Cold-sensitive dominant mutants scn1 and scn2 of Schizosaccharomyces pombe were isolated by their ability to suppress temperature-sensitive cut9-665 defective in an essential subunit (human Apc6/budding yeast Cdc16 ortholog) of anaphase promoting complex/cyclosome (APC/C). APC/C mutants were defective in metaphase/anaphase transition, whereas single scn mutants showed the delay in anaphase spindle elongation at 20°C. The scn mutants lost viability because of chromosome missegregation, and were sensitive to a tubulin poison. To understand the scn phenotypes, mutant genes were identified. Surprisingly, scn1 and scn2 have the same substitution in the anticodon of two different tRNA-Ala (UGC) genes. UGC was altered to UGU so that the binding of the tRNA-Ala to the ACA Thr codon in mRNA became possible. As cut9-665 contained an Ala535Thr substitution, wild-type Cut9 protein was probably produced in scn mutants. Indeed, plasmid carrying tRNA-Ala (UGU) conferred cold-sensitivity to wild-type and suppressed cut9-665 in a dominant fashion. The previously identified scn1+ (renamed as scn3+) turned out to be a high copy suppressor for scn1 and scn2. These are the first tRNA mutants that cause a mitotic defect.
APC (anaphase promoting complex)/cyclosome (hereafter APC/C) is a 20S protein complex that plays a pivotal role in the transition from mitotic metaphase to anaphase in eukaryotic cells by promoting ubiquitin-mediated anaphase proteolysis (King et al., 1995; Sudakin et al., 1995; Yanagida et al., 1999; Peters, 2002). The complex is evolutionarily conserved, present in yeast and human, consists of more than ten different subunits and acts as a ubiquitin ligase (E3) for the polyubiquitination of mitotic cyclins and securin (Funabiki et al., 1996; Yu et al., 1998; Zachariae et al., 1998; Yoon et al., 2002). Fission yeast Cut9 (orthologue of human APC6 and budding yeast Cdc16) is an essential subunit of APC/C (Yamashita et al., 1996; Yamada et al., 1997). Its temperature-sensitive (ts) mutant cut9-665 produces the mitotically hypercondensed chromosomes with the short metaphase spindle and the high Cdc2 kinase activity. Sister chromatid separation did not take place at all in cut9-665 cells at the restrictive temperature (Hirano et al., 1986; Samejima and Yanagida, 1994).
Cold-sensitive (cs) mutations designated scn (suppressors for cut nine) were isolated as extragenic suppressors for the ts phenotype of cut9-665 (Samejima and Yanagida., 1994). Genetic analyses enabled them to be classified into two genetic loci, scn1 and scn2. The cut9 scn1 and cut9 scn2 double mutants produced colonies at 33°C (a restrictive temperature for cut9 mutant) but not at 22°C (a restrictive temperature for scn mutants). Single mutant segregants of scn1 and scn2 obtained by crossing with the wild-type showed the cs phenotype but not the ts phenotype. These single scn1 and scn2 mutants revealed notable properties. First, all of the scn1 and scn2 mutants obtained were dominant: heterozygous diploids showed the cs phenotype. Second, the cellular phenotypes of scn1 and scn2 mutants cultured at 22°C revealed an apparent chromosome segregation defect in late mitosis.
To understand how these dominant cs mutations could suppress the ts phenotype of cut9-665, we attempted to characterize scn mutant phenotypes in detail and to identify mutant genes and the mutation sites. The results presented in this report clearly showed late mitotic defects particularly in spindle elongation. The resulting chromosome segregation defect seemed to be the cause of cell lethality at the restrictive temperature. Synchronous analysis indicated that cell viability remained high in G1, S and G2. Both scn1 and scn2 mutants were hypersensitive to a tubulin inhibitor, thiabendazole (TBZ) (Umesono et al., 1983) at the permissive temperature, which is consistent with the defect in spindle elongation. The destruction of cyclin observed in living cells occurred apparently at a normal timing in scn mutant cells so that the destruction of mitotic cyclin was normal in single scn mutants.
