https://orcid.org/0000-0002-1050-3934
Figures
Abstract
Clb1 and Clb2 are functionally redundant B-type cyclins, and the clb1Δ clb2Δ double mutant is lethal. In normal mitotic growth, Clb2 plays the central role in the G2-M progression. We previously demonstrated that the RNA-binding protein Puf5 positively regulates CLB1 expression by downregulating expression of the repressor Ixr1. The decreased expression of CLB1 by the puf5Δ mutation caused a severe growth defect of the puf5Δ clb2Δ double mutant. On the contrary, CLB2 expression was unchanged between wild-type strain and puf5Δ mutant in unsynchronized cultures, and the puf5Δ clb1Δ double mutant did not show growth retardation. Therefore, we assumed that CLB1 is the main target of Puf5 in the previous study. However, considering that CLB1 and CLB2 reportedly undergo a similar expression pattern during the cell cycle, we re-examined CLB2 expression in the puf5Δ mutant in cell cycle-synchronized cultures and found that CLB2 expression was decreased in the puf5Δ mutant strain. Deletion of IXR1 restored the decreased expression of CLB2 caused by the puf5Δ mutation. Moreover, we clarified that the decreased expression of CLB2 caused by the puf5Δ mutation resulted in the growth defect in the S-phase cyclin deficient condition: the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant grew worse than clb1Δ clb5Δ clb6Δ triple mutant, and the slow growth of the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant was suppressed by CLB2 overexpression. Moreover, the ixr1Δ mutation is known to be synthetically lethal with deletion of the DUN1 gene encoding the checkpoint kinase. We found that the clb2Δ mutation restored the lethality of ixr1Δ dun1Δ double mutant. Our results suggest that Puf5 and Ixr1 regulate the cell cycle-specific expression of both CLB1 and CLB2, that Clb5 and Clb6 have overlapping roles with Clb1 and Clb2, and that the regulation of CLB1 and CLB2 expression by Puf5 and Ixr1 is related to the function of Dun1 kinase.
Citation: Sato M, Rana V, Suda Y, Mizuno T, Irie K (2025) The RNA-binding protein Puf5 and the HMGB protein Ixr1 regulate cell cycle-specific expression of CLB1 and CLB2 in Saccharomyces cerevisiae. PLoS ONE 20(2): e0316433. https://doi.org/10.1371/journal.pone.0316433
Editor: Reiko Sugiura, Kindai University: Kinki Daigaku, JAPAN
Received: September 11, 2024; Accepted: December 10, 2024; Published: February 3, 2025
Copyright: © 2025 Sato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This research was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 22K06074 (to KI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The eukaryotic cell cycle is a stringently regulated biological process that controls cellular growth and division, and maintains genomic integrity. The precise timing and coordination of the cell cycle are controlled by the periodic activity of the complex of cyclin-dependent kinases (Cdks) and cyclins [1]. In Saccharomyces cerevisiae, commonly known as the budding yeast, the cell cycle is primarily driven by a single Cdk called Cdc28 [1]. Nine periodically expressed cyclin proteins, broadly categorized into G1 cyclins and B-type cyclins, activate Cdc28 at different phases. The G1 cyclins include Cln1, Cln2, and Cln3, which are essential for surpassing START and transitioning the cell cycle from G1 to S-phase. In contrast, the B-type cyclins encompass six different cyclins viz., Clb1, Clb2, Clb3, Clb4, Clb5, and Clb6. These cyclins are further grouped into three unique pairs according to homology and transcriptional regulation, Clb1/Clb2, Clb3/Clb4, and Clb5/Clb6. The transcriptional induction of these pairs occurs in three distinct waves at different phases of the cell cycle [1–3]. For instance, CLB5/CLB6 peaks just before the initiation of the cell cycle or S-phase and promotes efficient initiation of DNA replication. CLB3/CLB4 peaks when the DNA replication is completed and promotes mitotic spindle formation at the G2/M-phase transition. CLB1/CLB2 peaks during the transition from G2 to M-phase and are essential for mitotic spindle elongation, spindle pole body separation, and mitotic exit [4–7]. The functional variation of the cyclins is attributed to the timing of the accumulation of cyclins. However, previous studies have shown plasticity and functional overlap amongst the cyclins expressed in different stages of the cell cycle. For example, in the absence of S-phase cyclins Clb5 and Clb6, other cyclins are able to facilitate origin firing and DNA replication, albeit with a delay [8,9]. In contrast, mitotic exit can only be achieved by Clb2, not by Clb5 [7,8]. Therefore, B-type cyclins somehow show functional redundancy, but some crucial functions seem to be specific.
Among the B-type cyclins, the Clb2 cyclin is the major cyclin and is critical for mitotic entry [4,5]. During the G2/M-phase, the transcription of G2/M cluster genes including CLB1 and CLB2 is induced by the transcriptional activator Mcm1-Fkh2-Ndd1 complex [2,10–13]. Interestingly, Clb2 acts in a positive feedback loop; the Clb2/Cdc28 kinase complex phosphorylates Fkh2 and Ndd1 during the G2 phase of the cell cycle, further promoting assembly and activation of the Mcm1-Fkh2-Ndd1 complex [11,13,14]. In addition, Clb2 also cooperates with the other G2/M cluster genes to inhibit Swi4, a component of the SBF transcription complex, and negatively regulates the expression of the SBF-regulated genes, including the G1 cyclins CLN1 and CLN2 [14]. This further reiterates the significance of G2/M cyclins in the regulation of not only mitosis but also other stages of the cell cycle. Thus, considering the importance of G2/M cyclins, elucidating G2/M regulators could further help in the understanding of the cell cycle progression.
RNA-binding proteins play an integral part of the transcriptional and post-transcriptional control machinery, thereby providing additional control over the gene expression. In the context of cell cycle regulation, RNA-binding proteins have emerged as potential regulators of cyclins and other cell cycle-related transcripts. Puf5 (also known as Mpt5 or Htr1) is one such RNA-binding protein that has been reported to be essential for the G2/M phase transition of the cell cycle at high temperatures [15,16]. Puf5 belongs to the PUF (Pumilio and FBF) family, which is one of the highly evolutionary conserved families of RNA-binding proteins. Puf5 binds to the short motifs in the 3’UTR of an mRNA via its Pumilio homology domain and regulates the expression, stability, localization, and efficiency of translation of the bound transcript [17–19], contributing to cell growth by regulating diverse phenomena including cell wall integrity [20,21], maintenance of longevity [22], and mating type switching [23]. Puf5 has been reported to bind to more than 1000 mRNAs, which constitute approximately 16% of the yeast transcriptome [24], but physiologically important targets of Puf5 have not been elucidated well.
Recently, we reported that Puf5 positively regulates the expression of CLB1 via the HMGB (High Mobility Group Box B) protein Ixr1 specifically in G2/M-phase. Mechanistically, the binding of Puf5 to the 3’UTR of IXR1 mRNA suppresses its expression, which subsequently increases the expression of CLB1 [25]. In this study, we further elaborated on the role of Puf5-Ixr1 in the regulation of cell cycle and cell growth. We found that under cell cycle-synchronized conditions, Puf5 positively regulates the expression of not only CLB1 but also CLB2. Ixr1 also acts as a negative regulator of the CLB2 expression. Moreover, we found that the deletion of Puf5 causes a synthetic growth defect with clb5Δ mutation and the clb5Δ clb6Δ double mutation, which resulted from a decreased expression of CLB2 caused by the puf5Δ mutation. We also found that Clb2 overexpression was able to compensate for the absence of G2/M-phase and S-phase cyclins. In addition, the proper level of CLB2 expression under the control by Ixr1 was necessary for cell growth under the DNA-damage uninducible dun1Δ mutation condition. Altogether, our study suggests that Puf5 and Ixr1 maintain appropriate expression of CLB1 and CLB2, thereby contributing to the specific utilization of Clb1/2 and Clb5/6 throughout the cell cycle.
