A Novel Gene, msa1, Inhibits Sexual Differentiation in Schizosaccharomyces pombe

Corresponding author: Makoto Kawamukai, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan., kawamuka{at}life.shimane-u.ac.jp (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sexual differentiation in the fission yeast Schizosaccharomyces pombe is triggered by nutrient starvation or by the presence of mating pheromones. We identified a novel gene, msa1, which encodes a 533-aa putative RNA-binding protein that inhibits sexual differentiation. Disruption of the msa1 gene caused cells to hypersporulate. Intracellular levels of msa1 RNA and Msa1 protein diminished after several hours of nitrogen starvation. Genetic analysis suggested that the function of msa1 is independent of the cAMP pathway and stress-responsive pathway. Deletion of the ras1 gene in diploid cells inhibited sporulation and in haploid cells decreased expression of mating-pheromone-induced genes such as mei2, mam2, ste11, and rep1; simultaneous deletion of msa1 reversed both phenotypes. Overexpression of msa1 decreased activated Ras1^Val17-induced expression of mam2. Phenotypic hypersporulation was similar between cells with deletion of only rad24 and both msa1 and rad24, but simultaneous deletion of msa1 and msa2/nrd1 additively increased hypersporulation. Therefore, we suggest that the primary function of Msa1 is to negatively regulate sexual differentiation by controlling the expression of Ste11-regulated genes, possibly through the pheromone-signaling pathway.

THE haploid cells of Schizosaccharomyces pombe mate and initiate meiosis during nutritional starvation; these cells subsequently form ascospores after undergoing karyogamy, premeiotic DNA synthesis, meiosis I, and meiosis II (YAMAMOTO et al. 1997 ). Starvation induces expression of the ste11 gene that encodes a key transcription factor, which, in turn, upregulates transcription of several genes involved in conjugation, meiosis, and sporulation (SUGIMOTO et al. 1991 ). One of these upregulated genes, mei2, encodes a well-characterized RNA-binding protein that is absolutely required for meiosis (WATANABE and YAMAMOTO 1994 ). Pat1/Ran1 kinase inhibits meiosis by negatively regulating both Ste11 and Mei2 through phosphorylation (LI and MCLEOD 1996 ; WATANABE et al. 1997 ). Phosphorylation of Mei2 inhibits its ability to bind meiRNA (SATO et al. 2002 ) and converts Mei2 into a substrate suitable for ubiquitin-dependent proteolysis (KITAMURA et al. 2001 ). In nitrogen-starved diploid cells, transcription of mei3 increases; its gene product, Mei3, inhibits Pat1/Ran1 through a pseudosubstrate mechanism, thereby allowing Mei2 to initiate meiosis (LI and MCLEOD 1996 ).

Sexual differentiation in S. pombe is regulated primarily by three signaling pathways: the cAMP pathway, the stress-responsive pathway, and the pheromone-signaling pathway (YAMAMOTO et al. 1997 ). Regulation of the cAMP pathway in S. pombe consists of many molecular interactions. For example, adenylyl cyclase, encoded by the cyr1 gene, generates cAMP from ATP (MAEDA et al. 1990 ; KAWAMUKAI et al. 1991 ); trimeric G protein controls the activity of adenylyl cyclase through a nutrient-sensing mechanism (ISSHIKI et al. 1992 ); and cAMP phosphodiesterase, encoded by the pde1 gene, downregulates the cAMP pathway by converting cAMP to AMP (MOCHIZUKI and YAMAMOTO 1992 ). The intracellular level of cAMP decreases in nutrient-starved S. pombe cells as they exit the vegetative cycle to enter the stationary phase (MAEDA et al. 1990 ; KAWAMUKAI et al. 1991 ), but an experimentally high level of protein kinase A (PKA) activity inhibits initiation of sexual differentiation (MAEDA et al. 1994 ; YAMAMOTO 1996 ). The cAMP-dependent PKA holoenzyme consists of a catalytic subunit encoded by pka1 (MAEDA et al. 1994 ) and a regulatory subunit encoded by cgs1 (DEVOTI et al. 1991 ). PKA regulates expression of meiosis-specific genes such as ste11 and, as a consequence, mei2 (DEVOTI et al. 1991 ; SUGIMOTO et al. 1991 ; WU and MCLEOD 1995 ). In vitro, PKA phosphorylates Rst2, a zinc-finger protein that binds the upstream region of ste11 (HIGUCHI et al. 2002 ). Low levels of intracellular cAMP during starvation decrease activity of cAMP-dependent PKA, thereby decreasing downregulation of transcription factor Rst2 and triggering expression of ste11 (KUNITOMO et al. 2000 ).

The stress-responsive pathway in S. pombe is required for initiation of mating and progression through meiosis. The primary stress-responsive pathway components are the following: two MAPKK kinases, Wis4/Wik1/Wak1 and Win1; one MAPK kinase, Wis1, and one mitogen-activated protein (MAP) kinase, Phh1/Sty1/Spc1 (WARBRICK and FANTES 1991 ; KATO et al. 1996 ; SAMEJIMA et al. 1997 , SAMEJIMA et al. 1998 ). Loss of function of wis1, phh1/sty1/spc1, or atf1/gad7 greatly reduces ste11 transcription (TAKEDA et al. 1995 ; KANOH et al. 1996 ; SHIOZAKI and RUSSELL 1996 ). MAP kinase Phh1/Sty1/Spc1 phosphorylates the heterodimeric transcription factor Atf1/Gad7-Pcr1 (KON et al. 1998 ), which is required to activate expression of ste11.