Fine genetic analyses followed by nucleotide sequencing established that the mutated genes in scn1 and scn2 strains were actually the tRNA genes that carried Ala with the anticodon UGC. There are two Ala-tRNA genes with the identical sequence containing anticodon UGC at different map locations in the Schizosaccharomyces pombe genome. These scn1 and scn2 mutants were found to contain single substitutions in the anticodon sequence that led to UGU from UGC so that the amino acid codon in mRNA for this tRNAala would be changed to Thr from Ala. The resulting mischarged Ala for the Thr codon seemed to be the true cause for the ability of scn mutations to suppress the phenotype of cut9-665. The previously identified scn1+ was actually a high copy suppressor for scn1 and scn2 mutations so that it was renamed as scn3+. We will discuss the implication of these unexpected findings on the mitotic defects of dominant tRNA mutations.
Materials and Methods
Strains, media and genetic method
Haploid and diploid fission yeast strains were used. scn1, scn2 and cut9-665 were isolated as described previously (Samejima and Yanagida, 1994). Deletion mutants Δmad2 and Δscn3 were made previously (Samejima and Yanagida, 1994). The culture media used were YPD (1% yeast extract, 2% polypeptone, and 2% glucose) and EMM2 (minimal medium; Mitchison, 1970). The YPD and EMM2 Media containing 1.5% agar were used for plating.
Plasmids and yeast transformation procedure
pSK248 was used as the vector carrying the S. pombe genes. About 500 bp long S. pombe genomic DNA fragments containing only Ala-tRNA(UGC) gene located near the ryh1+ gene (SPAC4C5.02C) were amplified by PCR from the wild-type and scn2-7 mutant, and those PCR products were inserted into pSK248 vector, and were named as pTRNAala.02 or pTRNAala.02(UGC>UGU). Transformation of S. pombe was performed by the lithium method (Ito et al., 1983). To isolate multicopy suppressors of scn2-7 mutant, an S. pombe genomic DNA library containing the Saccharomyces cerevisiae LEU2 gene as the selection marker (a gift from C. Shimoda, Osaka City University, Japan). Plasmids were recovered from Cs+ Leu+ transformants of scn2-7. pTRNAthr.06 and pTRNAthr.10 were respectively constructed by PCR amplification and insertion into pSK248 vector.
Light microscopy and time-lapse imaging
DAPI staining was carried out as previously described (Adachi and Yanagida, 1989). Indirect immunofluorescence microscopy was performed as previously described (Hagan and Hyams, 1988). Anti-tubulin (TAT-1) and anti-Sad1 antibodies were used as primary antibodies (Woods et al., 1989; Hagan and Yanagida, 1995). The secondary antibodies employed were the CY3-tagged anti-mouse antibody and Alexa488-tagged anti-rabbit antibody (Molecular probes). For observing living cells, confocal scanning system CSU21 (Yokogawa) was used. Cells were cultured in the liquid EMM2 media supplemented with leucine on glass base dishes (Iwaki). Images were taken every 15 seconds, 30 seconds or 1 minute. To visualize the SPB movement or the Cdc13 protein degradation in live cells, the pSad1-GFP plasmid or the strain containing GFP-tagged cdc13+ gene were used (Nabeshima et al., 1997; Tatebe and Yanagida, 2000).
The G1 arrest by nitrogen starvation
Nitrogen source starvation was done according to the procedures described previously (Saka and Yanagida, 1993). Cells (7.5×106 cells/ml) were cultured in EMM2 medium at 33°C, washed four times with the EMM2-N media (EMM2 without NH4Cl) and then cultured in the EMM2-N media for 36 hours at 33°C (the cell concentration, 6×107 cells/ml). The FACS analysis confirms that the cells were arrested in the G1 phase. Arrested cells were released into the YPD medium at 20°C. Progression of DNA replication was monitored by FACS, and mitotic progression was monitored by determining the septation index.
Cellular phenotypes of single scn1-17 and scn2-7 mutants
As shown in Fig. 1A, ts cut9-655 mutant defective in the mitotic transition from metaphase to anaphase failed to produce colonies at 33°C, whereas the double mutants cut9-665 scn1-17 and cut9-665 scn2-7 could form colonies at 33°C but not at 22°C. Single scn1 and scn2 mutants obtained as tetrad segregants after crossing with the wild-type strain also showed the cs phenotype. Thus, scn1-17 and scn2-7 were extragenic suppressors for cut9-665 mutant (Samejima and Yanagida, 1994). It was previously shown that all of the isolated scn1 and scn2 were dominant cs mutations.