Results
Expression of not only CLB1 but also CLB2 was decreased in the puf5Δ mutant
CLB1 and CLB2 are functionally redundant B-type cyclins, and clb1Δ clb2Δ double mutant is lethal. In normal mitotic growth, Clb2 plays the central role for the G2-M progression [4,5]. In our previous paper [25], we showed that Puf5 positively regulates CLB1 expression by binding to IXR1 mRNA and negatively regulating its expression. CLB1 expression was reduced in the puf5Δ mutant compared to the wild-type strain (Fig 1A). Referring to the data that the puf5Δ single mutant grew similarly to the wild-type strain (Fig 1B), this regulation by Puf5 solely does not have a phenotypic effect. However, when Clb2 was absent, the puf5Δ mutation caused a severe growth defect (Fig 1B, puf5Δ clb2Δ), indicating that Puf5 ensures CLB1 expression in the case where the function of Clb2 is lost.
(A) The mRNA levels of CLB1 and CLB2 and in the wild-type strain and the puf5Δ mutant. The cells were cultured in a YPD medium at 28°C until the log phase. The CLB mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the SCR1 reference gene. The data shows the mean ± SE (n = 3) of the fold change of CLB1 and CLB2 mRNA levels relative to the mRNA level in the wild-type strain. **P < 0.01 indicates statistical significance, and NS does no significant change. (B) The tetrad analysis of the strains that are heterozygous for the alleles of PUF5, CLB1, and CLB2. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days. The strains encircled in the wide frame show growth defect.
On the contrary, although CLB1 and CLB2 are under the same expressional control machinery, CLB2 expression was unchanged between wild-type strain and the puf5Δ mutant in unsynchronized cultures (Fig 1A). In addition, the puf5Δ clb1Δ double mutant grew similarly to the wild-type strain and the puf5Δ single mutant (Fig 1B). Although this result suggests that Puf5 is involved in the regulation of CLB1 but not CLB2 expression, it seems curious that regulation by Puf5 differs between the same cluster genes, CLB1 and CLB2. Therefore, we re-examined CLB2 expression in the puf5Δ mutant in cell cycle-synchronized cultures. In this experiment, we first arrested the cell cycle in the G1-phase using α-factor, then released, and collected samples every 10 minutes. The bar1Δ mutation background was utilized for inhibiting degradation of α-factor. The RNA levels of RNR1 and SIC1 were examined as cell cycle markers of S-phase and late M-phase, respectively. In the bar1Δ mutant, RNR1 expression was highest at 40 minutes (Fig 2A), and SIC1 expression was peaked at 80 minutes (Fig 2B). Regarding the bar1Δ puf5Δ double mutant, RNR1 expression was induced the most at 50 minutes (Fig 2A), and SIC1 at 110 minutes (Fig 2B), implying that cell cycle was delayed in the bar1Δ puf5Δ double mutant, especially in the G2-phase. Consistent with the data shown in Fig 1A, CLB1 expression was decreased in the bar1Δ puf5Δ double mutant compared to the bar1Δ mutant (Fig 2C). Interestingly, in cell cycle-synchronized cultures, a decrease in CLB2 expression was observed in the puf5Δ mutant (Fig 2D). However, the decrease in CLB2 expression in the puf5Δ mutant strain was milder than the decrease in CLB1 expression (Fig 2C and 2D).
(A-D) The cell cycle-dependent mRNA levels of RNR1, SIC1, CLB1, and CLB2 in the synchronized bar1Δ cell (black circle) and bar1Δ puf5Δ mutant (grey square). The levels of RNR1 mRNA (A), SIC1 mRNA (B), CLB1 mRNA (C), and CLB2 mRNA (D) were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the SCR1 reference gene. The vertical axis shows the fold change of mRNA level relative to that in the bar1Δ 0 min sample, and the horizontal axis shows the time after release from G1-phase. This experiment was performed repeatedly (n = 2), and the representative data is shown.
Next, we examined whether these decreases in the mRNA levels are also reflected in the protein levels. In this experiment, YCplac33-CLB1-3HA or YCplac33-CLB2-3HA plasmid was transformed into the bar1Δ mutant and the bar1Δ puf5Δ double mutant, and cell cycle-specific protein levels of Clb1-3HA and Clb2-3HA were investigated. Although the basal protein levels of Clb1-3HA were quite low, the levels were decreased in the bar1Δ puf5Δ double mutant compared to the control bar1Δ mutant at 60, 80, and 100 minutes (Fig 3A). The Clb2-HA protein levels were also lower in the bar1Δ puf5Δ double mutant than the bar1Δ mutant at 40 minutes, but, at 60, 80, 100 minutes, the Clb2-HA protein levels were similar between in the bar1Δ puf5Δ double mutant and the bar1Δ mutant (Fig 3B). The extent of the decrease in Clb2 protein levels was much milder than that of CLB2 mRNA levels (Figs 2D and 3B), but it seems certain that at least the induction of Clb2 protein expression was delayed in the puf5Δ mutant. The reason why the decrease in Clb2 protein levels was so much milder than the decrease in CLB2 mRNA levels is not clear, but may be due to differences in experimental conditions, or there may be another mechanism to refill protein levels. From these results, even though the effect on the Clb2 protein level was much weaker than that on the Clb1 protein level, it is suggested that Puf5 positively regulates the cell cycle specific expression of CLB2 at both mRNA and protein levels in addition to CLB1.
(A, B) The cell cycle-dependent Clb1 (A) and Clb2 (B) protein levels. The bar1Δ mutant and bar1Δ puf5Δ double mutant strains harboring YCplac33-CLB1-3HA-CLB1 3´UTR or YCplac33-CLB2-3HA-CLB2 3´UTR plasmid were synchronized by α-factor-induced G1 arrest. After releasing, samples were collected every 20 minutes, and proteins were extracted and immunoblotted with anti-HA antibody. This experiment was repeatedly performed (n = 2), and the representative blot image is presented. The band labeled as (*) presents non-specific band of anti-HA antibody. The total protein abundance loaded was confirmed to be at the same level by Ponceau staining. The graphs show the fold change of the density of the Clb1-3HA or Clb2-3HA bands relative to that in the bar1Δ 60 minutes.
Thus, the findings raise a key question whether the positive regulation of B-type cyclin genes by Puf5 is specific to the G2/M cyclin, CLB1 and CLB2. To clarify the effect of Puf5 on other B-type cyclin genes, we additionally examined the expression of CLB3, CLB4, CLB5 and CLB6. The CLB3 and CLB4 expression remained unchanged between the bar1Δ mutant and the bar1Δ puf5Δ double mutant in the synchronous culture (S1A and S1B Fig). Although the expression of CLB5 and CLB6 was induced more slowly and at a lower extent in the bar1Δ puf5Δ double mutant than in the bar1Δ mutant, the difference in their expression was far milder than that in CLB1 and CLB2 expression (Figs 2C and 2D, S1C and S1D). Thus, Puf5 appears to positively regulate CLB1 and CLB2 most significantly among B-type cyclin genes.