The pheromone-signaling pathway includes Ras1, Byr2 (a MAPKK kinase), Byr1 (a MAPK kinase), and Spk1 (a MAP kinase; FUKUI et al. 1986 ; NADIN-DAVIS and NASIM 1988 ; TODA et al. 1991 ; WANG et al. 1991 ; NEIMAN et al. 1993 ). Binding of the pheromone to its receptor transmits a signal to Gpa1 (OBARA et al. 1991 ), the -subunit of the trimeric G protein, which presumably activates Byr2 with the help of Ras1. Byr2 can also be activated by Ste4 (OKAZAKI et al. 1991 ; BARR et al. 1996 ), a leucine-zipper protein that is capable of self-activation through homodimerization (TU et al. 1997 ); Byr2 can be downregulated by 14-3-3 proteins (OZOE et al. 2002 ). Activated Byr2 in turn signals Byr1, which signals Spk1 to initiate conjugation and sporulation. Loss of function of any component of the pheromone-signaling pathway causes S. pombe cells to become sterile.

We previously developed nine distinguishable hypersporulating S. pombe mutants, sporulation abnormal mutant 1 (sam1)–sam9, by mutating wild-type strains using ethyl methanesulfonate; sam1–9 sporulate on nutrient-rich medium (YEA) and have been partially characterized (KATAYAMA et al. 1996 ).

In this study, we used a gene library to screen for a suppressor of hypersporulation in a sam1 mutant and isolated a new gene, msa1, which encoded a putative RNA-binding protein and also a known gene, msa2/nrd1 (TSUKAHARA et al. 1998 ). We report here that the msa1 gene controlled sexual differentiation through inhibition of transcription of meiosis-inducing genes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, media, and genetic manipulation:
The strains of S. pombe used in this study are listed in Table 1. Standard yeast culture media and genetic manipulations were used, as described previously (ALFA et al. 1993 ; KAISER et al. 1994 ). S. pombe strains were grown in complete YEA medium (0.5% yeast extract, 2% glucose, and 0.0075% adenine) or in the synthetic minimal medium, PM (0.3% potassium hydrogen phthalate, 0.22% sodium phosphate, 0.5% ammonium chloride, 2% glucose, vitamins, minerals, and salts), with added appropriate auxotrophic supplements (0.0075% adenine, leucine, uracil, and histidine) when required (ALFA et al. 1993 ). Electroporation was used to transform yeast cells (PRENTICE 1992 ). Sporulation was detected by iodine vapor staining (GUTZ et al. 1974 ). Escherichia coli DH5 grown in Luria broth medium (1% polypepton, 0.5% yeast extract, 1% sodium chloride) hosted all plasmid manipulations, and standard methods were used for DNA manipulations (SAMBROOK et al. 1989 ).

Isolation of the msa1 gene:
HS412 cells were transformed using a S. pombe genomic library that had been constructed in the pWH5 vector (KAWAMUKAI 1999 ). Cells were spread on PM medium agar plates and incubated at 30° for 5 days. Cells were exposed to iodine vapor and colonies that did not stain with iodine vapor (i.e., no spores were present) were selected from the transformants. From 10⁵ transformants, nonsporulating clones were screened and plasmids from these strains were rescued in E. coli. Partial DNA sequencing and subcloning analysis of the sam1 suppressor genes identified two types of clones, msa1 (SPAC13G7.13c) and msa2 (SPAC2F7.11). For genomic integration, a 5-kb BamHI-PstI fragment of the genomic region from pW1-12 was cloned into a pYC11 plasmid, which is the derivative of pBluescript KS(+) carrying the Saccharomyces cerevisiae LEU2 gene (TAKAHASHI et al. 1992 ), and the resulting plasmid was named pYC11-3601. The cDNA clone of msa1 was isolated from a cDNA library constructed in ZAPII (KAWAMUKAI et al. 1992 ) by plaque hybridization using the genomic fragment as a probe (Fig 1A).

View larger version (82K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Cloning, disruption, and the nucleotide sequence of the msa1 gene. (A) Restriction map, subcloning, and disruption of the msa1 gene. The arrow indicates the region and direction of the msa1 ORF. Restriction sites for PstI (P), EcoRV (RV), HindIII (H), and BamHI (B) are shown. The complementation ability of each subclone was examined using HT3 (h90 sam1 leu1-32). Two PstI sites are shown within the pWH5 vector. (B) Nucleotide sequence of msa1 and its predicted amino acid sequence. The conserved amino acid sequences of RNP1 and RNP2 are underlined. (C) The RRM regions of msa1 aligned with those of other RNA-binding proteins, which include S. cerevisiae Rim4, mouse RBM3, human Tra2, human SRp30c, and Arabidopsis SF2. Identical residues are shown as white letters on black background.

Construction of msa1 and msa2 deletion mutants:
A 4.6-kb HindIII-PstI genomic fragment containing msa1 from pW1-12 (Fig 1) was introduced into the plasmid vector pUC118. The 1-kb EcoRV-EcoRV region that contained 60% of the msa1 open reading frame (ORF) was replaced with the 1.8-kb HincII DNA fragment of the ura4 gene from pHSG398-ura4 (TANAKA et al. 1999 ) and the resulting plasmid was named pUC118-msa1::ura4⁺. A 5.4-kb PstI-HindIII fragment derived from pUC118-msa1::ura4⁺ was used to transform the SP870 or SP335 strain (KAWAMUKAI et al. 1992 ) to make msa1 deletion mutants. Stable Ura⁺ transformants were selected, and the msa1 locus was analyzed by Southern hybridization with the probes of both msa1 and ura4. Southern hybridization was done as previously described (SAMBROOK et al. 1989 ). Similarly, to make the msa2 deletion mutants, the 1.4-kb SphI-SphI region containing 85% of the msa2 ORF in pUC118-msa2 was replaced with the 1.8-kb SphI DNA fragment of the ura4 gene, yielding pUC118-msa2::ura4⁺. pUC118-msa2 was derived by inserting the 4-kb HindIII fragment from originally screened clone pW136-19 into pUC118. The plasmid pUC118-msa2::ura4⁺ was made linear to transform SP870 yielding HT211 (msa2::ura4⁺). Proper disruption in HT211 was confirmed by Southern hybridization. HT81 (msa1::ura4⁺ msa2::ura4⁺) was isolated by crossing HT12 (ade6-216 msa1::ura4⁺) with HT211 (ade6-210 msa2::ura4⁺) and subsequent dissection of tetrad.

