Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3’ UTR regions

Jastin Edrian Cocuangco Revilleza; Megumi Sato; Kaoru Irie; Yasuyuki Suda; Tomoaki Mizuno; Kenji Irie

doi:10.1371/journal.pone.0268283

Abstract

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5’ cap structure and the 3’ poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3’ untranslated region (3’ UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3’ UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3’ UTR of CLB6 mRNA.

Citation: Revilleza JEC, Sato M, Irie K, Suda Y, Mizuno T, Irie K (2022) Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3’ UTR regions. PLoS ONE 17(5): e0268283. https://doi.org/10.1371/journal.pone.0268283

Editor: Reiko Sugiura, Kindai University: Kinki Daigaku, JAPAN

Received: December 31, 2021; Accepted: April 26, 2022; Published: May 6, 2022

Copyright: © 2022 Revilleza 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. Microarray data is available from GEO accession number GSE198743.

Funding: This research was supported by JSPS KAKENHI Grant Number 18K06053 (to KI).

Competing interests: The authors have declared that no competing interests exist.

Introduction

The cells contain the same DNA, but each cell contains a different subset of expressed genes. There are different points at which cells determine which genes are copied into RNA and translated to proteins [1]. Once the mRNA has been made, it gradually moves to the ribosome where it is translated and the regulation via RNA-binding proteins and small regulatory RNA molecules is a common mechanism for developmental control [1]. Eukaryotic pre-mRNAs are initially modified at the 5’ and 3’ ends. At the 5’ end, several enzymes synthesize the 5’ cap, a 7-methylguanylate that protects an mRNA from enzymatic degradation and assist in cytoplasm transport. At the 3’ end, it is cleaved by an endonuclease to yield a free 3’-hydroxyl group wherein a sequence of adenylic acid residues is added by a poly(A) polymerase [1]. Gene expression can be regulated at many steps in the pathway from DNA to protein, one of which is selectively destabilizing certain mRNA molecules in the cytoplasm or called mRNA degradation control [1]. mRNA degradation is modulated by small RNA molecules or RNA-binding proteins [1,2].

RNases are responsible for mediating the processing, decay, and quality control of RNA [3]. The stability of mRNA largely depends on the mRNA sequence, which affects the accessibility of various RNA-binding proteins or small RNAs to the mRNAs [4]. Most mRNAs including the AU-rich element (ARE)-containing mRNAs involves 3’ untranslated region (UTR) destabilizing elements [5]. Most mRNAs undergo a deadenylation-dependent decay where the poly(A) tail is removed by a deadenylase activity of the Ccr4-Not complex [6]. The deadenylated mRNAs are further degraded by decapping enzymes and 5’-3’ exonuclease, or 3’-5’ exonucleases.

The Ccr4-Not complex is the main deadenylase in all eukaryotes including the budding yeast, Saccharomyces cerevisiae [5–8]. This is a multi-subunit protein complex which has a big contribution to regulate RNA metabolism from synthesis to decay [8]. Several studies have shown its role in mRNA decay in S. cerevisiae. First, Pumilio-homology domain Family (Puf) protein, which binds to the 3’ UTR of target mRNA, recruits the Ccr4-Not complex to the target mRNA and stimulates deadenylation [9]. Next, the Ccr4-Not complex contributes to septin organization via the deadenylation of the mRNAs encoding the septin regulators [10]. Our lab also reported the role of Ccr4 in the regulation of LRG1 mRNA in which Ccr4 regulates not only the mRNA level through poly(A) shortening, but also its translation [11]. Recently, we also reported that the polyA-binding protein (Pab1)-binding protein, Pbp1, mediates the growth defect caused by the deletion of ccr4Δ and pop2Δ [12]. Indeed, the Ccr4-Not complex is a global regulator of gene expression from yeast to human [8,13].

The cell cycle progression is regulated by cyclin-dependent protein kinases (Cdk) [1]. Cdk1 is present at a constant level during the cell cycle. Cyclin protein levels vary in concentrations and act as a signal for the transition between phases [1]. In S. cerevisiae, 6 Cyclin B genes (CLB) are classified into 3 types with different stages of accumulation: CLB1/CLB2 CLB3/CLB4, and CLB5/CLB6 accumulate during the G2 and M, S and G2 and late G1 phase, respectively [1,14,15]. Cyclin degradation is essential for the cell cycle progression [1]. It was previously reported that Cyclin B is degraded by the ubiquitin pathway which is the most selective degradation pattern in eukaryotic cells [1,16] and the proteolysis of cyclin potentially plays a role in proper cell cycle progression [17–19]. However, this claim is still unclear or unknown whether mRNA stability regulation is involved in Clb protein level. It was previously reported that CLB mRNA accumulation is dependent on the heterogeneous nuclear ribonucleoprotein arginine methyltransferase (Hmt1) [20]. Another study also showed that mRNA stability of CLB2 is controlled in its promoter-dependent manner [21]. Lastly, it was also demonstrated that the endoribonuclease MRP cleaves the CLB2 mRNA in its 5’ UTR for rapid 5’ to 3’ degradation by the Xrn1 nuclease [22].

In this study, we investigated the role of Ccr4 on the expression of CLB1-6 mRNAs. By creating multiple gene cassettes of CLB1-6 genes, we were able to determine which region is responsible for its recognition by Ccr4 for degradation. Our results show that CLB6 expression is regulated by Ccr4 through the coding sequence and 3’ UTR of CLB6 mRNA.

Materials and methods

Strains and media

Escherichia coli DH5α strain was used for DNA manipulations. The yeast strains used in this study are isogenic derivatives of the W303 strain and listed in S1 Table. Gene deletions were conducted to replace the target gene with resistance cassettes by homologous recombination using standard PCR-based method [23]. Colony PCR was conducted with forming clones to confirm complete deletion at the expected locus. The media used in this study included YPD (1% yeast extract, 2% peptone, and 2% glucose) and SC (synthetic minimal medium). SC media lacking amino acids or other nutrients (e.g. SC-ura corresponding to SC lacking uracil) were used to select the transformants. General procedures were performed as described previously [24].

Plasmids

Plasmids used in this study are listed in S2 Table. pCgLEU2, pCgHIS3, and pCgTRP1 are pUC19 carrying the Candida glabrata LEU2, HIS3, and TRP1 genes, respectively [25].

Plasmids YCplac33-CLBx-HA-CLBx 3’ UTR, YCplac33-CLBx-HA-ADH1 3’ UTR, and YCplac33-MCM2 promoter-GFP-CLBx 3’ UTR were created using standard PCR-based method previously described [26]. Primers used for plasmid construction are listed in S3, S4, and S5 Tables. The process of creating the 3xHA tag gene construct was done in two sequential steps. First, the CLBx-HA-ADH1 3’ UTR was constructed by using a pFA6-3xHA-kanMX6 plasmid (Addgene plasmid # 39292). Next, the CLBx-HA-CLB 3’ UTR is constructed by using the gene template from the first step where primers are prepared by pairing the 5’ UTR primers with the HA-CLBx 3’ UTR region after the stop codon.