Puf5 regulates CLB2 expression via Ixr1
Puf5 is found to be involved in the regulation of CLB2 expression as well as CLB1 expression. We have reported that Ixr1, a HMGB protein functioning as a transcriptional repressor, is involved in the regulation of the expression of CLB1 [25]. Therefore, we examined whether Ixr1 also contributes to the regulation of CLB2 expression. As a result, CLB2 expression, as well as CLB1 expression, was increased in the ixr1Δ mutant compared to wild-type strain even in the asynchronous culture (Fig 4A and 4B). Moreover, the expression levels of CLB1 and CLB2 were higher in the puf5Δ ixr1Δ double mutant than in the puf5Δ single mutant (Fig 4A and 4B). These results indicate that Ixr1 also mediates the regulation of CLB2 expression by Puf5. Next, we also examined cell cycle-specific expression of CLB2 in the ixr1Δ mutant. When culturing three strains, the bar1Δ mutant, the bar1Δ puf5Δ double mutant, and the bar1Δ puf5Δ ixr1Δ triple mutant, the induction of cell phase markers, RNR1 and SIC1, was delayed in the bar1Δ puf5Δ double mutant compared to the bar1Δ mutant, and this delay was recovered by the additional ixr1Δ mutation (Fig 5A and 5B). As reported in our previous paper [25], the expression of CLB1 was decreased in the bar1Δ puf5Δ double mutant and restored in the bar1Δ puf5Δ ixr1Δ triple mutant strain (Fig 5C). Regarding the CLB2 expression, the RNA level was decreased in the bar1Δ puf5Δ double mutant as shown in Fig 2D. When additionally deleted IXR1, the basal expression of CLB2 was increased (Fig 5D). These results suggest that Ixr1 functions downstream of Puf5 and regulates the CLB1 and CLB2 expression.
(A, B) The mRNA levels of CLB1 (A) and CLB2 (B) in the wild-type strain, the ixr1Δ mutant, the puf5Δ mutant, and the puf5Δ ixr1Δ mutant. The cells were cultured in a YPD medium at 28°C until the log phase. The CLB mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the SCR1 reference gene. The data shows the mean ± SE (n = 3) of the fold change of CLB1 mRNA (A) and CLB2 mRNA (B) relative to the mRNA level in the wild-type strain. *P < 0.05, **P < 0.01 as determined by Tukey’s test. NS indicates no significant change.
(A-D) The cell cycle-dependent mRNA levels of RNR1, SIC1, CLB1, and CLB2 in the synchronized bar1Δ cell (black circle), bar1Δ puf5Δ mutant (red square) and bar1Δ puf5Δ ixr1Δ (blue triangle). The levels of an S-phase marker RNR1 mRNA (A), a late M-phase marker SIC1 mRNA (B), CLB1 mRNA (C) and CLB2 mRNA (D) were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the SCR1 reference gene. The vertical axis shows the fold change of mRNA level relative to that in the bar1Δ 0 min sample, and the horizontal axis shows the time after release from G1-phase. These experiments were repeated (n = 2), and the representative data is presented.
Additionally, we investigated the cell cycle-dependent expression of CLB2 in the bar1Δ ixr1Δ double mutant. In this experiment, samples were collected every 20 minutes after the cell cycle blocking and releasing. The S-phase marker RNR1 expressed at most at the same time points in the bar1Δ ixr1Δ double mutant as the bar1Δ mutant at 20 minutes and 80 minutes, but the peaked expression of the second cell cycle was lower in the bar1Δ ixr1Δ double mutant (Fig 6A). The late M-phase marker SIC1 showed a similar expressional pattern between the two strains examined (Fig 6B). As for CLB1, its expression was mildly higher in the bar1Δ ixr1Δ double mutant than in the bar1Δ mutant after starting to be induced at 20 minutes (Fig 6C). As for CLB2, the basal expression of CLB2 at 0 minute was approximately 6-times higher in the bar1Δ ixr1Δ double mutant than in the bar1Δ mutant (Fig 6D). The CLB2 expression peaked at 60 minutes in both strains, and the maximum induction level did not show a significant change between the two strains (Fig 6D). Altogether, although it is suggested that Ixr1 regulates not only CLB1 but also CLB2 expression in a downstream of Puf5, Ixr1 does not have strong effects on the cell cycle-dependent induction of CLB2. Rather, Ixr1 seems to control the basal expression of CLB2 during the cell cycle.
(A-D) The cell cycle-dependent mRNA levels of RNR1, SIC1, CLB1, and CLB2 in the synchronized bar1Δ cell (black circle) and bar1Δ ixr1Δ mutant (blue square). The levels of an S-phase marker RNR1 mRNA (A), a late M-phase marker SIC1 mRNA (B), CLB1 mRNA (C), and CLB2 mRNA (D) were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the SCR1 reference gene. The vertical axis shows the fold change of mRNA level relative to that in the bar1Δ 0 min sample, and the horizontal axis shows the time after release from G1-phase. These experiments were repeated (n = 2), and the representative data is presented.
The effect of reduced expression of CLB2 by the puf5Δ mutation was observed in the clb1Δ clb5Δ clb6Δ triple mutant strains
In our previous study, we observed that the reduced CLB1 expression in the puf5Δ mutant resulted in the growth defect of the puf5Δ clb2Δ double mutant [25] (Fig 1B). On the other hand, the puf5Δ clb1Δ double mutant grew similarly to the wild-type strain (Fig 1B). Why did the puf5Δ clb1Δ double mutant grow as well as the wild-type strain, even though CLB2 expression was reduced by the puf5Δ mutation? We hypothesized that the other B-type cyclins, Clb3, Clb4, Clb5 and Clb6, fulfill the function of Clb1 and Clb2. To verify this hypothesis, we investigated the genetical interaction between PUF5 and each of the four cyclin genes. We found that the puf5Δ clb5Δ double mutant and the puf5Δ clb5Δ clb6Δ triple mutant exhibited markedly slow growth than the puf5Δ mutant (Fig 7A), while the puf5Δ clb3Δ clb4Δ triple mutant just showed only a slight growth retardation (S2 Fig). This observation suggests that the reduced CLB2 expression by the puf5Δ mutation has a marked physiological importance in the background where S-phase cyclins are absent. Considering that Puf5 positively regulates both CLB1 and CLB2, we next examined which regulation is more important, CLB1 or CLB2. The tetrad analysis revealed that clb1Δ clb5Δ clb6Δ triple mutant grew slightly slower than wild-type strain, and that the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant showed a severe growth defect or sometimes lethality (Fig 7B). These results imply that the decreased CLB2 expression level caused by the puf5Δ mutation is physiologically important in the clb5Δ clb6Δ double mutation background. Next, we examined whether the cell cycle-specific expression of CLB2 was actually decreased in the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant. For synchronously culturing these strains, we utilized the BAR1 positive background and used 100 times higher concentration of α-factor as reported previously [26]. It is confirmed that CLB2 expression was induced slower in the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant than in the clb1Δ clb5Δ clb6Δ triple mutant, and that the maximum expression of CLB2 was also lower in the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant (Fig 8A). Even though the clb1Δ clb5Δ clb6Δ triple mutant and the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant lack the S-phase cyclins, the expression of the S-phase marker gene, RNR1, peaked at 10–20 minutes in both mutants. However, the second peak of the RNR1 expression was only observed in the clb1Δ clb5Δ clb6Δ triple mutant at 110 minutes (Fig 8B), indicating that the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant could not complete the first cell cycle and re-enter the second one. Indeed, the expression of the late M phase marker gene, SIC1, that is induced in the late M-phase, had peaked at 100 minutes in the clb1Δ clb5Δ clb6Δ triple mutant. However, in the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant, the expression of SIC1 was not induced during cell cycle (Fig 8C), implying that the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant harbors a difficulty in the G2 to M-phase transition. To address this possibility, we observed the morphology of the two strains with nuclear staining by DAPI. The clb1Δ clb5Δ clb6Δ triple mutant cells showed mildly elongated shapes (S3A Fig). In this triple mutant strain, small buds were observed from 40 minutes, and the buds enlarged at 60 minutes. At 60–80 minutes, nuclei were observed near the bud neck, and nuclear divisions were seen at 100 minutes. At 120 minutes, cell divisions were completed (S3A Fig). This time course was consistent with the mRNA levels of the G2-phase-induced CLB2 and late M-phase-induced SIC1. In contrast, in the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant, some cells were severely elongated, and the others did just mildly. The former sometimes contained two nuclei in one cell, indicating the difficulty in the cell division in this mutant (S3B Fig). The latter contained one nucleus within each cell. As for these cells, small buds emerged from 40 minutes, and the growth of the buds was observed at 60 minutes. The nuclei were located near the bud neck at 60–80 minutes and nuclear divisions started to be observed at 80–100 minutes. However, cells could not complete cell division even at 120 minutes (S3B Fig). These results suggest that cell cycle synchronization was successfully performed in the clb1Δ clb5Δ clb6Δ triple mutant and the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant cells, and that the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant was not able to accomplish M-phase.