Construction of various double mutants:
The double mutant of mas1 with a variety of mutants, including pde1, cgs1, phh1, rad24, gpa1, byr1, byr2, ras1, spk1, and ste4 in Table 1, are all derived by crossing their parental strains that retain different ade markers and subsequent dissection of tetrads. In tetrads, only a nonparental segregant (2Ura⁺, 2Ura^–) was selected for isolating proper double mutants. Typically, HT11 (ade6-210 msa1::ura4⁺) and JZ666 (ade6-216 pde1::ura4⁺) were crossed, the diploids were allowed to sporulate, and spores were subjected to tetrad analysis. HT43 (msa1::ura4⁺ pde1::ura4⁺) was isolated as one of the nonparental tetrads. All other double mutants listed in Table 1 were derived in a similar way.

Plasmids:
Plasmid manipulation and bacterial transformation were performed using standard techniques (SAMBROOK et al. 1989 ). pALmsa1 was constructed by inserting a 5-kb BamHI-PstI fragment of genomic region from pW1-12. The msa1 gene was amplified by PCR using primer oligonucleotides (msa1FL) in Table 2. Amplified msa1 was digested with SalI and SmaI and then ligated with pREP1, pREP41, or pREP81 (MAUNDRELL 1993 ), which had previously been digested with SalI and SmaI to generate pREP1msa1, pREP41msa1, and pREP81msa1. pSLF273-msa1 was constructed by inserting the same fragment in the SalI and SmaI sites of pSLF273 (FORSBURG and SHERMAN 1997 ). The deletion derivatives from pSLF273-msa1 used in Fig 3 were constructed either by using a primer set described in Table 2 or by digestion with restriction enzymes. The nmt1 promoters and its derivatives were repressed by addition of 50 µg/ml thiamine to the media (MAUNDRELL 1990 ).

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Examining msa1 function in cell growth and conjugation. (A) The h– wild-type (SP319) cell and h– msa1 (HT21) cell were grown in PM medium to midlog phase. Each strain was then inoculated into PM medium at 105 cells/ml and incubated at 30°. A portion of each culture was removed at the indicated times, and cells were counted using a hemocytometer. (B) Conjugation efficiency of msa1 cells at various glucose concentrations. The h90 wild-type (SP66) cell and h90 msa1 (HT11) cell were grown in PM medium to midlog phase. Each strain was then inoculated in PM medium containing 2% glucose or in PM medium containing 0.5% glucose and was incubated at 30° for the indicated time; the number of zygotes was counted. Mating rates shown here were calculated by dividing the number of zygotes (one zygote counted as two cells) by the number of total cells. (C) Conjugation efficiency of msa1 cells at various nitrogen concentrations. The h90 wild-type cell and h90 msa1 cell were grown in PM medium to midlog phase. Each strain was inoculated into PM medium containing 2% glucose and the indicated concentrations of nitrogen and incubated at 30°. At the indicated times, a portion of the cells was removed and the number of zygotes formed was counted. (D) Overexpression of msa1 inhibits the efficiency of conjugation of the msa1 cell. The msa1 cells harboring pAL or pALmsa1 were incubated for the indicated times in nitrogen-free PM medium containing 0.5% glucose, and the number of zygotes were counted.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Deletion analysis and identification of functionally essential regions of msa1. A series of msa1 deletion mutants were cloned into the pSLF273 vector and HT11 (h90 msa1::ura4+) was transformed with these plasmids. Mating efficiency was calculated by dividing the number of zygotes by the number of total cells.

Genomic integration of 3HA epitopes:
Sequences of three hemagglutinin (3HA) epitopes were integrated into the genomic locus of msa1 at the C terminus by a PCR-based method using the pFA6a-3HA-kanMX6 modules (KRAWCHUK and WAHLS 1999 ). DNA fragments 600–700 bp and corresponding to the 5' and 3' region of the msa1 gene were amplified by PCR using oligonucleotides msa1(w) and msa1 (pFA6a-1,x) or msa1 (pFA6a-2,y) and msa1 (z) in Table 2. Both amplified fragments were used to attach with the ends of the kanMX6 cassette by PCR. SP66 was transformed with the resulting msa1-3HA-kanMX6 fragment. G418-resistant transformants were selected and protein expression was assessed by Western blot analysis.

Conjugation and sporulation efficiency assay:
Cells were grown to midlog phase in PM medium, washed with nitrogen-free or glucose-free PM medium, inoculated in PM medium with various concentrations of nitrogen and glucose, and incubated at 30°. After incubation for selected times, 1 ml of cell suspension was removed and sonicated gently, and the number of zygotes were counted under the microscope. Conjugation frequencies were calculated by dividing the number of zygotes (one zygote counted as two cells) by the total number of cells present.

To determine the sporulation efficiency of diploid cells, the wild-type and each mutant strain were incubated at 30° for 5 days in PM plates that contained 0.5% nitrogen and 2% glucose. A minimum of three individual colonies from each strain was resuspended in water, 1000 cells/colony were microscopically examined for presence of ascospores, and sporulation efficiency was calculated.

Measurement of viability in stationary phase:
Cells were grown to 10⁷ cells/ml in PM at 30°. The cultures were maintained at this density and at daily intervals an aliquot was removed and plated onto YEA medium for incubation at 30°. The colonies formed were counted after 3 days.

