produce unstable transcript, (ii) direct regulation of pre-mRNA stability, and (iii) regulation of mRNA stability itself through other factor(s). The first appears quite reasonable because the 210-nt region seems to have the ability for proper 3'-end formation of mRNA (Fig. 5B). In addition, the 210-nt region could confer 3'-end forming ability on a heterologous transcript when inserted downstream of poly(A) site (M.J.Kim and S.D.Park, unpublished data). Hence, it would be possible that a glucose-starvation signal might direct the 210-nt region to produce abnormally-processed messages which might be the targets of general mRNA degradation, called mRNA surveillance system (35). The 210-nt region might also exert its destabilizing effect regardless of its ability for mRNA 3'-end formation as the second model. The 210-nt region, however, does not contain any motifs or sequences shared with previously identified stability determinants. In these two models, there is a possibility that the 210-nt region may regulate the mRNA stability via a downstream gene. The downstream gene of rrg1+ is in the same orientation as rrg1+, and its translation start codon is located 273 bp from the 210-nt region. However, its transcript level did not show any response to glucose (data not shown). In addition, the deletion constructs of the 3'-flanking region of rrg1+ were inserted into an irrelevant chromosome locus (Fig. 5A and Materials and Methods). Thus, the possibility of a downstream gene effect seems implausible. These two models, however, could explain the decrease in the level of nascent transcripts containing the 210-nt region. Considering that the half-life of rrg1+ mRNA in the presence of glucose is almost 1 h (Fig. 3A), decay of the nascent transcripts is not sufficient to create the rapid decline in RNA level observed in the northern blot analyses (Fig. 1B). Thus, decay of the mature mRNAs should be affected by the 210-nt region, even though they no longer contain the 210-nt region. The only conceivable way would be to adopt other factor(s) to transmit the effect of the 210-nt region to the mature mRNA of rrg1+. For these reasons, the third model would be superior to the previous ones to explain the action model of the 210-nt region. The levels or the activities of the putative factor(s), influencing rrg1+ mRNA stability under the guidance of the 210-nt region, might be regulated by glucose and they appeared to be dependent on protein synthesis (Fig. 4B). A possible candidate for the putative factors could be poly(A)-binding protein. The poly(A)-binding protein in S.cerevisiae, Pab1p, is required for cytoplasmic mRNA degradation processes such as mRNA decapping (36), deadenylation by stimulation of a poly(A) nuclease, PAN (36,37). Recently, Pab1p has been found to be involved in mRNA 3'-end formation to control the poly(A) tail length (38). These imply that Pab1p is a factor attached to mRNA throughout the lifetime of mRNA from 3'-end processing until cytoplasmic degradation. In addition, it is involved in shortening of the poly(A) tail on the message, which controls the mRNA stability by regulating association of RNA with the degradation machinery (39). It would be interesting to find a trans-acting factor for the 210-nt region and investigate its interaction with poly(A)-binding protein in S.pombe for future studies.

Studies are underway to elucidate the exact mechanism of mRNA decay control mediated by the 210-nt region and to find an upstream signaling pathway regulating the rrg1+ expression. The cellular function of Rrg1 protein is under investigation to understand the significance of the glucose-dependent expression of rrg1+.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr M. Yamamoto for providing S.pombe cell strains. We also appreciate the critical reading and comments of Prof. Onyou Hwang and Dr Minkyu Kim. This research was supported by a grant from the Korea Research Foundation (1996-012-D0540). M.J.K., J.B.K. and S.D.P. are supported by a BK21 Research fellowship from the Korean Ministry of Education.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +82 2 880 6689; Fax: +82 2 887 6279; Email: sdpark{at}plaza.snu.ac.kr Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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