(A) The tetrad analysis of the strains that are heterozygous for the alleles of PUF5, CLB5, and CLB6. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days. The puf5Δ clb5Δ double mutant and the puf5Δ clb5Δ clb6Δ triple mutant were surrounded by the wide frame. (B) The tetrad analysis of the strains that are heterozygous for the alleles of PUF5, CLB1, CLB5, and CLB6. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days. The puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant is emphasized in a wide frame, and the clb1Δ clb5Δ clb6Δ triple mutant is in the thin frame.
(A-C) The cell cycle-dependent mRNA levels of CLB2, RNR1, and SIC1 in the synchronized clb1Δ clb5Δ clb6Δ triple mutant (black circle) and the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant (grey square). The levels of CLB2 mRNA (A), S-phase marker RNR1 mRNA (B), and a late M-phase marker SIC1 mRNA (C) were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the SCR1 reference gene. The vertical axis shows the fold change of mRNA level relative to that in the clb1Δ clb5Δ clb6Δ triple mutant 0 min sample, and the horizontal axis shows the time after release from G1-phase. These experiments were repeated (n = 2), and the representative data is presented.
As previously mentioned, CLB2 expression is downregulated in the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant, and the strain shows a severe growth defect. In this case, does the decreased expression of CLB2 cause the growth defect by itself? To clarify this question, we introduced a multi-copy CLB2 plasmid and a multi-copy PUF5 plasmid into the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant strain. We observed that the slow growth of the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant was restored by both plasmids (Fig 9). Thus, the growth retardation of the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant was associated with a decrease in CLB2 expression caused by the puf5Δ mutation. However, the suppression was stronger on the introduction of the multi-copy PUF5 plasmid than the multi-copy CLB2 plasmid. This implies the existence of other regulation targets of Puf5 on cell growth than CLB2. Nevertheless, our findings indicate that the appropriate expression of CLB2 under the Puf5 control is physiologically important in the strain which lacks Clb1, Clb5, and Clb6.
Cell growth of the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant harboring plasmids YEplac195, YEplac195-CLB2, or YEplac195-PUF5. The transformants were incubated on an SC-Ura medium at 25°C for 4 days.
Clb2 can shoulder the function of S-phase cyclins, Clb5 and Clb6
Based on the aforementioned experiments, we found that Clb2 is essential when S-phase cyclins, Clb5 and Clb6, are lacked, and vice versa (Fig 7A). Does this imply that the G2/M-phase cyclins and S-phase cyclins complement each other’s function? The puf5Δ mutation causes a severe growth defect in the background where Clb2 or Clb5, the major counterpart of the G2/M-phase cyclin pairs or S-phase cyclin pairs, is absent (Figs 1B and 7A). Therefore, we investigated the difference of the effect of each cyclin on the cell growth by making these cyclin genes overexpressed using multi-copy plasmids in the puf5Δ clb5Δ and the puf5Δ clb2Δ double mutant. The puf5Δ clb5Δ double mutant grew slowly even in the optimal temperature (Fig 7A), but this strain showed a remarkable growth defect at high temperature (Fig 10A). This slow growth was recovered by the multi-copy CLB2, CLB5, and CLB6 plasmids at 35°C (Fig 10A). At 37°C, the puf5Δ clb5Δ double mutant harboring a multi-copy CLB6 plasmid grew slightly better than cells with a vector, but the suppression by the multi-copy CLB6 was much weaker than by the multi-copy CLB2 and CLB5 (Fig 10A). Therefore, the effect of the growth suppression by multi-copy CLB2 and CLB5 plasmids was suggested to be stronger than multi-copy CLB6. The multi-copy CLB1 could not suppress the growth defect (Fig 10A). Next, we transformed the multi-copy CLB1, CLB2, CLB5, and CLB6 plasmids into the puf5Δ clb2Δ double mutant which showed growth retardation at high temperatures. The growth of the puf5Δ clb2Δ double mutant was recovered at higher temperatures by the multi-copy CLB1 and CLB2 plasmids, but not by the multi-copy CLB5 and CLB6 plasmids (Fig 10B). These results suggest that Clb2, yet not Clb1, can shoulder the function of S-phase cyclins, Clb5 and Clb6, whereas Clb5 and Clb6 cannot substitute for the function of G2/M-phase cyclins, Clb2 and Clb1.
(A) Cell growth of the puf5Δ clb5Δ double mutant harboring plasmids YEplac195, YEplac195-CLB1, YEplac195-CLB2, YEplac195-CLB5, or YEplac195-CLB6. The transformants were incubated on an YPD medium at 25°C, 35°C, or 37°C for 3 days. (B) Cell growth of the puf5Δ clb2Δ double mutant harboring plasmids YEplac195, YEplac195-CLB1, YEplac195-CLB2, YEplac195-CLB5, or YEplac195-CLB6. The transformants were incubated on an YPD medium at 25°C, 35°C, or 37°C for 3 days.
The appropriate expression of CLB2 was vital for the cell growth in the dun1Δ background
The above results suggest that Puf5 regulates cell cycle-specific expression of CLB1 and CLB2 via regulating the expression of Ixr1. Moreover, Clb2 is able to substitute for the S-phase cyclins, Clb5 and Clb6, and the expressional control of Clb2 by Puf5 is essential in an S-phase cyclin-deficient condition. From these, we hypothesized that the regulation of CLB1/2 expression by Puf5 and Ixr1 is related to the differential use of Clb1/2 and Clb5/6 functions. However, no growth inhibition is observed in the ixr1Δ mutant, despite the increased expression of CLB1 and CLB2 (Figs 4A, 4B, 6C and 6D). Therefore, we further analyzed the physiological importance of Ixr1 in this B-type cyclin regulation.