Northern blot analysis:
Total RNA was prepared and Northern blot analysis was performed as described previously (OZOE et al. 2002 ). S. pombe cells were grown in PM medium at 30° to a density of 5 x 10⁶ cells/ml. The cells were pelleted by centrifugation, washed with nitrogen-free or low-glucose PM medium, and resuspended in nitrogen-free or low-glucose PM medium at the same density. The cells were incubated for selected times and resuspended in 1 ml of isogen (RNA isolation reagent; Nippon Gene) and vigorously vortexed 6 min with glass beads. After centrifugation (10,000 x g for 15 min at 4°), the supernatant was precipitated with isopropanol. Approximately 10 µg of each sample of total RNA was applied to a 1% denaturing formaldehyde-agarose gel, electrophoresis was applied, and RNA was transferred to a hybridization membrane (Hybond-N+, Amersham Biosciences) in alkali transfer buffer (0.05 M sodium hydroxide) for 4 hr. The probes were labeled with [-³²P]dCTP (Amersham Biosciences) by using BcaBEST labeling kit (Takara Biomedicals, Berkeley, CA). The transcription on the blot was analyzed by autoradiography with a BAS1500-Mac image analyzer (Fuji Film).

The hybridization probes used were the 1.3-kb PvuII fragment for ste11 from pSX1 (SUGIMOTO et al. 1991 ), the 3.2-kb ClaI fragment for mei2 (WATANABE et al. 1988 ), the 3.5-kb HindIII fragment containing mam2 (KITAMURA and SHIMODA 1991 ), and the 1.9-kb cDNA fragment for rep1 amplified from the cDNA library using primers (rep1) described in Table 2 and the 1.7-kb cDNA fragment for msa1.

Western blot analysis:
The msa1-3HA genomic integrated strain (HT5: h⁹⁰ ade6-216 leu1-32 msa1-3HA<<kanMX6) was cultured to midlog phase in synthetic medium PM at 30°. Cells were then shifted to nitrogen-free medium, PM –N, and cell-free extracts were prepared at indicated times. About 1 x 10⁸ cells of S. pombe were harvested. Pellets were washed with STOP buffer [150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM NaN₃ (pH 8.0)] and stored at –80°. The pellets were diluted in 100 µl of dH₂O and boiled at 95° for 5 min. Then 120 µl of 2x Laemmli buffer [4% SDS, 20% glycerol, 0.6 M ß-mercaptoethanol, 0.12 M Tris-HCl (pH 6.8)] containing 8 M urea and 0.2% bromo phenol blue was added to the samples, which were vigorously vortexed with an equal volume of zirconia-silica beads for 3 min and then heated again at 95° for 5 min. The zirconia-silica beads and large debris were removed by centrifugation at 10,000 x g for 15 min. Approximately equal amounts of each sample were analyzed by SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel and then transferred to Immobilon transfer membranes (Millipore, Bedford, MA) using a semidry-type transfer system. For detection of 3HA fusion proteins, membranes were incubated with an anti-HA polyclonal antibody (Molecular Probes, Eugene, OR) diluted 1:3000 in 5% dry milk in TBS-T (15 mM Tris, 137 mM NaCl, 0.1% Tween20), washed, and incubated with horseradish-peroxidase-conjugated anti-rabbit secondary antibody (Bio-Rad Laboratories, Richmond, CA) diluted 1:5000 in 5% dry milk in TBS-T. The secondary antibodies were detected with the enhanced chemiluminescence (ECL) system as described by the manufacturer (Amersham Biosciences). For detection of Cdc2p, membranes were incubated with an anti-PSTAIRE polyclonal antibody (Santa Cruz Biotechnology) diluted 1:3000 in 2% dry milk in TBS-T, washed, and incubated with horseradish-peroxidase-conjugated anti-rabbit secondary antibody diluted 1:2000 in 2% dry milk in TBS-T. The secondary antibodies were detected with the ECL system (Amersham Biosciences).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of the msa1 gene:
To isolate the gene that suppressed hypersporulation in S. pombe sam1 cells (HS412; KATAYAMA et al. 1996 ), HS412 cells were transformed using a S. pombe genomic library. In this screening, two types of clones, msa1 (SPAC13G7.13c) and msa2 (SPAC2F7.11), were identified as multicopy suppressors of sam1. We began to investigate both genes, but because we later showed that msa2 was identical to the nrd1 gene (TSUKAHARA et al. 1998 ), we concentrated on msa1 in this study. The initial clone pW1-12 with the msa1 gene contained a DNA fragment of 5.3 kb. The region responsible for the suppressor activity in strain HT3 (sam1) was then isolated and sequenced (Fig 1). Although integration of plasmid pYC11-3601, which bears wild-type msa1, into the HT3 genome by homologous recombination was conducted, the genomic integration of pYC11-3601 did not suppress hypersporulation in the HT3 (sam1) strain, which indicated that the pW1-12 plasmid contains only a multicopy suppressor and not the sam1 allele. We also sequenced the msa1 locus of the HT3 (sam1) mutant after PCR cloning of the corresponding region but did not find alteration of the msa1 sequence compared with the wild-type strain. It was for this reason that we tentatively named this suppressor gene multicopy suppressor of sporulation abnormal mutant (msa1).