Deletions of CLB6 coding sequence and 3’ UTR were prepared by using the gene constructs of CLB6-HA-CLB6 3’ UTR and MCM2 promoter-GFP-CLB6 3’ UTR for the coding region and 3’ UTR, respectively. Primers used for plasmid construction are listed in S6 and S7 Tables. Starting from the second base pair, 50 amino acids were deleted per sequence for the coding region, overlapping the middle 25^th for the consecutive number (e.g. deletion 1 is from amino acid 2 to 50 while deletion 2 is from amino acid 25 to 75) with a total of 15 deletions. For the 3’ UTR, 30 bases were deleted per sequence, overlapping the middle 5^th for the consecutive number (e.g. deletion 1 is from 2 to 12 while deletion 2 is from 7 to 17) with a total of 6 deletions.

Cell sampling, sample preparation, and RNA extraction

Yeast cells without plasmids were grown overnight at 28°C using YPD medium. It is then inoculated into 30 ml fresh YPD medium to 0.5 OD. Cell samples of OD₁₀ were collected after 4 hours. Yeast cells harboring plasmids were grown overnight at 28°C using SC-ura medium. It is then inoculated into 30 ml fresh SC-ura medium to 0.5 OD. Cell samples of OD₁₀ were collected after 4 hours. The cells are washed, spun down and is ready for sample preparation.

The RNA isolation procedure are as follows. After washing, the cells were immersed using ISOGEN reagent (Nippon Gene, Toyama, Japan). It was then mashed using a Micromash MS-100R (Tomy, Japan) for 2x. Chloroform is added, mixed and centrifuged. The aqueous top layer was collected (with the RNA sample) and was precipitated with Isopropanol. The RNA samples were placed at 4°C overnight. On the next day, the cDNAs were prepared using a Primescript RT reagent Kit with gDNA Eraser (Takara, Japan). The cDNAs are finally ready for qRT-PCR analysis.

qRT-PCR analysis and microarray data

For qRT-PCR, the QuantStudio 5 real-time RT-PCR systems (Applied Biosystems, MA, USA) with SYBR Premix Ex Taq (Takara, Shiga, Japan) was used. The mRNA fold changes in mRNA level were computed using the 2^-ΔΔCt method and normalized against the ACT1 reference gene. Primers are listed on S8 Table. The microarray analysis was performed by the KURABO Bio-Medical Department (Osaka, Japan) using the Agillent-016322 Yeast (V2) Gene Expression 8X15K Microarray. Microarray data sets are available at the Gene Expression Omnibus at http://www.ncbi.nlm.nih.gov/geo (GEO accession number GSE198743).

Western blot analysis

Yeast cells were cultivated first overnight (16-hours), then a solution having OD₆₀₀ = 0.5 was prepared. For every sampling time, 10 OD of cells was collected from the cultured liquid media to be used for protein extraction. The cells (OD₆₀₀ = 10) of the collected cells were treated with sodium hydroxide for protein extraction [27]. Protein samples were loaded onto an 8% SDS-PAGE gel for protein electrophoresis and then transferred to a PDVF membrane (Merck Millipore, Molsheim, France) for Western blot analysis. The Biomolecular imager LAS-4000 (Fuji Film, Tokyo, Japan) and ODYSSEY CLx (LI-COR, Japan) were used to capture the samples Protein levels were quantified by immunoblotting with anti-HA, anti-GFP and anti-Pgk1 antibodies. The loading control used for the entire study was Pgk1. The protein fold changes in protein level were measured by normalizing with the wild-type.

Cell cycle synchronization by α-factor block and release

For synchronizing cell cycle, we performed the pheromone-induced cell cycle synchronization procedure previously reported [28]. MATa bar1Δ strains were used to prevent degradation of α-factor. After the overnight culture in YPD medium at 28°C, yeast strains were transferred into fresh YPD medium, and cultured until mid-exponential phase. Then, α-factor was added into the medium and strains were incubated for 2 hours. After the 2-hour exposure to α-factor, 0-minute sample was collected and cells remaining were washed with fresh YPD medium by centrifuge and released by transferring into fresh YPD medium and incubated at 28°C. After start releasing, samples were collected by centrifuge every 10 minutes to 120 minutes.

Determination of half-lives of CLB mRNAs

The half-lives (t_1/2) of CLB mRNAs were investigated by thiolutin-induced inhibition of transcription. We followed the procedure previously reported [29,30]. The bar1Δ and the bar1Δ ccr4Δ mutant cells were pre-cultured in YPD medium at 28°C overnight, and transferred into fresh YPD medium, and cultured until mid-exponential phase. Then, thiolutin (2 mg/ml dissolved in DMSO) was added, and samples were collected by centrifuge at 0, 1, 2, 5, 10, 20, 40, 80 minutes after exposure to thiolutin. The mRNA levels at each time point were determined by qRT-PCR. The half-lives (t_1/2) were calculated using Microsoft Excel.

Results

The ccr4Δ mutation showed a synthetic growth defect with the mih1Δ mutation

It has been suggested that Ccr4 is involved in the cell cycle progression [10,31]. To investigate a possible involvement of Ccr4 in the cell cycle progression, we first examined genetic interactions between CCR4 and the genes involved in G2-M transition, MIH1 (mitotic inducer homolog 1), and SWE1 (Saccharomyces wee1) [32,33]. Since the growth defect of the ccr4Δ single mutant strain is difficult to detect, we used the ccr4Δ khd1Δ double mutant strain [34,35] (Fig 1). Tetrad analysis revealed that the mih1Δ mutant had no obvious phenotype at room temperature, while the ccr4Δ mutant grew slowly. We found that the ccr4Δ mih1Δ double mutant showed slower growth than the ccr4Δ single mutant (Fig 1A). The ccr4Δ khd1Δ double mutant strain also showed slower growth than the ccr4Δ single mutant, and the ccr4Δ khd1Δ mih1Δ triple mutant strain was never germinated (Fig 1A). On the other hand, the ccr4Δ khd1Δ swe1Δ triple mutant showed better growth than the ccr4Δ khd1Δ double mutant strain (Fig 1B). The ccr4Δ swe1Δ double mutant showed a similar growth with the ccr4Δ single mutant (Fig 1B). These results suggest that Ccr4 may have a role in G2-M progression together with Mih1.

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Fig 1. The ccr4Δ mutation showed a synthetic growth defect with the mih1Δ mutation.