Wild-type, scn1-17 and scn2-7 grown at 33°C were shifted to 20°C, and their increase in cell number was measured. Approximately two rounds of cell division occurred before the cease of cell number increase in the cultures of scn1 and scn2 (Fig. 1B). In addition, cell elongation was observed at 20°C (Fig. 1C). The average cell length of scn1 mutant cells after 24 hours at 20°C increased from 10.7 μm to 14.1 μm, 32% longer than that of wild-type. Basically the same cell elongation was observed for scn2 mutant cells (data not shown).
A prominent feature of scn1 mutant cells cultured at 20°C and stained by DAPI was the accumulation of cells revealing late mitosis (Fig. 1D). Divided daughter nuclei often displaced from the middle of cell were seen. Chromosomes appeared to be still condensed. These results showed late mitotic defects with some pleiotropic effects in scn1 mutant. Similar results were obtained for scn2 mutant cells (data not shown).
Defects in spindle elongation, nuclear position and cell division
Immunofluorescence microscopy using anti-tubulin antibodies (red) for the spindle and DAPI (blue) for nuclear chromatin staining was done for scn1-17 mutant cells that were cultured at 20°C for 9-12 hours, and shown in Fig. 2A. Cells designated A are those in late anaphase. Divided daughter nuclei were often displaced from the middle of cell (indicated as B). The spindle that was not fully extended was frequently seen in the mutant cells, suggesting that mutant cells might be retarded in late anaphase.
The frequency of late anaphase cells increased from ∼3% in exponentially growing wild-type cells and 5.9% in mutant cells at the permissive temperature to 13.7% in mutant cells after 9 hours at the restrictive temperature (Fig. 2B). In Fig. 2C, quantitative data for those containing the displaced daughter nuclei and those lacking the nucleus are shown. Cells lacking the nucleus increased from a negligible level at the permissive temperature to approximately 20% after 24 hours at 20°C in mutant cells. The displaced nuclei increased to ∼7% in mutant cells, while they were at a negligible level in the wild-type cells. The temporary decrease of late anaphase cells after 3 hours in the wild-type and mutant cells was probably due to the shift to 20°C. The displaced daughter nuclei, mostly connected with the spindle, were frequently seen and seemed to be characteristic of scn mutant cells at 20°C, as the same cellular phenotypes were seen in scn2 mutant cells. These results indicated that sister chromatid separation took place but full segregation of the resulting daughter nuclei failed. Because nuclear positioning was abnormal, cytokinesis would produce two cells, one of which lacked the nucleus. The loss of nucleus was hence another characteristic of scn mutant cells.
The loss of cell viability corresponds to the timing of aberrant mitosis
To examine when scn1 lost the cell viability, mutant cells were first arrested in the G1 phase at the permissive temperature (33°C) by nitrogen source starvation, and then released to the complete YPD medium at 20°C to advance cell cycle progression. The occurrence of DNA replication (Fig. 3A) was slightly delayed in scn1 mutant cells. The duration of the S phase is indicated by the vertical bar. In this S phase period, however, the cell viability was completely maintained. The loss of viability occurred after 15 hours in scn1 mutant cells. This decrease corresponded to the passage through the first mitosis and the time when the improper chromosome segregation began to be seen in the scn1 mutants (Fig. 3B).
In 72% of the scn1 mutant cells with the septum, sister chromatids were not fully apart. These results showed that the late step of nuclear division in combination with septation and cytokinesis was not properly coordinated in scn1 mutants. We concluded that scn1 mutant cells did not lose viability until mitosis, but they had some defects in other events, including the delay in DNA replication. The cause of cell death seemed to be the occurrence of abnormal chromosome segregation followed by cell division.
Normal timing of cyclin destruction and abnormal spindle dynamics
To determine the defects that would lead to improper chromosome segregation, mitotic phenotypes of scn1 and scn2 were further examined. No significant increase of the spindle index was observed after transferring the cells to 20°C, suggesting that scn1 did not arrest in metaphase (Fig. 4A). We then examined the timing of cyclin destruction using the strain of scn1-17 carrying the chromosomally integrated Cdc13-GFP gene (Tatebe and Yanagida, 2000). Hoechst H33342 was used for staining DNA in order to distinguish anaphase cells. The GFP signal disappeared in all of the anaphase cells (indicated by the arrows in Fig. 4B) observed at the restrictive temperature (20°C). The cyclin destruction thus seemed to be normal in the scn1-17 mutant.