No intron was found in the sequence of the msa1 gene after sequencing and comparison of cDNA and genomic DNA. Translation of the msa1 gene revealed that the msa1 gene encodes a 533-amino acid (aa) protein (Fig 1B). Homology searches using the DDBJ and GenBank databanks revealed no strong similarity with other proteins or genes, except for two putative RNA-recognition motifs (RRMs; Fig 1C). The most homologous protein (37% identity in 200 aa around the RNP motifs region) is S. cerevisiae Rim4, which is known to be a putative RNA-binding protein involved in meiosis (SOUSHOKO and MITCHELL 2000 ).

msa1 cells mate without nitrogen starvation:
To determine the function of the msa1 gene, we made a chromosome deletion mutant of the msa1 gene. The resulting msa1 deletion mutants (msa1) of homothallic and heterothallic strains were named HT11 (h⁹⁰ msa1::ura4⁺) and HT21 (h^– msa1::ura4⁺), respectively. No distinguishable difference in cell growth or morphology was seen between heterothallic msa1 (HT21) and wild-type cells (SP319) cultured in PM medium (Fig 2A, Fig 8C). No aberrant spore formation as found in the pat1^ts mutant was observed in HT21.

View larger version (61K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Expression of msa1. (A) Northern blot analysis of msa1 mRNA. Midlog phase cultures of SP66 (h90) and HT11 (msa1) were incubated in nitrogen-free medium for the periods indicated. Total RNA was extracted from each sample and analyzed with a hybridization probe for msa1. Ribosomal RNAs stained with ethidium bromide were used as equal loading controls. (B) Western blot analysis of Msa1-3HA fusion protein. A midlog phase culture of HT5 (h90 msa1-3HA<<kanMX6) was incubated in nitrogen-free medium. Cell-free extracts were subjected to SDS-PAGE and probed with anti-HA antibody. The Msa1-3HA fusion protein was detected as a band of 70 kD. Immunodetection with anti-Cdc2 antibody was used as a loading control.

View larger version (89K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Mating-pheromone-induced genes are derepressed in msa1 cells and repressed by msa1 overexpression. (A) Northern blot analysis of cellular mRNA shows the time course for induction of ste11, mei2, mam2, and rep1 transcripts in the wild-type cell (SP66) and the msa1 deletion mutant (HT11) upon glucose starvation. Cells were grown in PM medium to midlog phase, washed, inoculated into low-glucose (0.5%) PM medium, and incubated for the indicated times. (B) Mating-pheromone-induced genes are repressed by msa1 overexpression. The msa1 deletion mutants harboring pAL or pALmsa1 were incubated for the indicated times in the nitrogen-free PM medium and cellular RNAs were prepared. The expression level of transcript was analyzed by Northern blotting. The equality of RNA loading was confirmed by staining with ethidium bromide (Et-Br).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 6. msa1 function is independent of the cAMP pathway. (A) Wild-type and cyr1 deletion mutant cells were transformed with pREP81 or pREP81msa1. Transformed cells were grown in PM medium to midlog phase, washed, inoculated in nitrogen-free PM medium, incubated for the indicated times, and mating rates were calculated. (B) Wild-type and pka1 disruptant cells were transformed with pREP81 or pREP81msa1. Transformed cells were grown in PM medium to midlog phase, washed, inoculated in nitrogen-free PM medium, incubated for the indicated times, and mating rates were calculated. (C and D) Wild type and each disruptant were grown in PM medium to midlog phase, washed, inoculated in nitrogen-free PM medium, incubated for the indicated times, and mating rates were calculated. Cells used were the wild type (SP66), msa1 (HT11), pde1 (JZ666), cgs1 (JZ858), msa1 pde1 (HT43), and msa1 cgs1 (HT58). (E and F) Cells were grown to saturation (1 x 107 cells/ml; day 0) and incubated for an additional 4 days (days 1–4) in PM medium. A portion of the culture was removed each day and plated onto YEA plates for cultivation at 30°. The colonies formed were counted after 3 days.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Figure 7. msa1 functions independently of the phh1-stress MAP kinase pathway. (A) The wild-type (SP66), msa1 (HT11), phh1 (TK105), and msa1 phh1 (HT76) cells were grown in PM medium to midlog phase. Each strain was then inoculated into nitrogen-free PM medium and incubated at 30° for the indicated times, and the number of zygotes was counted. (B) Expression of ste11 during nitrogen starvation in the wild-type, msa1, phh1, or msa1 phh1 cells. Each strain was inoculated into nitrogen-free PM medium and incubated at 30° for the indicated times. Total RNA was prepared, and 10 µg was applied to each lane for Northern blot analysis. Et-Br, ethidium bromide.

View larger version (112K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Loss of msa1 can bypass the function of ras1. (A) The level of mam2 transcript was examined 6 hr after nitrogen starvation in SP66 (wild type), SPRU (ras1), SPSU (byr2), SPBU (byr1), SPKU (spk1), SPFU (ste4), HT11 (msa1), HT95 (ras1 msa1), HT93 (byr2 msa1), HT91 (byr1 msa1), HT97 (spk1 msa1), and HT99 (ste4 msa1) strains. Total RNA was prepared from each strain and analyzed by Northern blot. (B) Wild-type cells and strain ras1val17 were transformed with either pREP41 or pREP41msa1. Transformed cells were cultured in nitrogen-free PM liquid medium for 6 hr and total RNA was analyzed by Northern blot. (C) Photomicrographs of homothallic diploid wild-type and msa1-null mutants. Arrows indicate sporulated diploid cells.

We next examined the mating efficiencies of homothallic msa1 cells under various culture conditions. Neither wild-type nor msa1 (HT11) cells conjugated in growth medium containing 2% glucose and 0.5% ammonium chloride as the sole carbon and nitrogen sources. When the glucose concentration in the medium was decreased to 0.5%, msa1 cells conjugated with 30% efficiency and wild-type cells conjugated with 1.5% efficiency (Fig 2B). The msa1 cells conjugated very efficiently in nitrogen-free and glucose-rich medium (Fig 2C). Overexpression of msa1 in the msa1 cells significantly inhibited the efficiency of conjugation under severe nutrient starvation (Fig 2D). Because the cells that lacked msa1 conjugated with markedly increased efficiency under conditions of either glucose or nitrogen starvation, we concluded that the ability of msa1 to inhibit sexual differentiation was not specifically associated with nutrient conditions of glucose and nitrogen.