The strains that were heterozygous for favorite alleles were sporulated, and tetrads were dissected onto YPD plate. The growth of segregants after 4 days at 30°C is shown. Genotypes are indicated on both sides. More than 50 tetrads were dissected, and representative data are shown. (A) Tetrad analysis of strain 10BD-c4k1m1 that was heterozygous for ccr4Δ, khd1Δ, and mih1Δ alleles. The ccr4Δ khd1Δ double mutant strain showed slower growth than the ccr4Δ single mutant, and the ccr4Δ khd1Δ mih1Δ triple mutant strain was never germinated. (B) Tetrad analysis of strain 10BD-c4k1s1 that was heterozygous for ccr4Δ, khd1Δ, and swe1Δ alleles. The ccr4Δ khd1Δ swe1Δ triple mutant showed better growth than the ccr4Δ khd1Δ double mutant strain.

https://doi.org/10.1371/journal.pone.0268283.g001

The CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant

To investigate a possible role of Ccr4 in G2-M progression, we examined endogenous mRNA levels of CLB1-6 in wild-type and the ccr4Δ mutant cells. All CLB1-6 mRNA levels were increased in the ccr4Δ mutant compared to those in wild-type cells, but its ratio differed in the genes (Fig 2, Table 1). The CLB2 and CLB6 mRNA levels were increased about 7.5-fold; the CLB1, CLB3, and CLB4 mRNA levels were increased about 5-fold; the CLB5 mRNA level was increased 3-fold (Fig 2, Table 1). In contrast, the PGK1 mRNA level was similar in wild-type and the ccr4Δ mutant cells. Our previous microarray analyses [12] also support our current results (Table 1): CLB1, CLB2, CLB4, and CLB6 mRNA levels are increased in the ccr4Δ mutant compared to wild-type, while CLB3 and CLB5 mRNA levels are not (Table 2). The mRNA levels of G1 cyclin genes, CLN1-3, are not increased in the ccr4Δ mutant (Table 2). Thus, Ccr4 contributes to the levels of CLB mRNAs, but not those of CLN mRNAs.

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Fig 2. Expression of CLB1, CLB2, CLB3, CLB4, CLB5, and CLB6 in wild-type and ccr4Δ mutant cells.

The mRNA levels of CLB1 (A), CLB2 (B), CLB3 (C), CLB4 (D), CLB5 (E), and CLB6 (F) in ccr4Δ mutant strain growing in YPD media relative to the wild-type strain. PGK1 (G) was used as a control. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 h of culture in YPD. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g002

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Table 1. Summary of endogenous CLB mRNA levels in various mutant.

All values written are standardized to their respective wild-type values.

https://doi.org/10.1371/journal.pone.0268283.t001

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Table 2. Comparative expression levels of CLB and CLN genes from the microarray data (GEO accession number GSE198743).

The data show the relative microarray values of each gene obtained from wild-type and the ccr4Δ mutant strains. These values were normalized against their corresponding wild-type value which reflects the fold change in expression (values in parenthesis).

https://doi.org/10.1371/journal.pone.0268283.t002

Ccr4 is a catalytic subunit of the Ccr4-Not deadenylase complex [8]. To investigate whether other components of the Ccr4-Not complex are involved in the increase of the CLB mRNA levels, we examined endogenous mRNA levels of CLB1-6 in the pop2Δ, not1 (cdc39), not2 (cdc36), and not4Δ mutant cells. Pop2, also known as Caf1, is an exonuclease of the DEDD super family that is another catalytic subunit of the Ccr4-Not deadenylase complex, Not1/Cdc39 is a scaffold protein, Not2/Cdc36 is one of core proteins, and Not4 is an E3 ubiquitin ligase [8]. As expected from the observation that the pop2Δ mutant is similar to the ccr4Δ mutant [34,35], all CLB1-6 mRNA levels were increased in the pop2Δ mutant compared to those in wild-type cells (Fig 3). Similar to the ccr4Δ mutant, the increase of CLB6 mRNA level, approximately 18-fold increase, was most prominent. The CLB1, CLB2, and CLB5 mRNA levels were increased about 4 to 5-fold, and the CLB3 and CLB4 mRNA levels were increased about 3-fold. On the other hand, some CLB mRNA levels were slightly increased in the not4Δ mutant, and no CLB mRNA level was increased in the not1Δ or not2Δ mutant (Fig 3, Table 1). Thus, two catalytic subunits of the Ccr4-Not complex, Ccr4 and Pop2, are mainly involved in the levels of CLB mRNAs.

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Fig 3. Expression of CLB1, CLB2, CLB3, CLB4, CLB5, and CLB6 in wild-type, pop2Δ, not1, not2, and not4Δ mutant cells.

The mRNA levels of CLB1 (A), CLB2 (B), CLB3 (C), CLB4 (D), CLB5 (E), and CLB6 (F) in pop2Δ, not1Δ, not2Δ, and not4Δ mutant strains growing in YPD media relative to the wild-type strain. PGK1 (G) was used as a control. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 h of culture in YPD. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g003

The CLB6 mRNA level was significantly increased in the ccr4Δ mutant in synchronized culture

CLBs expression is known to be regulated during the cell cycle [1,14,15]. The increase in CLBs mRNA levels by the ccr4Δ mutation may be due to the ccr4Δ mutant being delayed in a particular phase during cell cycle. To examine this possibility, we analyzed a cell cycle progression of the ccr4Δ mutant and also examined the CLB1-6 mRNA levels in synchronized cultures. For this purpose, we utilized the MATa bar1Δ and MATa bar1Δ ccr4Δ cells, in which α-factor protease Bar1 was absent [28]. The MATa bar1Δ and MATa bar1Δ ccr4Δ cells were arrested in G1 phase with α-factor. The cells were then released from the G1 arrest by several washes with fresh media and allowed to progress into the S phase. In the bar1Δ cell, the S phase marker RNR1 peaked at 30 minutes, which was considered to be the S phase (Fig 4A). CLB5 and CLB6 also peaked at this time (Fig 4G and 4H). The peak of RNR1 of the bar1Δ ccr4Δ mutant was slightly delayed compared to that of the bar1Δ strain and reached the peak at about 50 minutes (Fig 4A). The peak of the M phase marker, SIC1, in the bar1Δ ccr4Δ mutant was also delayed in comparison with that in the bar1Δ strain (Fig 4B).

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Fig 4. The cell-cycle regulated expression of CLBs in the ccr4Δ mutant.

The qRT-PCR analysis data of RNR1 mRNA, SIC1 mRNA, and CLB mRNAs in the cell cycle synchronized bar1Δ mutant (black circle) and bar1Δ ccr4Δ mutant (gray square). Cell cycle was arrested in G1 phase by α-factor, and, after release, cells were collected from 0min (just before releasing) to 120min. The fold change of RNR1 mRNA, S phase marker, (A) and SIC1 mRNA, late M phase marker, (B) show cell cycle successfully progress both in the bar1Δ mutant and the bar1Δ puf5Δ mutant. The fold change of B-type cyclin mRNAs, CLB1 mRNA (C), CLB2 mRNA (D), CLB3 mRNA (E), CLB4 mRNA (F), CLB5 mRNA (G), CLB6 mRNA (H) are presented.

https://doi.org/10.1371/journal.pone.0268283.g004

In these synchronous cultures, the CLB6 mRNA level, which had the largest increase in the ccr4Δ mutant in the asynchronous culture (Fig 2F), was significantly increased in the bar1Δ ccr4Δ mutant compared to those in the bar1Δ cells over the cell cycle (Fig 4H). The CLB4 mRNA level was a little higher in the bar1Δ ccr4Δ mutant than those in the bar1Δ cells (Fig 4F). On the other hand, there were no significant increases in the CLB1, CLB2, CLB3, and CLB5 mRNA levels in the synchronous bar1Δ ccr4Δ mutant (Fig 4C, 4D, 4E and 4G). Thus, the increase in CLBs mRNA levels by the ccr4Δ mutation in asynchronous culture seemed to be partly due to the ccr4Δ mutant being delayed in cell cycle. However, since the mRNA levels of CLB6 and CLB4 were elevated in the bar1Δ ccr4Δ mutant over the cell cycle, it was also likely that mRNA degradation by Ccr4 regulates CLB levels.