The ixr1Δ mutation alone does not affect proliferation, but the ixr1Δ dun1Δ double mutant is reported to be lethal [27]. DUN1 gene encodes a checkpoint kinase functioning downstream of Mec1-Rad53. When cell cycle checkpoint is induced, Mec1 (ATR) and Rad53 (CHEK2) are activated. This Mec1-Rad53 pathway then activates Dun1 kinase to regulate the dNTP level via upregulating the expression of RNR genes encoding subunits of the ribonucleotide reductase [28–30]. It is reported that in the ixr1Δ mutant, RNR1 expression is decreased, which results in the low level of dNTP pool, and this regulation contributes to the lethality of the ixr1Δ dun1Δ double mutant [27]. However, in our data, the RNR1 expression remains preserved in the ixr1Δ mutant during the first cell cycle, and the decrease was only observed in the second cell cycle (Fig 6A). Therefore, there are supposed to be other causes of the growth retardation of the ixr1Δ dun1Δ double mutant. We then considered and tried to verify the possibility that elevated expression of CLB1 and CLB2 in the ixr1Δ mutant was involved in the lethality of the ixr1Δ dun1Δ double mutant. As previously reported [27], the ixr1Δ dun1Δ double mutant was lethal (Fig 11A). As we expected, this lethality was also restored by the deletion of CLB2 (Fig 11A). Nevertheless, the clb1Δ deletion did not recover the growth of ixr1Δ dun1Δ double mutant (S4 Fig). These results suggest that elevated CLB2 expression, but not CLB1, caused by the ixr1Δ mutation may be associated with the lethality of the ixr1Δ dun1Δ double mutant. We next introduced a single copy or a multi-copy CLB2 plasmid into the ixr1Δ dun1Δ clb2Δ triple mutant. Although a single-copy CLB2 plasmid only slightly inhibited the growth of the ixr1Δ dun1Δ clb2Δ triple mutant, the growth repression effect of a multi-copy CLB2 plasmid was much significant (Fig 11B). Therefore, the high dosage of CLB2 expression seems to be harmful for the cell growth in the dun1Δ mutation background.
(A) The tetrad analysis of the strains that are heterozygous for the alleles of IXR1, DUN1, and CLB2. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days. The ixr1Δ dun1Δ double mutant was emphasized with the wide frame, and the ixr1Δ dun1Δ clb2Δ triple mutant was in the thin frame. (B) Cell growth of the ixr1Δ dun1Δ clb2Δ triple mutant harboring plasmids, YCplac33, YCplac33-CLB2, or YEplac195- CLB2. The transformants were incubated on an SC-Ura medium at 25°C for 4 days.
In a previous study [27], one of the causes of the lethality caused by the ixr1Δ dun1Δ double mutation was explained by the reduced RNR1 expression, and the deletion of SML1 gene encoding a ribonucleotide reductase inhibitor was reported to suppress the lethality of the ixr1Δ dun1Δ double mutant. Thus, we compared the suppression by the sml1Δ deletion with that by the clb2Δ deletion. We observed that the ixr1Δ dun1Δ sml1Δ triple mutant grew better than the ixr1Δ dun1Δ clb2Δ triple mutant (Fig 12A). Furthermore, the additional deletion of SML1 further recovered the growth of the ixr1Δ dun1Δ clb2Δ triple mutant strain (Fig 12A). Thus, even though the effect of the clb2Δ deletion was weaker than that of the sml1Δ deletion, each of them independently functions in this suppression. Originally, the sml1Δ mutation was identified as a suppressor mutation which restored the lethality caused by mec1Δ deletion [29–31]. Therefore, we next examined whether the clb2Δ deletion also suppresses the lethality caused by mec1Δ deletion. Tetrad analysis showed that the mec1Δ single mutant was lethal, and the mec1Δ sml1Δ double mutant was viable as reported [31]. In contrast, the mec1Δ clb2Δ double mutant was lethal (Fig 12B). Thus, the effect by the clb2Δ deletion seemed to be different from the effect by the sml1Δ deletion.
(A) The tetrad analysis of the strains that are heterozygous for the alleles of IXR1, DUN1, CLB2, and SML1. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days. The ixr1Δ dun1Δ double mutant was emphasized with the wide frame. (B) The tetrad analysis of the strains that are heterozygous for the alleles of MEC1, CLB2, and SML1. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days. The mec1Δ single mutant and the mec1Δ clb2Δ double mutant was emphasized in a wide frame.
To further analyze the difference between the clb2Δ mutation and the sml1Δ mutation, we examined the expression of RNR1 and RNR3 genes in the ixr1Δ dun1Δ clb2Δ triple mutant and the ixr1Δ dun1Δ sml1Δ triple mutant. RNR1 expression is known to be regulated by both Ixr1 and Sml1: Ixr1 positively and Sml1 negatively regulate its expression. In contrast, the expression of RNR3 encoding the functional partner of Rnr1 is under control of Mec1-Rad53 pathway, but not under Ixr1 or Sml1 [27]. The expression of RNR1 was not increased in the ixr1Δ mutant compared to wild-type strain but was increased in the dun1Δ mutant (Fig 13A). As for the triple mutants of interest, RNR1 expression was significantly decreased in both the ixr1Δ dun1Δ clb2Δ triple mutants and the ixr1Δ dun1Δ sml1Δ triple mutants compared to the dun1Δ mutant (Fig 13A). Regarding RNR3, the expression was highly upregulated in the ixr1Δ dun1Δ clb2Δ triple mutant compared to wild-type strain and each single mutant, ixr1Δ mutant or dun1Δ mutant (Fig 13B). In contrast, in the ixr1Δ dun1Δ sml1Δ triple mutant, the RNR3 level was not induced (Fig 13B). Even though RNR1 expression levels showed a similar pattern between the ixr1Δ dun1Δ clb2Δ triple mutant and the ixr1Δ dun1Δ sml1Δ triple mutant, the converse expression of RNR3, a gene controlled by Mec1-Rad53 checkpoint pathway, implies the difference in the intracellular condition of these mutants.
(A, B) The mRNA levels of RNR1 (A), and RNR3 (B) in the wild-type strain, the ixr1Δ mutant, the dun1Δ mutant, the ixr1Δ dun1Δ sml1Δ triple mutant, and the ixr1Δ dun1Δ clb2Δ triple mutant. The cells were cultured in a YPD medium at 28°C until the log phase. The RNR mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the ACT1 reference gene. The data shows the mean ± SE (n = 3) of the fold change of RNR1 (A) and RNR3 (B) relative to the mRNA level in the wild-type strain. *P < 0.05, **P < 0.01 as determined by Tukey’s test. NS indicates no significant change.