RNA-binding domains are essential for Msa1 function:
To investigate the region essential for the function of msa1, several deletion mutants of the msa1 gene were constructed and examined for their ability to suppress the msa1 deletion mutants. Deletion of 30 amino acids from the N terminus or deletion of 45 amino acids from the C terminus only slightly decreased the ability of Msa1 to suppress the high mating efficiency of msa1 deletion mutants. However, when half of the N terminus RRM (pSLF273-DM2) or half of the C terminus RRM (pSLF273-DM3) was deleted, the function of Msa1 was completely inactivated (Fig 3). These results indicate that both RRMs are essential for the function of Msa1.

Expression of msa1:
To examine the expression pattern of msa1 mRNA, we performed Northern blot analysis, which showed that msa1 mRNA was present in vegetatively growing cells, but the expression level was very low and was further reduced by nitrogen starvation (Fig 4A).

The level of the msa1 product was examined by Western blotting. The msa1 gene, tagged with 3HA at the C terminus, was integrated into the msa1 genomic locus; expression of mas1-3HA was controlled by the authentic promoter. The h⁹⁰ msa1-3HA strain (HT5) was incubated in PM medium and then shifted to nitrogen-free medium. Similar to mRNA level, the level of the Msa1-3HA fusion protein was also reduced during nitrogen starvation, but the timing of reduction is 3–4 hr later than that in mRNA expression, which we presumed to be the lag time of mRNA turnover (Fig 4B). These results indicate that the expression of msa1 is inhibited by nitrogen starvation.

Meiosis-induced genes are derepressed in msa1 cells and repressed by msa1 overexpression:
Because signals from nutrient starvation and pheromones are essential for both meiosis and conjugation in fission yeast (KITAMURA and SHIMODA 1991 ; OBARA et al. 1991 ; SUGIMOTO et al. 1991 ; TANAKA et al. 1993 ), we examined the mechanism used by the msa1 gene product to interfere with signals from nutrient starvation and mating pheromones. We tested the regulation of expression of mei2, rep1, ste11, and mam2 in wild-type and msa1 cells transferred to glucose-starved (0.5%) medium. These tested genes all positively regulate sexual differentiation; mei2 encodes an RNA-binding protein essential for induction of meiosis (WATANABE and YAMAMOTO 1994 ); rep1 encodes a zinc-finger protein required for onset of premeiotic DNA synthesis (SUGIYAMA et al. 1994 ); mei2 and rep1 are upregulated by ste11; and mam2 encodes a mating-pheromone receptor and is upregulated by both ste11 and mating pheromone (KITAMURA and SHIMODA 1991 ; SUGIMOTO et al. 1991 ; OZOE et al. 2002 ). Although ste11 and mei2 genes were expressed in both cell types after transfer to glucose-starved medium, expression was somewhat higher in msa1 cells than in wild-type cells (Fig 5A). Expression of both mam2 and rep1 was markedly higher and faster in msa1 cells than in wild-type cells.

Similarly, on the nitrogen-free medium, ste11, mei2, and mam2 were induced significantly faster in the msa1 cells than in the wild-type cells (data not shown). These same genes were markedly repressed in the msa1 cells when msa1 was overexpressed (Fig 5B). These data suggest that Msa1 represses both Ste11-regulated genes and the mating-pheromone signaling pathway.

The msa1 function is independent of the cAMP pathway:
We next examined whether the function of msa1 was related to the cAMP pathway. Loss of function of cyr1 (which encodes adenylate cyclase) usually results in hypersporulation (KAWAMUKAI et al. 1991 ); however, overexpression of msa1 under the nmt promoter in cyr1-disruptant cells suppressed hypersporulation of the cyr1 disruptants. The vector alone did not suppress hypersporulation (Fig 6A). A similar result was obtained with pka1 disruptant cells (Fig 6B). Thus, overexpression of the msa1 gene suppressed the effect of the loss of function of either cyr1 or pka1.

Cellular cAMP levels regulate cell survival in stationary phase and cgs1 codes for the regulatory subunit of PKA (DEVOTI et al. 1991 ). We therefore developed cgs1 disruptant (cgs1) and msa1-cgs1 double-disruptant cells (msa1 cgs1) and examined their phenotype. The msa1 cgs1 cells exhibited cgs1^– phenotype and scarcely conjugated during nitrogen starvation (Fig 6D). In addition, cgs1 and msa1 cgs1 cells died after 3 days at G₀; conversely, msa1 and wild-type cells survived equally well at G₀ (Fig 6F). The same results, poor sporulation during nitrogen starvation and death after 3 days at G₀, were obtained with pde1 disruptants (pde1) and msa1-pde1 double disruptants (msa1 pde1; Fig 6C and Fig E). Thus, overexpression of msa1 can reverse the hypermating phenotype of cyr1 and pka1 mutants, but deletion of msa1 does not reverse the sterile phenotype of cgs1 and pde1 mutants. Sterility from msa1 overexpression in pka1 cells is thought to be caused by inhibition downstream of Pka1. If Msa1 acted upstream of Pka1, Msa1 could not affect pka1 cells. Because msa1 cgs1 and msa1 pde1 double mutants have the same phenotype as cgs1 and pde1 (single) mutants, loss of functional Msa1 cannot overcome the hyperactive protein kinase A. These combined results suggest that Msa1 acts either downstream or independently of the cAMP pathway.

msa1 is independent of the spc1/sty1/phh1 pathway:
The Wis1-Spc1/Sty1/Phh1 pathway primarily mediates stress signals and also is partly required for ste11 induction during the onset of sexual differentiation (YAMAMOTO et al. 1997 ). We investigated the possible relationship between msa1 and this pathway. We constructed a homothallic msa1 and phh1 double mutant and compared it with each singly mutated cell for ability to perform conjugation. The phh1 single mutant is not completely sterile but is nearly sterile (KATO et al. 1996 ), whereas the msa1 deletion mutants conjugated efficiently even in nutrient-rich medium. The msa1 phh1 cells had an intermediate level of mating frequency and ste11 expression, a result that indicated that msa1 functions independently of the phh1 pathway (Fig 7).