To monitor the time course of spindle extension in living cells, scn1-17 cells carrying multicopy plasmid expressing the GFP-tagged Sad1 protein were used. Sad1 is an essential SPB (spindle pole body) component and, when mildly overproduced, distributed in the nuclear envelope (Nabeshima et al., 1997). We found that anaphase spindle elongation was considerably delayed and often compromised in scn1 mutant cells (Fig. 4C,D). Furthermore, the nuclear envelope breakdown did not seem to take place in mutant cells in the late anaphase stage (Fig. 4C, right panel). The nuclear envelope revealed complex movements during late anaphase, suggesting that the resolution of the nuclear envelope into the two daughter nuclei might be aberrant in scn1 mutant cells. The loss of cell viability was possibly due to the cytokinesis-induced breakage of the thin nuclear envelope channel connecting the two daughter nuclei. The rate of spindle extension in anaphase B was greatly reduced in scn1 mutants. The SPBs did not reach the opposite poles even after 102 minutes (Fig. 4D).
scn2 is alanine-tRNA(UGC) mutant
In the previous study, the scn2+ gene was not isolated because the cs scn2 mutation was dominant so that plasmids introduced into mutant cells by transformation failed to rescue the cs phenotype (Samejima and Yanagida, 1994). To determine the precise map location in the S. pombe genome, we conducted a series of genetic mapping by crossing scn2-7 with strains carrying various marker genes. Ultimately, it was found that scn2-7 was tightly linked to the ryh1 locus (PD:NPD:TT=56:0:0). The ryh1+ gene (SPAC4C5.02C) in scn2 mutant was isolated by PCR, but no nucleotide change was found in the ryh1+ gene sequence obtained. Then PCR amplification was made for the ∼2 kb long genomic DNA around the ryh1+ gene in scn2-7 mutant (corresponding to cosmid c4C5 2425-4520). The resulting PCR product was sequenced (Fig. 5A). Surprisingly, a single nucleotide substitution was found within the adjacently located Ala-tRNA(anticodon UGC) gene sequence (SpaTRNAala.02). In scn2-7 mutant cells, the anticodon UGC of this tRNA gene was substituted to UGU. It was further found that scn2-27, another allele of scn2, contained the identical anticodon substitution, and otherwise the same sequence to the wild-type. The scn2 mutant Ala-tRNA(UGC>UGU) should mis-interact with the Thr codon in mRNA instead of Ala codon (Fig. 5B).
To confirm that the Ala-tRNA(UGC>UGU) mutant gene was the real cause for the phenotype seen in scn2 mutant cells, the ∼500 bp long DNA fragment containing the Ala-tRNA(UGC>UGU) mutant gene was cloned into the vector pSK248. Resulting plasmid (pTRNAala.02 (UGC>UGU)) was transformed into the wild-type or cut9-665 mutant. Plasmid containing the Ala-tRNA(UGC>UGU) gene suppressed the ts phenotype of cut9-665 (Fig. 5C). In addition, the same plasmid conferred the cold-sensitivity if introduced into wild-type cells. Control plasmid containing the wild-type Ala-tRNA(UGC) (pTRNAala.02) neither suppressed the ts phenotype of cut9-665 mutant nor caused any cs effect to wild-type cells (Fig. 5C). The wild-type cells carrying the mutant Ala-tRNA(UGC>UGU) plasmid showed the cellular phenotypes similar to that of scn1 and scn2 mutant at 20°C (Fig. 5D). We therefore concluded that scn2 was an anticodon allele of the Ala-tRNA(UGC).02 gene.
The strain scn1 is also Ala-tRNA(UGC) mutant
The genome contains generally multiple copies of the tRNA genes for each amino acid. If there are many copies for the Ala-tRNA(UGC) gene in the S. pombe genome, how does a substitution mutation in one copy strongly affect cell multiplication in a dominant fashion? We examined the genomic tRNA database of S. pombe (http://rna.wustl.edu/GtRDB/Sp/) to find out how many Ala-tRNA(UGC) genes existed in the S. pombe genome and found that S. pombe has only two copies of the Ala-tRNA(UGC) gene (there are nine other Ala-tRNA genes but they have different anticodons). Furthermore, we noticed that the second Ala-tRNA(UGC) gene was present only 16.7 kb apart from the scn1+ (SPAC688.13) gene previously identified (Samejima and Yanagida, 1994). This gene was previously isolated by its ability to suppress the cs phenotype of scn1 mutant cells. The previous study showed that the LEU2 marker integrated with the SPAC688.13 gene was tightly linked to the cs scn1 locus (PD:TT:NPD=16:0:0). This was the reason that the cloned gene was assigned to be the actual scn1+ gene. However, we failed to find any nucleotide change in the SPAC688.13 gene cloned from the scn1 mutant genomic DNAs (corresponding to cosmid c688 27458-28872) so that the gene assignment could be false, although we confirmed that plasmid carrying the SPAC688.13 gene could suppress the cs phenotype of both scn1 and scn2 mutant cells. By searching the conserved domain database (NCBI) we found that the gene product of SPAC688.13 has a TatD-related nuclease domain (pfam01026) (Holm and Sander, 1997; Wexler et al., 2000).