To examine whether Ccr4 is involved in mRNA degradation of CLB mRNAs, we analyzed the half-lives of CLB mRNAs after transcription inhibition with thiolutin (Fig 5). The half-lives of CLB1-6 mRNAs in wild-type cells were 3 to 7 min, and those were clearly extended in the ccr4Δ mutant (Fig 5). It was surprising that the effects of the ccr4Δ mutation on the mRNA half-lives was similar in all CLB1-6 mRNAs. The significant increase of the CLB6 mRNA levels may be caused by the multiple effects of the ccr4Δ mutation, including mRNA degradation, cell cycle delay, or transcription.

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Fig 5. The half-lives of CLB1-CLB6 mRNAs in wild-type and the ccr4Δ mutant.

The bar1Δ and the bar1Δ ccr4Δ mutant cells were pre-cultured in YPD medium at 28°C overnight, and transferred into fresh YPD medium, and cultured until mid-exponential phase. Then, thiolutin was added, and samples were collected by centrifuge at 0, 1, 2, 5, 10, 20, 40, 80 minutes after exposure to thiolutin. The mRNA levels at each time point were determined by qRT-PCR. The half-lives (t_1/2) were calculated using Microsoft Excel. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g005

CLB6 expression is reported to be changed by various stresses, such as replication stress [30]. Then we examined whether the ccr4Δ mutation affects the CLB6 mRNA levels in the replication stress condition. The MATa bar1Δ and MATa bar1Δ ccr4Δ cells synchronized in G1 with α-factor were synchronously released into the S phase in the presence of hydroxy urea (HU), a reagent that generates replication stress by depleting the pool of dNTPs. As shown in Fig 6C, the CLB6 mRNA level peaked at 30 min in the bar1Δ cells and was kept at a high level. The CLB6 mRNA levels were increased more in the bar1Δ ccr4Δ mutant (Fig 6C). While the CLB5 mRNA level was not increased in the bar1Δ ccr4Δ mutant without replication stress (Fig 4G), the CLB5 mRNA level was also increased in the HU-treated bar1Δ ccr4Δ mutant cells (Fig 6B). Thus, Ccr4 seems to contribute the CLB5 and CLB6 mRNA levels in replication stress condition.

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Fig 6. The cell-cycle regulated expression of CLBs in the ccr4Δ mutant.

The qRT-PCR analysis data of RNR1, CLB5, and CLB6 mRNAs in the cell cycle synchronized bar1Δ mutant (black circle) and bar1Δ ccr4Δ mutant (gray square). Cell cycle was arrested in G1 phase by α-factor, and, after releasing in the presence of HU, cells were collected from 0 min (just before releasing) to 120 min. The fold change of RNR1 mRNA, S phase marker, (A) and SIC1 mRNA, late M phase marker, (B) show cell cycle successfully progress both in the bar1Δ mutant and the bar1Δ puf5Δ mutant. The fold change of B-type cyclin mRNAs, CLB1 mRNA (C), CLB2 mRNA (D), CLB3 mRNA (E), CLB4 mRNA (F), CLB5 mRNA (G), CLB6 mRNA (H) are presented.

https://doi.org/10.1371/journal.pone.0268283.g006

The Clb protein levels are not significantly increased in the ccr4Δ mutant

To examine whether the increased CLB mRNA levels by the ccr4Δ mutation confer the increased Clb protein levels, we next constructed the CLBx-HA-CLBx 3’ UTR plasmids (Fig 7). Using these plasmids, we measured the CLBx-HA mRNA levels and the Clbx-HA protein levels in wild-type and the ccr4Δ mutant cells. We used a specific primer to detect CLBx-HA mRNA, but not endogenous CLBx mRNA. Generally, all the CLB1-6-HA mRNA levels were increased in the ccr4Δ mutant compared to those in wild-type cells (Fig 7A, CLBx-HA mRNA, Table 3). The ratios were somewhat different from the results of endogenous CLB mRNAs. The CLB6-HA mRNA level was increased about 7-fold; the CLB1-HA, CLB2-HA, and CLB5-HA mRNA levels were increased about 4-fold; the CLB3-HA and CLB4-HA mRNA levels were increased less than 2-fold. In contrast to the results of the increased mRNA levels, we found that the Clb1-6-HA protein levels were not significantly increased in the ccr4Δ mutant compared to those in wild-type cells (Fig 7B, Clb1-6-HA protein, Table 3). The Clb6-HA protein level was increased about 2-fold; the Clb1-HA and Clb4-HA protein levels were increased about 1.5-fold; the Clb2-HA, Clb3-HA. and Clb5-HA protein levels were not increased. These results indicated that Ccr4 contributes the levels of CLB mRNAs, but the Clb protein levels are not increased in the ccr4Δ mutant (summarized in Table 3).

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Fig 7. Expression of CLB1-HA, CLB2-HA, CLB3-HA, CLB4-HA, CLB5-HA, and CLB6-HA in wild-type and ccr4Δ mutant cells harboring the CLBx-HA-CLBx 3’-UTR plasmid.

The mRNA (A) and protein (B) levels of CLB1-HA, CLB2-HA, CLB3-HA, CLB4-HA, CLB5-HA, and CLB6-HA in wild-type (WT) and ccr4Δ mutant cells growing in SC-ura media. The strains harboring the CLBx-HA-CLBx 3’ UTR plasmids were grown at 28°C. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 H of culture in SC-ura. Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-HA and anti-Pgk1 antibodies where Pgk1 was used as the loading control. The data show mean ± SEM (n = 3) of fold change of pr level from wild-type cells at 4 H of culture in SC-ura. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g007

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Table 3. Summary of ccr4Δ/wild-type ratio of mRNA and protein levels on different gene constructs.

All values written are standardized to their respective ccr4Δ/wild-type ratio.

https://doi.org/10.1371/journal.pone.0268283.t003

The coding sequences of CLB mRNAs are involved in the Ccr4-dependent regulation

Although it was reported that mRNA decay is usually mediated at the 5’ UTR and 3’ UTR ends [36], we examined whether the coding sequences of CLB genes are involved in mRNA levels. This was done by constructing the CLBx-HA-ADH1 3’ UTR plasmids (Fig 8). The same method of estimating the mRNA level by qRT-PCR using the HA primers and protein level by HA tagging were done.