Loss of msa1 can bypass the function of ras1:
Msa1 significantly repressed the expression of mam2 and rep1 genes (Fig 5), genes that are induced by the pheromone-response pathway. To monitor negative regulation of the pheromone-response pathway, we examined the phenotype of msa1 null cells, which also lacked genes for the pheromone-response pathway. Because mam2 encodes the P-factor pheromone receptor in h^– cells and expression of mam2 requires components of the pheromone-response pathway (XU et al. 1994 ), we constructed cells with mutations in both the msa1 gene and different genes known to be important along the Ras-MAPK pathway and compared their expression of mam2 by Northern blot analysis (Fig 8A). The levels of mam2 expression in the disruptant for ras1, byr2, byr1, spk1, and ste4 were very low, but the level of mam2 expression in the msa1 ras1 and msa1 ste4 double mutants was higher than that of any of the single mutants. Interestingly, transcription of the mam2 gene in the msa1 ras1 double mutant was similar to the transcription level in the wild-type cell (Fig 8A). In addition, the induction of mam2 expression by the activated ras1^val17 gene was repressed by overexpression of msa1 (Fig 8B). These results suggest that loss of msa1 can bypass the function of ras1 and that Msa1 negatively controls sexual differentiation (possibly) downstream of Ras1.

Because mating pheromone signaling is essential for meiosis in fission yeast (KITAMURA and SHIMODA 1991 ; TANAKA et al. 1993 ), we analyzed whether msa1 can bypass the function of ras1 in meiosis. All mutants were constructed in diploid form: msa1 ras1 cells sporulated, ras1 cells scarcely sporulated, and msa1 byr2 cells and byr2 cells appeared sterile (Fig 8C). The gpa1, byr1, spk1, and ste4 mutants behaved in the same manner as the byr2 mutant (data not shown). As shown in Table 3, the sporulation efficiency of msa1 ras1 diploid cells peaked 90-fold higher than that of the ras1 diploid cell. However, deletion of msa1 did not affect deletion-mutant gpa1, byr2, byr1, spk1, or ste4 diploid cells. These results indicate that the loss of msa1 can bypass the function of ras1 in sporulation but not that of gpa1, byr2, byr1, spk1, or ste4, which suggests that Msa1 acts on the pheromone-response pathway downstream from Ras1.

Epistatic analysis of msa1 and rad24:
Rad24 acts as a negative regulator of the pheromone-response pathway by physically interacting with Byr2; this interaction affects the timing of Byr2 translocation in response to sexual differentiation signal (OZOE et al. 2002 ). We performed epistatic analysis to examine the relations of msa1 and rad24. A homothallic msa1-rad24 double mutant was constructed and was compared with each single mutant for mating efficiency (Fig 9A). The double mutant had a hypersporulation phenotype of rad24 in nitrogen-free medium. We compared expression of mam2 between wild-type, msa1, rad24, and msa1 rad24 cells using Northern blot analysis (Fig 9B). The mam2 mRNA began to appear in wild-type cells 6 hr after nitrogen starvation, 2 hr after nitrogen starvation in msa1 cells, and before nitrogen starvation in rad24 cells. The induction pattern of mam2 mRNA in msa1 rad24 cells was similar to that of rad24 cells, a result that suggests that msa1 function is dependent on that of rad24.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 9. Epistatic analysis of msa1 and rad24. (A) Wild-type, msa1, rad24, and mas1 rad24 cells were grown to log phase in PM medium, transferred into nitrogen-free PM medium, and mating rates were calculated. (B) Expression of mam2 during nitrogen starvation in the wild-type, msa1, rad24, or mas1 rad24 cells. Each strain was inoculated into nitrogen-free PM medium and incubated at 30° for the indicated times. Total RNA was prepared and Northern blot analysis was performed. Et-Br, ethidium bromide. (C) Wild-type, msa1, and rad24 strains were transformed with pREP81 or pREP81msa1. Transformed cells were inoculated into nitrogen-free PM liquid medium and then cultured for 24 hr at 30°, and mating rates were calculated. Three independent samples were measured. (D) The msa1 cells were transformed with pREP41 or pREP41rad24. Transformed cells were inoculated into nitrogen-free PM liquid medium and then cultured for the times indicated, and mating rates were calculated.

Because the loss of function of rad24 in cells showed a hypersporulation phenotype (OZOE et al. 2002 ), we next examined whether the function of msa1 is related to the function of rad24. Wild type, msa1, and rad24 were transformed with pREP81 or pREP81msa1 and mating frequencies were assayed (Fig 9C). Overexpression of the msa1 gene under the nmt1 promoter did not suppress well the hypersporulated phenotype of the rad24 disruptant. Conversely, overexpression of rad24 under the nmt1 promoter suppressed the hypersporulated phenotype of a deletion mutant of msa1 (Fig 9D).