The dun1Δ mutation shows a synthetic growth defect with the clb5Δ clb6Δ double mutation
As mentioned in the former section, we hypothesize that Puf5 and Ixr1 play an important role for the proper utilization of G2/M-phase cyclins and S-phase cyclins. The increased level of CLB2 encoding a major G2/M cyclin caused by the ixr1Δ mutation is found to be toxic in the dun1Δ mutation background. Then, does this effect extend to the S-phase cyclins? Previously, genetical interactions among DUN1 and S-phase cyclin genes, CLB5 and CLB6, have been reported [32]. Therefore, we next examined these genetic interactions. Tetrad analysis revealed that the dun1Δ clb5Δ clb6Δ triple mutant had a poor growth (Fig 14A). Contrarily, the dun1Δ clb5Δ double mutant showed only a moderate growth retardation, and the dun1Δ clb6Δ double mutant grew as well as wild-type strain (Fig 14A). From these results, the expression of CLB5 and CLB6, in contrast to CLB2, seem to have a positive effect on cell growth under the dun1Δ mutation background. Next, we investigated how CLB2 expression affected the growth of the dun1Δ clb5Δ clb6Δ triple mutant by additionally deleting PUF5 gene. As shown in Fig 7A, the puf5Δ clb5Δ clb6Δ triple mutant showed a severe growth defect, and this defect was more remarkable than in the dun1Δ clb5Δ clb6Δ triple mutant (Fig 14B). The puf5Δ dun1Δ double mutant grew as well as wild-type strain, but the puf5Δ mutation significantly accelerated the growth retardation of the dun1Δ clb5Δ double mutant and the dun1Δ clb5Δ clb6Δ triple mutant (Fig14B). Although the excessive expression of CLB2 worsens the growth of dun1Δ mutant, proper CLB2 expression seems to be needed to maintain cell growth in the absence of S-phase cyclins. To further clarify the difference among the effects of CLB5/CLB6 and CLB2 on cell growth in the dun1Δ background, we next examined the expression of RNR1 and RNR3 genes in the dun1Δ clb5Δ clb6Δ triple mutant, together with the puf5Δ clb5Δ clb6Δ triple mutant. The expression of RNR1 did not change significantly in the dun1Δ clb5Δ clb6Δ triple mutant compared to the dun1Δ mutant (S5 Fig), while the expression of RNR3 was significantly increased about 15 times in the dun1Δ clb5Δ clb6Δ triple mutant and 8 times in the dun1Δ clb5Δ double mutant compared to wild-type strain (Fig 15). The increased expression of RNR3 were also significant compared to the dun1Δ mutant (Fig 15). Similarly, the expression of RNR1 did not change significantly between the puf5Δ clb5Δ clb6Δ triple mutant and other single or double mutants (S5 Fig). In contrast, RNR3 mRNA level was significantly higher about 2.5 times in the puf5Δ clb5Δ double mutant and 3 times in the puf5Δ clb5Δ clb6Δ triple mutant compared to wild-type strain (Fig 16). Together with the shown data in Fig 13B, the expression of RNR3 regulated by the Mec1-Rad53 checkpoint pathway is highly induced when G2/M-phase cyclins or S-phase cyclins are absent, and this induction is particularly remarkable in the dun1Δ mutation conditions.
(A) The tetrad analysis of the strains that are heterozygous for the alleles of DUN1, CLB5, and CLB6. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days. The dun1Δ clb5Δ clb6Δ triple mutant was surrounded by the wide frame. (B) The tetrad analysis of the strains that are heterozygous for the alleles of PUF5, DUN1, CLB5, and CLB6. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days.
The mRNA levels of RNR3 in the wild-type strain, the clb5Δ mutant, the clb6Δ mutant, the dun1Δ mutant, the dun1Δ clb5Δ mutant, the dun1Δ clb6Δ mutant, the clb5Δ clb6Δ mutant, and the dun1Δ clb5Δ clb6Δ mutant. The cells were cultured in a YPD medium at 28°C until the log phase. The RNR3 mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the ACT1 reference gene. The data shows the mean ± SE (n = 3) of the fold change of RNR3 relative to the mRNA level in the wild-type strain. **P < 0.01 as determined by Tukey’s test. NS indicates no significant change.
The mRNA levels of RNR3 in the wild-type strain, the puf5Δ mutant, the puf5Δ clb5Δ mutant, the clb5Δ clb6Δ mutant, and the puf5Δ clb5Δ clb6Δ mutant. The cells were cultured in a YPD medium at 28°C until the log phase. The RNR3 mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the ACT1 reference gene. The data shows the mean ± SE (n = 3) of the fold change of RNR3 relative to the mRNA level in the wild-type strain. *P < 0.05, **P < 0.01 as determined by Tukey’s test. NS indicates no significant change.
Discussion
We previously showed that Puf5 positively regulates CLB1 expression via the post-transcriptional regulation of IXR1 mRNA. This IXR1 mRNA encodes a repressor protein Ixr1 which appears to function as a negative regulator of CLB1 expression [25]. In that paper, in an asynchronous culture, the expression of CLB2 encoding a redundant cyclin of Clb1 is not decreased in the puf5Δ mutant. However, regarding that CLB1 and CLB2 are under the same expressional control machinery in mitotic cell proliferation, it would be reasonable that Puf5 also regulates the expression of CLB2 in addition to CLB1. Thus, in this study, we re-examined whether Puf5-Ixr1 is also involved in the control of the expression of CLB2. As a result, we observed a decrease in cell cycle-specific expression of CLB2 mRNA and protein in the puf5Δ mutant in synchronized cultures (Figs 2D and 3B). The degree of the decrease in CLB2 expression, however, seemed to be milder than that in CLB1 expression in both mRNA and protein levels (Figs 2D and 3B). This difference also affects the physiological importance of these two regulations. In detail, the decreased expression of CLB1 by the puf5Δ mutation caused a severe growth defect in the clb2Δ mutation background, whereas the puf5Δ clb1Δ double mutant grew like wild-type strain (Fig 1B). These results imply two possibilities: one is that the remaining CLB2 expression, albeit at the decreased level, in the puf5Δ mutant maintained cell proliferation, the other is that the other B-type cyclins, such as Clb5 and Clb6, compensate for Clb1 and Clb2. Since our results indicated that Clb5 and Clb6 were not able to substitute for the G2/M-phase cyclins (Fig 10B), the former possibility is more reasonable for explaining the growth of the puf5Δ clb1Δ double mutant.
Seeking the physiological importance of the regulation of CLB2 expression by Puf5, we uncovered that the decrease in the CLB2 expression causes a growth retardation in the absence of S-phase cyclins. The puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant exhibited a severe growth defect or sometimes lethality (Fig 7B), and this remarkably poor growth was caused by the decreased CLB2 expression by the puf5Δ mutation. However, when we introduced a multi-copy CLB2 or a multicopy PUF5 plasmid into the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant, the suppression effect of the multi-copy CLB2 plasmid was milder than that of multicopy PUF5 plasmid (Fig 9). Since Puf5 binds to more than 1,000 mRNAs [24] and regulates diverse phenomena, Puf5 is supposed to regulate more targets than specified so far. Therefore, this difference in the suppression effect is implied to originate from other targets of Puf5. Nevertheless, Puf5 does positively regulate CLB2 expression of physiological importance in the S-phase cyclin-deficient condition. Considering that Clb2, but not Clb1, could compensate for the loss of S-phase cyclin Clb5 (Fig 10A), Puf5 controls the CLB2 expression at a proper level to ensure the functional redundancy of two major B-type cyclins, Clb2 and Clb5. The B-type cyclins in S. cerevisiae were supposed to evolve from common B-type cyclin like ancestors and diverge into six types during evolution [33]. The ancestors are believed to harbor both replicational and mitotic activity. Therefore, the functional redundancy between Clb2 and Clb5 is possibly an evolutionary trace. Actually, it has been reported that in the clb5Δ clb6Δ double mutant, the entry into the S-phase was delayed severely, but origin firing was performed normally, suggesting that there is a functional overlap between Clb1-4 and Clb5/Clb6 [34]. However, Clb2 under the control of endogenous CLB5 promoter was not able to suppress the origin firing defect in the clb5Δ clb6Δ double mutant and had only a partial effect on the recovering the entry into the S-phase [35]. Considering our data that the multi-copy CLB2 plasmid suppressed the growth defect of the puf5Δ clb5Δ double mutant (Fig 10A), two possibilities are suggested: one is that the high dosage of Clb2 is essential to shoulder the Clb5 function, and the other is that only a partial functional redundancy is sufficient for cell growth. We regard the former possibility more plausible, since the expression level of Clb2 is critical for maintaining cell growth in the dun1Δ mutation background.