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Fig 8. Expression of CLB1-HA, CLB2-HA, CLB3-HA, CLB4-HA, CLB5-HA, and CLB6-HA in wild-type and ccr4Δ mutant cells harboring the CLBx-HA-ADH1 3’ UTR plasmid.

The mRNA (A) and protein (B) levels of CLB1-HA, CLB2-HA, CLB3-HA, CLB4-HA, CLB5-HA and CLB6-HA in wild-type (WT) and ccr4Δ mutant strain growing in SC-ura media. The strains harboring the CLBx-HA-ADH1 3’ UTR plasmid were grown at 28°C. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 h of culture in Sc-ura. Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-HA and anti-Pgk1 antibodies where Pgk1 was used as the loading control. The data show mean ± SEM (n = 3) of fold change of protein level from wild-type cells at 4 H of culture in SC-ura. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g008

First, we examined the CLBx-HA-ADH1 3’ UTR mRNA levels in wild-type and the ccr4Δ mutant cells (Fig 8). All the CLB1-6-HA-ADH1 3’ UTR mRNA levels were increased in the ccr4Δ mutant compared to those in wild-type cells (Fig 8A, CLBx-HA mRNA). The CLB1-HA-ADH1 3’ UTR mRNA level was increased about 5-fold; the CLB2-HA-ADH1 3’ UTR, CLB4-HA-ADH1 3’ UTR, and CLB6-HA-ADH1 3’ UTR mRNA levels were increased about 3-fold; the CLB3-HA-ADH1 3’ UTR and CLB5-HA-ADH1 3’ UTR mRNA levels were increased with less than 2-fold.

When we compared the results of the CLBx-HA-CLBx 3’ UTR plasmids and the CLBx-HA-ADH1 3’ UTR plasmids (Figs 7A and 8A, Table 3), the increased fold of CLB5 and CLB6 mRNAs in ccr4Δ mutant was different. In the CLBx-HA-CLBx 3’ UTR plasmids, the CLB6-HA mRNA level was increased about 7-fold; CLB5-HA mRNA levels was increased about 4-fold (Fig 7A, Table 3). However, in the CLBx-HA-ADH1 3’ UTR plasmids, CLB6-HA-ADH1 3’ UTR mRNA levels was increased about 3-fold; CLB5-HA-ADH1 3’ UTR mRNA level was increased with less than 2-fold (Fig 8A, Table 3). These results suggest that the coding sequence of CLB6, but not that of CLB5, may have a role in mRNA level and that the 3’ UTRs of CLB5 and CLB6 may have a role in mRNA level.

Then we examined the Clbx-HA protein levels from the CLBx-HA-ADH1 3’ UTR plasmids in wild-type and the ccr4Δ mutant cells (Fig 8B, Clb1-6-HA protein). The Clb6-HA protein level was increased about 3-fold; the Clb4-HA protein level was increased about 1.7-fold; the Clb1-HA, Clb2-HA, Clb3-HA and Clb5-HA protein levels were not increased.

These results indicated that the coding sequences of some CLB mRNAs are involved in the Ccr4-dependent regulation, but again the Clb protein levels are not simply dependent on the mRNA levels (summarized in Table 3).

The 3’ UTR sequences of CLB mRNAs are involved in the Ccr4-dependent mRNA destabilization

The 3’ UTR of an mRNA is reported to be crucial for degradation, and this is conserved throughout eukaryotes [36]. Here, we assumed that the 3’ UTRs of CLB1-6 are also a key for its stability. To confirm this assumption, we constructed the MCM2 promoter-GFP-CLBx 3’ UTR plasmids which express the GFP-CLBx 3’ UTR mRNAs and GFP proteins from MCM2 promoter (Fig 9). Mcm2 protein is present through the cell cycle [37], the MCM2 promoter activity is not strong [38], and the changes in mRNA and protein levels are easy to detect. The different CLBx 3’ UTRs were compared with ADH1 3’ UTR by using GFP primers for qRT-PCR and GFP antibody for Western blot.

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Fig 9. Expression of GFP mRNA and protein in wild-type strain harboring the MCM2 promoter-GFP-CLBx 3’-UTR plasmids.

The mRNA (A) and protein (B) levels of in wild-type (WT) strains harboring the MCM2 promoter-GFP-CLBx 3’ UTR plasmids were grown at 30°C in SC-ura media. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 H of culture in Sc-ura. Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-GFP and anti-Pgk1 antibodies where Pgk1 was used as the loading control. The data show mean ± SEM (n = 3) of fold change of protein level from wild-type cells at 4 H of culture in SC-ura. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g009

First, we compared the GFP-CLB1-6 3’ UTR mRNA levels with the GFP-ADH1 3’ UTR mRNA level in wild-type cells. Generally, all the GFP-CLB1-6 3’ UTR mRNA levels were decreased than the GFP-ADH1 3’ UTR mRNA level, but its ratio differed in the genes (Fig 9A, GFP mRNA, Table 4). The CLB1, CLB2, and CLB6 3’ UTRs gave about 0.30-fold decrease; the CLB3, CLB4, and CLB5 3’ UTRs gave about 0.60-fold decrease. Then we examined the GFP protein levels from the MCM2 promoter-GFP-CLBx 3’ UTR plasmids in wild-type (Fig 9B, GFP protein, Table 4). In this case, it is consistent with the results of mRNA levels, the GFP protein levels from the CLB1-6 3’ UTR constructs were decreased than that of the ADH1 3’UTR construct. The CLB1, CLB2, and CLB6 3’ UTRs gave about 0.20-fold decrease of GFP protein level; the CLB3, CLB4, and CLB5 3’ UTRs gave about 0.60-fold decrease of GFP protein level. These decreases of GFP protein levels were correlated with GFP mRNA levels (Fig 9A and 9B, Table 4). These results indicated that the CLBx 3’UTRs have destabilizing sequence and the instability caused by the 3’ UTR sequence simply contributes the decrease of protein level in the GFP reporter construct.

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Table 4. Summary of GFP mRNA and protein levels of different 3’-UTRs of CLB.

All gene constructs were transformed into wild-type strain and standardized on ADH1 3’-UTR.