The second copy of the Ala-tRNA(UGC) (SpaTRNAala.03) gene 16.7 kb apart from the SPAC688.13 gene has the same sequence to the first one (Fig. 5E). If its expression level is similar to that of scn2 Ala-tRNA(UGC), the same anticodon mutation may lead to the same phenotype as that of scn2. Indeed, a single substitution was found in the second Ala-tRNA(UGC) gene in scn1 mutant cells (Fig. 5E). The anticodon UGC was again changed to UGU, in an identical manner to the scn2 mutant. The previous gene assignment is therefore clearly false. We propose to call the former scn1+ as scn3+. It acts as a high copy suppressor for both scn1 and scn2 mutants. These results established that both scn1 and scn2 were the same Ala-tRNA(UGC) anticodon mutants. We found that scn1 and scn2 double mutants were synthetic lethal. This was easily explained as the S. pombe genome has only two Ala-tRNAs with this anticodon UGC and therefore the GCU codon would not be properly translated at all in the double scn1 scn2 mutant cells.
Hypersensitivity to thiabendazole and cycloheximide
As scn1 and scn2 mutants were defective in protein synthesis and also in the completion of spindle elongation, we examined whether these tRNA mutants were sensitive to a protein synthesis inhibitor cycloheximide (CYH) and a tubulin poison thiabendazole (TBZ). As shown in Fig. 6A,B, they were found to be hypersensitive to these drugs. At the permissive temperature (26-30°C), scn1-17 and scn2-7 mutants were hypersensitive to the presence of 5-20 μg/ml CYH. They were also sensitive to TBZ. In the presence of 10-15 μg/ml TBZ, scn1-17 and scn2-7 grew poorly, whereas wild-type cells grew nearly normally. These are the first RNA mutants sensitive to tubulin inhibitor.
Scn3 and tRNA-Thr, multicopy suppressors of scn1 and scn2 mutants
An S. pombe genomic DNA library was used to screen for multicopy plasmids that could suppress the cs phenotype of scn2 mutant. Plasmids carrying three different genomic DNAs were isolated. One of them contained the scn3+ gene (SPAC688.13, described above). Its product showed sequence similarity to Escherichia coli TatD, S. cerevisae Ymr262p and Candida albicans Orf6.8887p, containing a nuclease motif, and E. coli TatD shows DNase activity in vitro (Holm and Sander, 1997; Wexler et al., 2000). Plasmid expressing only the open reading frame of scn3+ with the inducible promoter nmt1 in the presence of thiamine also suppressed both scn1 and scn2 mutants (Fig. 6C). This putative nuclease gene scn3+ was nonessential for cell viability as gene-disrupted cells grew normally but, unexpectedly, scn3+-disrupted cells were resistant to cycloheximide (Fig. 6A). If Scn3 had the ribonuclease activity, the high dosage Scn3 might suppress scn1 and scn2 mutant cells by cleaving the mutated Ala-tRNAs.
The remaining two high copy suppressor plasmids contained the Thr-tRNA(UGU) gene (SpbTRNAthr.06 and SpaTRNAthr.10). This Thr-tRNA(UGU) might be able to suppress scn2 by competing against the mischarged mutant tRNA. The plasmids could also rescue the scn1 mutant (pTRNAthr.06 and pTRNAthr.10 in Fig. 6D). Suppression of scn1 and scn2 by Thr-tRNA(UGU) expression indicates that the phenotypes of scn1 and scn2 are due to misincorporation of Ala to the position of Thr during translation, which could make mal-functional proteins.