In this study, we presented that Puf5 regulates CLB2 expression in an Ixr1-dependent manner. Then, what is the significance of Ixr1 regulating the expression of CLB1 and CLB2? Previously it had been reported that the ixr1Δ mutation causes the decreased RNR1 expression level and results in the lethality in the dun1Δ mutation background, one of the checkpoint kinase-deficient condition [27]. Here, we found that the clb2Δ mutation, but not the clb1Δ mutation, suppressed the lethality of the ixr1Δ dun1Δ double mutant (Figs 11A and S4). The deletion of SML1, a negative regulator of RNR1, also restored the growth defect of the ixr1Δ dun1Δ double mutant. However, the sml1Δ mutation could additionally recover the growth of the ixr1Δ dun1Δ clb2Δ triple mutant (Fig 12A), and the suppression effects of the mec1Δ mutant between the sml1Δ mutation and the clb2Δ mutation were far different: the sml1Δ mutation could, but the clb2Δ mutation could not restore the lethality (Fig 12B). From these results, it is hypothesized that the growth suppression by the clb2Δ mutation is independently performed of Sml1. Moreover, the growth of the ixr1Δ dun1Δ clb2Δ triple mutant was inhibited by the multi-copy CLB2 plasmid, whereas the inhibitory effect of the single-copy CLB2 plasmid was not sufficient as that of the multi-copy CLB2 plasmid (Fig 11B). Altogether, the ixr1Δ dun1Δ double mutant or CLB2-overexpressed ixr1Δ dun1Δ clb2Δ triple mutant showed growth defect or lethality, and the ixr1Δ dun1Δ clb2Δ triple mutant or single-copy CLB2 expressed ixr1Δ dun1Δ clb2Δ triple mutant was viable (Fig 11A and 11B). From these results, we suppose that the high dosage of Clb2 is critical to the dun1Δ mutant. In other words, it is crucial for cell proliferation to maintain the proper level of CLB2 expression under the control by Ixr1 in the dun1Δ mutant. Regarding that CLB1 expression does not have physiological importance in the dun1Δ mutation background (S4 Fig), the functions of Ixr1 on CLB1 or CLB2 seem to be different. Ixr1 regulates CLB1 expression specifically to G2/M-phase, while regulating CLB2 expression at the global level rather than in a cell cycle-specific way (Figs 5D and 6D). It is possible that this global effect of Ixr1 on CLB2 expression ensures the proper level of Clb2 and contributes to the cell viability under the DNA-damage uninducible dun1Δ mutation background.
In contrast to CLB2, the expression of CLB5 and CLB6 positively affected the growth of the dun1Δ mutant: the dun1Δ clb5Δ clb6Δ triple mutant strain grew slowly (Fig 14A). Moreover, the puf5Δ dun1Δ clb5Δ clb6Δ quadruple mutant strain was also lethal (Fig 14B). Therefore, in addition to CLB2, the adequate expression of B-type cyclins seem to be necessary for the dun1Δ mutant to survive. Thus, what does happen when the expression of B-type cyclins were lost in the dun1Δ mutant? From the mRNA levels of RNR3, we hypothesize that DNA damage response is highly induced in the condition. The expression of RNR3, a gene encoding subunits of the ribonucleotide reductase, is generally induced by Mec1-Rad53-Dun1 checkpoint pathway and also directly induced Mec1-Rad53 in a Dun1-independent manner [28,29]. Since its expression is quite low under the non-stressed condition, the induction of RNR3 expression is usually used as a marker for DNA damage [36]. As a result, RNR3 expression was mildly induced in the dun1Δ mutant and highly induced in the ixr1Δ dun1Δ clb2Δ triple mutant and the dun1Δ clb5Δ clb6Δ triple mutant (Figs 13B and 15). This induction compared to the wild-type strain was more prominent in the dun1Δ clb5Δ clb6Δ triple mutant than in the ixr1Δ dun1Δ clb2Δ triple mutant (Figs 13B and 15). These results imply that Mec1-Rad53 dependent DNA damage response is activated when B-type cyclins are absent in the dun1Δ mutation background. Previously, it was reported that DNA damage activates the checkpoint pathway more strongly in the S phase than in other cell cycle phases [37]. Thus, the abnormality of DNA damage induction in the dun1Δ mutant is possibly highly accelerated by the absence of S-phase cyclins. In addition, RNR3 expression was significantly increased in the puf5Δ mutant, the puf5Δ clb5Δ double mutant, and the puf5Δ clb5Δ clb6Δ triple mutant, while RNR3 expression was not induced in the clb5Δ clb6Δ double mutant (Fig 16). Therefore, we assume that the deficiency of G2/M-phase cyclins causes DNA damage responses by themselves, and that this response is spurred by the additional deletion of CLB5/CLB6. This induced DNA damage responses possibly contributes to the growth retardation of the puf5Δ clb5Δ double mutant and the puf5Δ clb5Δ clb6Δ triple mutant. In this study, we analyzed the regulation mechanism of a B-type cyclin gene CLB2 by Puf5 and Ixr1. We speculate that Puf5 and Ixr1 finetune expression of CLB2 and contribute to the maintenance of the sufficient function of cyclins in the G2/M-phase and S-phase. Furthermore, Ixr1-mediated regulation maintains adequate expression levels of CLB2 and cell proliferation under DNA damage conditions. From our data, the deficiency of G2/M-phase cyclins is suggested to cause DNA damage by itself, so the finetuning of CLB2 expression is assumed to be a vital process for cells to survival.
Materials and methods
Strains and media
The Saccharomyces cerevisiae strain W303 was used as the background yeast strain for the study. Escherichia coli, DH5α strain, was used to manipulate the DNA. The genetic manipulation of yeast strains was performed using standard procedures as previously described [38]. All W303-derived strains used in this study are described in detail in S1 Table. For standard culture, Saccharomyces cerevisiae was grown in YPD medium (2% Glucose, 2% bactopeptone, and 1% yeast extract) and synthetic complete medium (SC) [38]. To culture yeast strains that require alleviation of osmolarity stress, 10% sorbitol was supplemented to the media. SC medium lacking amino acids (e.g., SC-Ura medium i.e., SC medium lacking uracil) were used to select transformants.
Plasmids
The plasmids used in this study are described in S2 Table. For the construction of YEplac195-PUF5 plasmid, the fragment containing the PUF5 gene together with upstream and downstream regions was amplified by PCR of genomic DNA. The fragment was inserted between SalI and EcoRI sites of YEplac195 plasmid. YEplac195-CLB1, YEplac195-CLB2, YEplac195-CLB5, and YEplac195-CLB6 plasmids were constructed following similar procedure. For the construction of YCplac33-CLB2 plasmid, the fragment containing the CLB2 gene together with upstream and downstream regions was amplified by PCR and inserted between SalI and EcoRI sites of YCplac33 plasmid.
The pCgLEU2, pCgHIS3, and pCgTRP1 plasmids, which were pUC19 carrying the Candida glabrata LEU2, HIS3, and TRP1 genes, respectively, were used to delete genes [39]. The pKl-URA3 plasmid, pUC19 carrying the Kluyveromyces lactis URA3 were also used for gene deletion.