https://doi.org/10.1371/journal.pone.0268283.t004

To investigate mRNA stability control of CLB1-6 3’UTR sequence involves Ccr4, we examined the GFP-CLBx 3’ UTR mRNA levels and the GFP protein levels in wild-type and the ccr4Δ mutant cells (Fig 10, Table 3). All GFP-CLBx 3’ UTR mRNA levels were increased in the ccr4Δ mutant compared to those in wild-type cells, while the GFP-ADH1 3’ UTR mRNA level was not (Fig 10A, GFP mRNA, Table 3). The GFP-CLB6 3’ UTR was about 6.5-fold increase in the ccr4Δ mutant compared to that in wild-type cells; the GFP-CLB2 3’ UTR was about 4-fold increased; the GFP-CLB1 3’ UTR and GFP-CLB5 3’ UTR were about 3-fold increased; the GFP-CLB3 3’ UTR and GFP-CLB4 3’ UTR were about 2.5-fold increased. Consistent with the results of mRNA levels, the GFP protein levels from the MCM2 promoter-GFP-CLBx 3’ UTR constructs were increased in the ccr4Δ mutant than those in wild-type cells (Fig 10B, GFP protein). The GFP protein level from the GFP-ADH1 3’ UTR construct was similar in the ccr4Δ mutant and wild-type cells (Fig 10B, GFP protein, Table 3). The GFP protein levels derived from GFP-CLB1 3’ UTR and GFP-CLB6 3’ UTR were about 15-fold-increased in the ccr4Δ mutant compared to that in wild-type cells; the GFP protein levels derived from GFP-CLB2 3’ UTR and GFP-CLB4 3’ UTR were about 12-fold-increased; the GFP protein level derived from GFP-CLB5 3’ UTR was about 9-fold-increased; the GFP protein level derived from GFP-CLB3 3’ UTR was about 6-fold-increased (Fig 10B, GFP protein, Table 3). These results indicated that mRNA instability mediated by CLB1-6 3’ UTR involves Ccr4 and that the instability caused by the 3’ UTR sequence simply contributes the decrease of protein level in the GFP reporter construct (summarized in Table 3). This result is different from that of the CLBx-HA plasmid (summarized in Table 3).

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Fig 10. Expression of GFP mRNA and protein in wild-type and ccr4Δ mutant cells harboring the MCM2 promoter-GFP-CLBx 3’ UTR plasmids.

The mRNA (A) and protein (B) levels of GFP in wild-type (WT) and ccr4Δ strains harboring the MCM2 promoter-GFP-CLBx 3’ UTR plasmids were grown at 28°C in SC-ura media. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 H of culture in Sc-ura. Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-GFP and anti-Pgk1 antibodies where Pgk1 was used as the loading control. The data show mean ± SEM (n = 3) of fold change of protein level from wild-type cells at 4 H of culture in SC-ura. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g010

The CLB6 coding sequence contains the three regions for mRNA destabilization

From the above results of the CLB6-HA-ADH1 3’ UTR plasmid, we have shown that CLB6 mRNA and protein levels were most increased in the ccr4Δ mutant (Fig 8A and 8B, Table 3). We suspected that a certain sequence within the coding region is responsible for the destabilization of the CLB6 mRNA. To determine the sequence for mRNA instability, we constructed a series of deletions in the CLB6-HA-CLB6 3’ UTR gene construct, deletions of 150 base pairs corresponding 50 amino acids starting from the 2^nd bp after the start codon with a total of 15 deletions (Fig 11A). We then compared the mRNA level by qRT-PCR and protein level by western blot using the HA tag.

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Fig 11. Deletion analysis of the CLB6 coding sequence.

Deletion sequences were done by deletion 10 amino acid base pairs per region (Deletion 1 to 15) relative to the ND (no deletion). The Cyclin Domain 1 and 2 are highlighted as grey (A). The mRNA (B) and protein (C) levels comparing the full-length and deletion strains transformed in wild-type strain grown in SC-ura media at 30°C. Protein levels are prepared on two gels from D1-7 and 8–15 and were quantified relative to the ND gene. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 H of culture in SC-ura. Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-HA and anti-Pgk1 antibodies where Pgk1 was used as the loading control. The data show mean ± SEM (n = 3) of fold change of protein level from wild-type cells at 4 H of culture in SC-ura. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g011

First, we investigated the mRNA level. Our results showed that certain deletion sequences significantly increased the CLB6 mRNA by 1.5 to 2-fold. These were the sequences, D4 and D5, D9 and D10, and D14 (Fig 11B). As for the other deletions, we suspect that they are not crucial for destabilization as they showed incomparable results with that of the complete sequence.

Next, we investigated the protein level. Unsurprisingly, the same deletion sequences with the increase in mRNA level also showed an increase in protein level. That is, D4 and D5, D9 and D10, and D14 by 3 to 9-fold increase (Fig 11C). The other deletions did not show any significant difference with the complete sequence. From these data, we were able to deduce the certain sequences within the coding region have an immense effect on the destabilization of CLB6.

The 3’ UTR sequence is crucial for CLB6 expression

Comparing the CLB1-6 3’ UTR, we have shown that CLB6 3’ UTR was significantly decreased by Ccr4 both on the mRNA and protein level (Fig 10A and 10B, Table 4). This implicated that CLB6 3’ UTR contains a sequence responsible for proper degradation. To test this hypothesis, we constructed multiple deletions at the 3’ UTR of CLB6. We used the MCM2 promoter-GFP-CLB6 3’ UTR plasmid and prepared deletions of 30 base’s starting from the 2^nd bp after the start of 3’ UTR (Fig 12A). Then we measured both the mRNA level and protein level by qRT-PCR and western blot, respectively. Comparing the mRNA level among the deletion constructs, there was no significant difference in all deletion constructs (Fig 12B). Though, D3 and D4 showed slightly higher mRNAs which were almost close to being significant. The lowest was deletion 1 which was close to the undeleted sequence. However, the increase was shown at the protein level on D3 and D4 (Fig 12C). This may further explain that the control mediated by the 3’ UTR only happens at the protein level and not on the mRNA. This elucidates that those regions have a specific sequence critical for the degradation of CLB6.

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Fig 12. Deletion analysis of the CLB6 3’ UTR.

Deletion sequences were done by ~30 base pairs deletion per region (Deletion 1 to 6) starting from the 5’ end after the GFP relative to the full-length. The mRNA (B) and protein level (C) in wild-type strain harboring gene construct of MCM2-GFP-CLB6 3’ UTR were grown at 30°C in SC-ura media. mRNA levels were quantified by qRT-PCR analysis and were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-GFP and anti-Pgk1 antibodies where Pgk1 was used as the loading control. It was plotted as the fold change relative to the ‘no deletion strain’ cells at 4H of culture. The data show mean ± SEM (n = 3) *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g012

The increase of CLB6 expression in the ccr4Δ mutant is not dependent on the Puf protein

Puf family RNA-binding proteins are known to recruit the Ccr4-Not deadenylase complex to the mRNAs promote decay [9,39,40]. From the previous results, we have shown that CLB6 was significantly destabilized by Ccr4 on both the coding and 3’ UTR regions. Using the MCM2 promoter-GFP-CLB6 3’ UTR plasmid, we investigated the GFP mRNA and protein level on the deletion strains of PUF1-6 genes and compare them with the ccr4Δ mutant. Results showed that there was no significant increase for both the mRNA (Fig 13A) and protein level (Fig 13B) on the puf1-6Δ mutants. These results suggest that Puf proteins are not involved in the Ccr4-dependent mRNA destabilization of CLB6.

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Fig 13. Expression of GFP on gene construct of MCM2-GFP-CLB6 3’-UTR with pufΔ mutants.