The delay of spindle elongation was independent of Mad2
To determine whether the spindle elongation delay in scn mutant cells was due to the activation of spindle checkpoint, the double mutant scn1-17 Δmad2 was constructed. Mad2 is essential for establishing the spindle checkpoint (Li and Murray, 1991). It was cultured at 20°C for 9 hours, and cells stained by antibodies against tubulin (red) and Sad1 (green) were observed with DAPI (blue) as counterstaining for DNA (Fig. 7A). The appearances and frequencies of late anaphase cells were not affected in scn1 Δmad2. To confirm that the delay in late anaphase was independent of Mad2, the spindle length in the double mutant cells was measured by the pole-to-pole distance in mitotic cells of the wild-type, scn1 and scn1 Δmad2 at 20°C for 9 hours (Fig. 7B). The filled arrowheads indicate the position of the wild-type metaphase spindle length. The frequencies of anaphase cells and the size of the anaphase spindle were not affected in the double mutant. Hence scn1 Δmad2 double mutant showed the same spindle elongation delay as the single scn1 mutant cells, suggesting that the spindle elongation delay in scn1 was not caused by the activation of Mad2-dependent spindle checkpoint.
In this study we show that scn1 and scn2, extragenic suppressors for cut9-665, are substitution mutants in the two tRNA genes, SpaTRNAala.03 and SpaTRNAala.02, respectively, both of which contain the UGC anticodon. The S. pombe genome has a total of 11 tRNA-Ala genes but only two contain the UGC anticodon. Nucleotide sequences of SpaTRNAala.03 and SpaTRNAala.02 were identical, making the same tRNA. The scn1 and scn2 mutations, both of which occurred in the anticodon sequence in these two tRNA genes, were also identical (from UGC to UGU), so that the mutant tRNAs should be identical. Although we have no direct evidence, it is most likely that the resulting altered tRNA-Ala made in scn1 and scn2 mutants should bind to the ACA (Thr) codons in mRNA and misincorporate Ala into proteins at the site of protein synthesis on the ribosome. Hydrophilic Thr and hydrophobic Ala are distinct in terms of their properties, including the ability to be phosphorylated. It is hence possible that many proteins made in scn1 and scn2 mutants have altered properties from the wild-type cells, and some may even become poisonous. Note, however, that one wild-type Ala-tRNA(UGC) gene still exists in mutant cells, producing the normal tRNA-Ala (UGC), and that the activity of aminoacyl-tRNA synthetase often requires the correct anticodon sequence (Ibba et al., 1997; Ibba and Söll, 2000) so that the level of malfunctionally altered proteins made in scn1 and scn2 mutants may not be high. Indeed, these mutants normally grew at the permissive temperature. It is hence consistent with the fact that the double mutant scn1 and scn2 is synthetic lethal.
It was surprising that the scn1 and scn2 mutations in the Ala-tRNA (UGC) genes affect late mitosis rather strongly and that the tRNA mutation can cause improper chromosome segregation and lead to aneuploidy in a dominant fashion. As the mutant tRNA-Ala (UGU) introduced by plasmid into the wild-type cells was also found to cause similar mitotic phenotypes in a dominant manner, the mitotic defects seemed to be a consistent feature by this anticodon mutation. If chromosome missegregation has any implication in evolution through altering the genome organization, the anticodon change in the Ala-tRNA(UGC) gene described in this paper might have contributed to evolution. These tRNA mutants were sensitive to a tubulin inhibitor so that microtubule organization seemed to be somewhat impaired in mutant cells.
Other aspects that should be considered for interpreting the results is that the phenotypes seen in scn1 and scn2 mutants at 20°C might be produced by a kind of cell cycle checkpoint (Hartwell and Weinert, 1989). The accumulation of cells delayed in late mitosis might be caused by a checkpoint control that is hitherto unknown. The delay was independent of the presence of Mad2. In an earlier study by Mitchison and his colleagues protein synthesis was shown to be reduced during mitosis and restored after mitosis (Creanor and Mitchison, 1984). In scn1 and scn2 mutant cells, the protein synthesis checkpoint that monitored the presence of mutant tRNA might block cell cycle progression and delay the re-initiation of protein synthesis during late mitosis in scn1 and scn2 mutant cells.