Gene deletion
Deletions of PUF5, IXR1, CLB1, CLB2, CLB3, CLB4, CLB5, CLB6, and BAR1 were constructed by a PCR-based gene-deletion method as previously described [39–41]. The primer sets used in this study are listed in S3 Table. The fragments amplified by PCR were transformed into the wild-type strain and the transformants were selected on the SC medium lacking the corresponding amino acids.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Yeast cells were pre-cultured overnight in appropriate liquid medium at 28°C. The overnight culture was then diluted to OD600 = 0.5 (optical density measured at a wavelength of 600 nm) in fresh medium, and further cultured for 4 hours. Following this, the cells were then collected by centrifuge and total RNA was extracted using ISOGEN reagent (Nippon Gene). From the extract, genomic DNAs were removed using RNeasy Mini kit (Qiagen), and reverse transcription was performed using the Prime Script RT reagent Kit (Takara). The cDNA levels were quantified by qRT-PCR using QuantStudio 5 (Thermo Fisher Scientific) with TB Green Ex Taq (Takara). The primers used for the qRT-PCR were listed in S4 Table. The fold change of the mRNAs was calculated using SCR1 or ACT1 as internal control genes and statistically analyzed using Microsoft Office Excel.
Cell cycle synchronization by α-factor block and release
For the pheromone-induced cell cycle synchronization procedure MATa bar1Δ strains were used to prevent degradation of α-factor and the cell cycle synchronization procedure was followed as previously reported [26]. Yeast cells were pre-cultured overnight in YPD medium at 28°C, then transferred into a fresh YPD medium, and cultured for 4 hours.
After the 4-hour culture, α-factor was added into the culture, and incubated for 2 hours. Following the incubation, after collecting the 0-minute sample, cells were washed with a fresh YPD medium by centrifuge, transferred into a fresh YPD medium, and incubated at 28°C. Samples were collected by centrifuge every 10 minutes from the time at release.
Observation of cell morphology and nuclei
Following the cell cycle arrest using the α-factor as previously discussed, the cells were released and collected at an interval of 20 minutes from the time of release up to 120 minutes. The collected cells were fixed in formaldehyde solution for 1 hour at room temperature. After fixation, cells were washed with 1X Phosphate-buffered saline (PBS) and incubated with 4’,6-diamidino-2-phenylindole (DAPI) in a ratio of 1:1 at room temperature. The stained cells were then mounted on glass slides and visualized using a fluorescence microscope (Keyence BZ-X710; Keyence Corporation, Japan) equipped with a 100× oil immersion, under the appropriate excitation and emission settings for DAPI fluorescence. Fluorescent images were acquired using a Keyence BZ-X Viewer software (Keyence Corporation, Japan) and processed with ImageJ version 2.14.0.
Protein extraction and western-blot analysis
Yeast cells harboring YCplac33-CLB1-3HA or YCplac33-CLB2-3HA were precultured in SC-Ura medium overnight, and then cells were collected and dissolve into the same amount of YPD medium and cultured for 2 hours. After that, cell cycle synchronization procedures were performed as described above. After releasing, cells (OD600 = 10) were collected every 20 minutes and reacted with sodium hydroxide for protein extraction [42]. Protein samples were loaded onto a 10% SDS-PAGE gel, and electrophoreses were performed. After transferring to a PDVF membrane (Millipore), the membrane was treated with Ponceau and then reacted with the primary antibody, the anti-HA monoclonal antibody HA11, at 4°C overnight. Reacted with the secondary antibody, the anti-mouse IRDye® 800CW secondary antibodies and IRDye® 680RD secondary antibodies (LI-COR), for 1 hour at room temperature, the Clb1-3HA and Clb2-3HA proteins were visualized and quantified using ODYSSEY CLx (LI-COR).
Statistical analysis
Microsoft Office Excel was used to perform statistical analyses and generation of graphs. The data was represented as mean ± standard error (SE). Statistical significance was analyzed by One-way ANOVA, followed by Tukey’s test. For comparison between two sample groups, t-test was performed. **P < 0.01 or *P < 0.05 were considered statistically significant.
Supporting information
S3 Table. Primers used for the gene deletion.
https://doi.org/10.1371/journal.pone.0316433.s003
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S1 Fig. The cell cycle-regulated expression of CLB3, CLB4, CLB5, and CLB6 was not significantly changed in the puf5Δ mutant.
(A-D) The cell cycle-dependent mRNA levels of CLB3, CLB4, CLB5, and CLB6 in the synchronized bar1Δ cell (black circle) and bar1Δ puf5Δ mutant (grey square). The levels of CLB3 mRNA (A), CLB4 mRNA (B), CLB5 mRNA (C), and CLB6 mRNA (D) were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the SCR1 reference gene. The vertical axis shows the fold change of mRNA level relative to that in the bar1Δ 0 min sample, and the horizontal axis shows the time after release from the G1-phase.
https://doi.org/10.1371/journal.pone.0316433.s005
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S2 Fig. The puf5Δ clb3Δ double mutant, the puf5Δ clb4Δ double mutant, and the puf5Δ clb3Δ clb4Δ triple mutant showed no growth retardation.
The tetrad analysis of the strains that are heterozygous for the alleles of PUF5, CLB3, and CLB4. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days.
https://doi.org/10.1371/journal.pone.0316433.s006
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S3 Fig. The puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant is defective to complete the M-phase.
Morphology and nuclear images of the clb1Δ clb5Δ clb6Δ triple mutant (A) and the puf5Δ clb1Δ clb5Δ clb6Δ quadruple mutant (B). Cells were synchronously cultured and collected as described in the material and method section. Bright-field (left) and the overlayed (right) were shown. The scale bar represents 2 μm.
https://doi.org/10.1371/journal.pone.0316433.s007
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S4 Fig. The clb1Δ mutation did not suppress the lethality of the ixr1Δ dun1Δ double mutant.
The tetrad analysis of the strains that are heterozygous for the alleles of IXR1, DUN1, and CLB1. The cells were sporulated, dissected on a YPD plate, and cultured at 30°C for 3 days.
https://doi.org/10.1371/journal.pone.0316433.s008
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S5 Fig. The expression of RNR1 was unchanged in the dun1Δ clb5Δ clb6Δ triple mutant strain as compared to the dun1Δ mutant.
The mRNA levels of RNR1 in the wild-type strain, the dun1Δ mutant, the clb5Δ mutant, the clb6Δ mutant, the dun1Δ mutant, the dun1Δ clb5Δ mutant, the dun1Δ clb6Δ mutant, the clb5Δ clb6Δ mutant, and the dun1Δ clb5Δ clb6Δ mutant. The cells were cultured in a YPD medium at 28°C until the log phase. The RNR1 mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the ACT1 reference gene. The data shows the mean ± SE (n = 3) of the fold change of RNR1 in relative to the mRNA level in the wild-type strain. *P < 0.05, **P < 0.01 as determined by Tukey’s test. NS indicates no significant change.
https://doi.org/10.1371/journal.pone.0316433.s009
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S6 Fig. The expression of RNR1 was unchanged in the puf5Δ clb5Δ clb6Δ triple mutant.
The mRNA levels of RNR1 in the wild-type strain, the puf5Δ mutant, the puf5Δ clb5Δ mutant, the clb5Δ clb6Δ mutant, and the puf5Δ clb5Δ clb6Δ mutant. The cells were cultured in a YPD medium at 28°C until the log phase. The RNR1 mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using the ACT1 reference gene. The data shows the mean ± SE (n = 3) of the fold change of RNR1 relative to the mRNA level in the wild-type strain. NS indicates no significant change.
https://doi.org/10.1371/journal.pone.0316433.s010
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Acknowledgments
We thank all the members of the Molecular Cell Biology Laboratory for valuable discussions and suggestions.
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