The mRNA (A) and protein (B) levels of GFP in wild-type (WT), puf1Δ, puf2Δ, puf3Δ, puf4Δ, puf5Δ, puf6Δ, and ccr4Δ strains harboring the gene constructs of MCM2-GFP-CLB6 3’UTR grown at 28°C in SC-ura. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 H of culture in SC-ura. Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-GFP and anti-Pgk1 antibodies where Pgk1 was used as the loading control. The data show mean ± SEM (n = 3) of fold change of protein level from wild-type cells at 4 H of culture in SC-ura. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g013

The deletion of WHI3 showed an increased level of endogenous CLB mRNA

To further investigate the possible involvement of other RNA-binding proteins on the expression of CLB genes, we prepared several deletion strains, whi3Δ, caf20Δ, and eap1Δ mutants. Whi3 and Caf20 are reported to interact with CLB mRNA [41,42]. Eap1 was also selected because Eap1 is an eIF4E-binding protein, similar to Caf20 [43]. Using the same primers for the endogenous mRNA of CLB1 to CLB6, we measured the mRNA levels. Comparing the different deletion strains, we found that the whi3Δ mutant showed a significant increase in all CLB mRNAs (Fig 14, Table 1). These results suggest that Whi3 may have a role in mRNA destabilization of CLB mRNAs.

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Fig 14. Expression of CLB1, CLB2, CLB3, CLB4, CLB5, and CLB6 in whi3Δ, caf20Δ, eap1Δ and caf20Δ eap1Δ strains.

The mRNA levels of CLB1 (A), CLB2 (B), CLB3 (C), CLB4 (D), CLB5 (E), and CLB6 (F) in ccr4Δ mutant strain growing in YPD medium relative to the wild-type strain. PGK1 (G) was used as positive control. mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using 2^-ΔΔCt method normalized to ACT1 reference gene. The data show mean ± SEM (n = 3) of fold change of mRNA level from wild-type cells at 4 H of culture in YPD. *P < 0.05, **P < 0.01 as determined by Tukey’s test.

https://doi.org/10.1371/journal.pone.0268283.g014

Discussion

Ccr4 is involved in the expression of CLB genes

In this study, we examined CLB1-6 mRNA levels in the ccr4Δ mutant. The first experiment was to investigate the significance of Ccr4 on the endogenous CLB1-6 mRNAs. The data in Fig 1 showed that all CLB1-6 mRNA levels were increased in the ccr4Δ mutant. Although the increase in mRNA happens for all the CLB1-6 genes, it was most prominent in CLB6. While the levels of CLB1-6 mRNAs are increased in the ccr4Δ mutant compared to wild-type, the mRNA levels of G1 cyclin genes, CLN1-3, are not increased in the ccr4Δ mutant (Table 1). Recent analyses [44] showed that poly A tail lengths of CLB1-6 mRNA are increased in the ccr4Δ mutant (poly A tail lengths in wild-type/ccr4Δ, CLB1: 36.5/48.4, CLB2: 38.8/ 46.8, CLB3: 50.7/ 49.7, CLB4: 33.0/ 49.7, CLB5: 38.1/ 54.4, CLB6: 0.01/ 51.7). These data are consistent with our observation that CLB1-6 mRNA levels are increased in the ccr4Δ mutants and that CLB6 mRNA level is most affected by the ccr4Δ mutation. The CLB6 mRNA level was also significantly increased in the ccr4Δ mutant in synchronized culture (Fig 3). On the other hand, the half-lives of CLB1-CLB6 mRNAs are similar: 3 to 7 mins in wild-type, 10–15 mins in the ccr4Δ mutant. The significant increase of the CLB6 mRNA level may be caused by the multiple effects of the ccr4Δ mutation, including mRNA degradation, cell cycle delay, or transcription.

We also examined whether the Clb1-6 protein levels were also affected by the ccr4Δ mutation. Unexpectedly, although the CLB1-6 mRNA levels were increased in the ccr4Δ mutant, only Clb2, Clb4, and Clb6 protein levels showed a slight increase in the ccr4Δ mutant. Since we have previously shown that Ccr4 plays more important role in the regulation of gene expression in the stationary phase than in the log phase [11], elevated mRNA levels may not be important in the log phase but may be more important in the stationary phase. Since it is reported that translation efficiency is coupled with mRNA stability [45], the translation efficiency of CLB mRNAs may be involved in both mRNA and protein levels. Indeed, codon adaptation index of Clb proteins is relatively low: Clb1 0.14, Clb2, 0.12, Clb3 0.13, Clb4 0.13, Clb5 0.13, Clb6 0.17, Pgk1 0.81. Inefficient translation would account for the observation that increased mRNA levels do not confer the increase in the Clb protein levels. Recent study shows that the Ccr4-Not complex monitors the translating ribosome for codon optimality and links the decoding efficiency with mRNA stability [46]. Thus, Ccr4 may be directly involved in not only mRNA degradation but also translation efficiency.

Both coding sequence and 3’ UTR of CLB6 mRNA are involved in gene expression

We investigated the role of the coding region of CLBs in mRNA expression using the CLBx-HA-ADH1 3’ UTR plasmid and the CLBx-HA-CLBx 3’ UTR plasmid. Since all the CLB1-6-HA-ADH1 3’ UTR mRNA levels were increased in the ccr4Δ mutant compared to those in wild-type cells, the coding region of CLBs seems to have a role in mRNA expression. We suspected that a certain sequence within the coding region is responsible for the destabilization of the CLB mRNAs. Then we examined the region responsible for mRNA destabilization within CLB6 coding sequence. We found that the CLB6 coding sequences have three regions, 76–150 (the regions of D4 and D5), 201–278 (the regions of D9 and D10), and 201–278 (the region of D14) (Fig 7). Of these regions, 76–150 (the regions of D4 and D5) are different from the regions that encode the cyclin domain. The regions 201–278 (the regions of D9 and D10) and 201–278 (the region of D14) are the regions that encode the cyclin domain. The region encoding the cyclin domain may be involved in degradation at the mRNA level as well as at the protein level.

From the comparison of the results of the CLBx-HA-CLBx 3’ UTR plasmids and the CLBx-HA-ADH1 3’ UTR plasmids (Figs 7 and 8, Table 3), we found that the 3’ UTRs of CLB5 and CLB6 may have a role in mRNA level. This was further confirmed with the experiments using the MCM2 promoter-GFP-CLBx 3’ UTR plasmids. Since the CLB6 3’ UTR has the strongest effect, we examined the region responsible for mRNA destabilization within CLB6 3’ UTR. We found that the CLB6 3’ UTR has a region 37–83 (the regions of D3 and D4) (Fig 12). The results of D3 and D4 showed that the mRNA level did not rise so much, but the protein level rose significantly (Fig 12). Therefore, CLB6 3’ UTR controls both mRNA stability and translation efficiency. Our results suggest that the presence of sequences that control the amount of mRNA in both the coding region and the 3’ UTR determines the total amount of mRNA.