The mutation in cut9-665 was reported to be caused by the nucleotide change from G to A in the 535th codon (Samejima and Yanagida, 1994) that caused the amino acid change from Ala to Thr; the altered codon could be bound to the mutant tRNA-Ala (UGU). As the Ala residue is expected to be incorporated into the 535th position of Cut9 in scn1 and scn2 mutant cells, the cause of suppression for cut9-665 is now resolved. This interpretation is consistent with the failure of suppression for cut9 null mutant cells and the failure of suppression for other APC/C mutant nuc2-663 in the background of scn1 and scn2 mutations (Hirano et al., 1988; Samejima and Yanagida, 1994). This mode of suppression mediated by tRNA mutations is well known in prokaryotes as the missense suppression, but there have been only a few reports in eukaryotes (Su et al., 1990; Chiu and Morris, 1997; Kim et al., 1999). Dominant tRNA mutation has been reported for the case of PRP2 (see below).
The mitotic phenotypes of scn1 and scn2 at 20°C were somewhat reminiscent to that seen in α1-tubulin mutant, nda2-KM52 (Toda et al., 1983). A hypothesis that tubulin functions in scn1 and scn2 mutants may be impaired due to misincorporation of Ala at the Thr positions might be consistent with the result that scn1 and scn2 mutants were hypersensitive to TBZ. To estimate how many Thr residues may be affected, we examined the ACA codon usage in the S. pombe tubulins. The α1-tubulin, Nda2, has three ACA codons in 455 residues, whereas the α2-tubulin, Atb2/Tub1 has two ACA codons in 449 residues (Toda et al., 1984). The β-tubulin, Nda3, has three ACA codons in 448 residues (Hiraoka et al., 1984). Because the wild-type tRNA-Ala (UGC) is present, the actual misincorporation should be lower than the values indicated here. According to the codon usage database for S. pombe (http://www.kazusa.or.jp/codon/), the percentage of ACA codon in all codons is 1.44%, indicating that 6.4 ACA codons are used in a 450 amino acid protein on average. This value is higher than those found in tubulins.
In budding yeast, a dominant cs mutation in the anticodon sequence has been reported (Kim et al., 1999). The PRP2 gene encodes an RNA-dependent ATPase that activates spliceosomes (Kim et al., 1992). A dominant, allele-specific suppressor of the ts prp2-1 mutation was identified to be the mutation in the elongation methionine tRNA gene EMT1. The EMT1-201 mutant seemed to suppress prp2-1 by alleviating the splicing block. EMT1-201 mutant displayed defects of pre-mRNA splicing and cold-sensitive growth. The mutation in EMT1-201 exists in the anticodon, changing CAT to CAG, which presumably allowed EMT1-201 suppressor tRNA to recognize CUG Leu codons instead of AUG Met codons. Curiously, the prp2-1 allele has a point mutation that changes Gly to Asp, indicating that EMT1-201 is not a classical missense suppressor. The scn1 and scn2 are the first examples of classical missense suppressors in S. pombe, although various nonsense suppressor tRNAs of S. pombe have been reported by Söll and his colleagues (Kohli et al., 1979; Wetzel et al., 1979). We did not examine whether scn1 and scn2 had splicing defects, but it is possible that scn1 and scn2 have slight defects in various cellular activities. However, the spindle elongation defects appear to be a most strong phenotype at low temperature and crucial for maintaining the cell viability of scn1 and scn2.
The scn3+ gene was cloned by transformation followed by tetrad dissection. It was falsely assigned as the scn1+ gene in the previous report – it was actually a high copy suppressor present near the true scn1+ tRNA gene. The ability of scn3+ to suppress both scn1 and scn2 mutations suggested that Scn3 has a role in downregulating dominant mutant tRNA. Recently, it was noticed in the conserved domain database that Scn3 has a nuclease motif that is even conserved in bacteria (E. coli). We postulate that Scn3 might preferentially cleave scn1 and scn2 mutant tRNAs at the restrictive temperature. Note that the level of the Scn3 transcript is stress inducible and increased at the high temperature (Chen et al., 2003).
In summary, the present report is the first to relate tRNA mutations to mitosis and regulation of anaphase. Mutations in the tRNA anticodon should produce a broad effect, but unexpectedly our phenotypic characterizations strongly indicated that mitotic defect was the principal outcome in scn1 and scn2 tRNA mutant cells. Translational regulation during mitosis has been poorly understood, and the present study suggests that the late anaphase step may be dependent on the quality control of protein synthesis. It is of considerable interest whether the same mutation causes similar cellular effects on other organisms.
We are greatly indebted to Itaru Samejima who contributed at an early stage of the present study. We thank Kazuki Kumada and Jun Morishita for cloning the scn1+ and scn2+ gene. This work was supported by the COE Special Promotion grant from the Ministry of Education, Culture, Sports, Science and Technology.