Whi3, but not Puf proteins, is involved in the expression of CLB mRNA

Puf family RNA-binding proteins are known to recruit the Ccr4-Not deadenylase complex to the mRNAs promote decay [9,39,40]. However, our results using the MCM2 promoter-GFP-CLBx 3’ UTR plasmids suggested that Puf1-6 proteins are not involved in the Ccr4-dependent mRNA destabilization of CLB6. We found that Whi3 has a role in mRNA destabilization of CLB mRNAs. Whi3 is identified to have a role in stress-dependent RNA processing on various stress conditions [47] and a post-transcriptional control regulator of genes involved in cell division [48]. Whi3 is also known to bind to the CLN3 mRNA and can cause sterility in old yeast cells [49]. Our results show that there was a significant increase in endogenous CLB mRNA with the deletion of this protein. Whi3 is known to have a protein-mRNA interaction with CLB6 and this is a possible candidate as a regulatory-binding protein which interacts with Ccr4.

In this study, we have demonstrated the role of Ccr4 on the regulation of CLB expression. We have shown that this protein is a rate-limiting step on post-transcriptional regulation. Of all the CLB genes, it was CLB6 which showed a potential to be degraded by Ccr4 by recognizing both its coding region and 3’ UTR. For further study, we plan to detect how exactly Ccr4 stabilizes the initiation of DNA synthesis stage, specifically the activation of Cdc28 via CLB6.

Supporting information

S1 Fig. Expression of Clb1-HA, Clb2-HA, Clb3-HA, Clb4-HA, Clb5-HA, and CLb6-HA proteins in wild-type and ccr4Δ mutant cells harboring the CLBx-HA-CLBx 3’ UTR plasmid.

https://doi.org/10.1371/journal.pone.0268283.s001

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S2 Fig. Expression of Clb1-HA, Clb2-HA, Clb3-HA, Clb4-HA, Clb5-HA, and CLb6-HA proteins in wild-type and ccr4Δ mutant cells harboring the CLBx-HA-ADH1 3’ UTR plasmid.

https://doi.org/10.1371/journal.pone.0268283.s002

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S3 Fig. Expression of GFP protein in wild-type strain harboring the MCM2 promoter-GFP-CLBx 3’ UTR plasmids.

https://doi.org/10.1371/journal.pone.0268283.s003

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S4 Fig. Expression of GFP protein in wild-type and ccr4Δ mutant cells harboring the MCM2 promoter-GFP-CLBx 3’ UTR plasmids.

https://doi.org/10.1371/journal.pone.0268283.s004

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S5 Fig. Deletion analysis of the CLB6 coding sequence using the CLB6-HA-CLB6 3’ UTR plasmid.

Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-HA and anti-Pgk1 antibodies where Pgk1 was used as the loading control.

https://doi.org/10.1371/journal.pone.0268283.s005

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S6 Fig. Deletion analysis of the CLB6 3’ UTR using the MCM2 promoter-GFP-CLB6 3’-UTR plasmid.

Protein levels were quantified by preparing cell extracts collected at log phase (4 H) for immunoblotting with anti-GFP and anti-Pgk1 antibodies where Pgk1 was used as the loading control.

https://doi.org/10.1371/journal.pone.0268283.s006

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S1 Table. Strains used in this study.

https://doi.org/10.1371/journal.pone.0268283.s007

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S2 Table. Plasmids used in this study.

https://doi.org/10.1371/journal.pone.0268283.s008

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S3 Table. Primers used for cloning of CLBx 3’ UTR.

https://doi.org/10.1371/journal.pone.0268283.s009

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S4 Table. Primers used for constructing CLBx-HA-ADH1 3’ UTR plasmid.

https://doi.org/10.1371/journal.pone.0268283.s010

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S5 Table. Primers used for constructing CLBx-HA-CLBx 3’ UTR plasmid.

https://doi.org/10.1371/journal.pone.0268283.s011

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S6 Table. Primers used for the deletion sequences of CLB6 coding region of 50 amino acids each.

https://doi.org/10.1371/journal.pone.0268283.s012

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S7 Table. Primers used for the deletion sequences of CLB6 3’ UTR of 30 bases each.

https://doi.org/10.1371/journal.pone.0268283.s013

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S8 Table. Primers for RT-PCR used in this study.

https://doi.org/10.1371/journal.pone.0268283.s014

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Acknowledgments

We thank the members of Molecular Cell Biology Laboratory for valuable discussions.

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Figures

Abstract

Introduction

Materials and methods

Strains and media

Plasmids

Cell sampling, sample preparation, and RNA extraction

qRT-PCR analysis and microarray data

Western blot analysis

Cell cycle synchronization by α-factor block and release

Determination of half-lives of CLB mRNAs

Results

The ccr4Δ mutation showed a synthetic growth defect with the mih1Δ mutation

The CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant

The CLB6 mRNA level was significantly increased in the ccr4Δ mutant in synchronized culture

The Clb protein levels are not significantly increased in the ccr4Δ mutant

The coding sequences of CLB mRNAs are involved in the Ccr4-dependent regulation

The 3’ UTR sequences of CLB mRNAs are involved in the Ccr4-dependent mRNA destabilization

The CLB6 coding sequence contains the three regions for mRNA destabilization

The 3’ UTR sequence is crucial for CLB6 expression

The increase of CLB6 expression in the ccr4Δ mutant is not dependent on the Puf protein

The deletion of WHI3 showed an increased level of endogenous CLB mRNA

Discussion

Ccr4 is involved in the expression of CLB genes

Both coding sequence and 3’ UTR of CLB6 mRNA are involved in gene expression

Whi3, but not Puf proteins, is involved in the expression of CLB mRNA

Supporting information

S1 Fig. Expression of Clb1-HA, Clb2-HA, Clb3-HA, Clb4-HA, Clb5-HA, and CLb6-HA proteins in wild-type and ccr4Δ mutant cells harboring the CLBx-HA-CLBx 3’ UTR plasmid.

S2 Fig. Expression of Clb1-HA, Clb2-HA, Clb3-HA, Clb4-HA, Clb5-HA, and CLb6-HA proteins in wild-type and ccr4Δ mutant cells harboring the CLBx-HA-ADH1 3’ UTR plasmid.

S3 Fig. Expression of GFP protein in wild-type strain harboring the MCM2 promoter-GFP-CLBx 3’ UTR plasmids.

S4 Fig. Expression of GFP protein in wild-type and ccr4Δ mutant cells harboring the MCM2 promoter-GFP-CLBx 3’ UTR plasmids.

S5 Fig. Deletion analysis of the CLB6 coding sequence using the CLB6-HA-CLB6 3’ UTR plasmid.

S6 Fig. Deletion analysis of the CLB6 3’ UTR using the MCM2 promoter-GFP-CLB6 3’-UTR plasmid.

S1 Table. Strains used in this study.

S2 Table. Plasmids used in this study.

S3 Table. Primers used for cloning of CLBx 3’ UTR.

S4 Table. Primers used for constructing CLBx-HA-ADH1 3’ UTR plasmid.

S5 Table. Primers used for constructing CLBx-HA-CLBx 3’ UTR plasmid.

S6 Table. Primers used for the deletion sequences of CLB6 coding region of 50 amino acids each.

S7 Table. Primers used for the deletion sequences of CLB6 3’ UTR of 30 bases each.

S8 Table. Primers for RT-PCR used in this study.

Acknowledgments

References