msa1 is independent of msa2/nrd1:
Of the two independent clones (msa1⁺ and msa2⁺) that were identified as multicopy suppressors of sam1, one gene (msa2) was identical to the nrd1 gene. Msa2/Nrd1 is an RNA-binding protein that blocks commitment to conjugation until cells reach a critical level of nutrient starvation (TSUKAHARA et al. 1998 ). We independently constructed a msa2/nrd1-deleted strain and confirmed that it behaved as reported (TSUKAHARA et al. 1998 ). Cells that lack Msa2/Nrd1 resemble those that lack Msa1 in that they conjugate without starvation. The phenotypic similarity led us to perform epistatic analysis of the two genes. A homothallic msa1-msa2 double mutant was constructed by crossing and was compared with each single deletion mutant for mating efficiency. The double-mutant cells had greater conjugation efficiency in nitrogen-free medium than either of the single-mutant strains (Fig 10), suggesting that msa1 functions independently of msa2/nrd1.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 10. msa1 is independent of msa2/nrd1. Wild-type, msa1, msa2/nrd1 deletion mutant, and msa1 msa2 cells were grown to log phase in PM medium, transferred into nitrogen-free PM medium for the times indicated, and mating rates were calculated.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We isolated two genes (msa1 and msa2/nrd1) that negatively regulate sexual differentiation of S. pombe. Nrd1 has been characterized (TSUKAHARA et al. 1998 ) and Msa1 is analyzed in this study. Both proteins have the RNA-binding motifs that are essential for their functions (Fig 3; TSUKAHARA et al. 1998 ). In the regulation of sexual differentiation in fission yeast, Mei2, which is essential for meiosis and binds to meiRNA, is the best-characterized RNA-binding protein (WATANABE and YAMAMOTO 1994 ). Sla1, another RNA-binding protein, was recently characterized as the inducer of sexual differentiation when truncated (TANABE et al. 2003 , TANABE et al. 2004 ). Thus, at least four RNA-binding proteins are known to function as regulators of sexual differentiation in fission yeast. S. cerevisiae Rim4 (SOUSHOKO and MITCHELL 2000 ) is the most homologous protein of Msa1. It is interesting to note that Rim4 is a presumed RNA-binding protein that positively regulates meiosis in opposition to the function of Msa1, which negatively regulates meiosis.

The msa1 deletion mutant conjugated with great efficiency in either nitrogen-free or low-glucose medium compared with the wild-type cell (Fig 2). The ability of S. pombe cells to sense nitrogen and glucose levels and to thus regulate sexual differentiation is mediated partly by the cAMP pathway and partly by the stress-responsive MAP kinase pathway. Overexpression of msa1 largely controlled hypersporulation in cyr1 and pka1 cells, and epistatic analysis showed that the function of msa1 is either downstream or independent of the cAMP pathway (Fig 6). The inability of msa1 deletion to suppress the phenotype of cgs1 and pde1 mutants suggested that msa1 functions independently of the cAMP pathway. In the homothallic wild-type cell, the msa1 gene is consistently and immediately expressed after cells are shifted from nitrogen-rich to nitrogen-free medium, but this expression was repressed after conjugation started (Fig 4A and Fig B, Fig 6 hr after nitrogen starvation). Because we did not observe phenotypes other than involvement of sexual differentiation in the msa1 deletion mutant and its combination with several mutants, the primary function of Msa1 is thought to be limited to sexual differentiation. A hypothetical function of Msa1 is as the threshold sensor, sensing the critical nutrient conditions independently of the cAMP pathway and transferring this signal to some factor or factors involved in sexual differentiation.

Msa1 controls expression of several genes that are necessary for the induction of sexual differentiation. Expression of genes usually induced by nutritional starvation, ste11 and mei2, is mildly increased in msa1 cells compared with wild-type cells. Expression of genes usually induced by Ste11 and the mating-pheromone signals, mam2 and rep1, is significantly increased in msa1 cells compared with wild-type cells (Fig 5). Msa1 influenced signaling of both nutritional starvation and mating pheromones. These results suggest to us that the primary function of Msa1 is to control the expression of Ste11-regulated genes through the pheromone-response pathway. DNA microarray experiments to compare gene expression level in wild-type and msa1 cells indicated that the pheromone-inducible genes like mfm1, mfm3, and map2 were highly induced in the msa1 cells compared with wild type (our preliminary observation). Although wild-type diploid cells commonly formed azygotic spores, we observed that the zygotic spores increased in the diploid msa1 mutants compared with the wild-type diploid cells (data not shown). This also suggests that loss of functional Msa1 deregulates pheromone signaling.

The mating pheromone-response signal is transferred by the MAP kinase cascade, which consists of Byr2, Byr1, and Spk1 (NADIN-DAVIS and NASIM 1988 ; TODA et al. 1991 ; WANG et al. 1991 ; NEIMAN et al. 1993 ). Many proteins positively regulate this cascade, including Ras1, Gpa1, and Ste4 (WANG et al. 1991 ; XU et al. 1994 ; BARR et al. 1996 ; TU et al. 1997 ), and recently two negative regulators, Rad24 and Rad25, were reported (OZOE et al. 2002 ). Because the deletion of msa1 reversed the phenotype seen in ras1-deletion mutants (Fig 8) and increased the expression of mating-pheromone-induced genes, and because the expression of msa1 reversed the hypersporulation seen with Ras1^Val17 (Fig 8), we further suggest that Msa1 acts as a negative regulator in the mating pheromone-response pathway.

Similar to cells with combined rad24 and ras1 deletion (OZOE et al. 2002 ), deletion of msa1 in the ras1 cells rescues mam2 expression. But msa1 is not epistatic to rad24. Because the 14-3-3 homologs, Rad24 and Rad25, have multiple targets that include Cdc25, Chk1, Plc1, Mei2, Ste11, Cap1, and Byr2 (ANDOH et al. 1998 ; CHEN et al. 1999 ; LOPEZ-GIRONA et al. 1999 ; ZHOU et al. 2000 ; KITAMURA et al. 2001 ; OZOE et al. 2002 ; SATO et al. 2002 ), it is difficult to elucidate the relations between msa1 and rad24. However, deletion of msa1 did not increase hypersporulation in the rad24 mutant, a result that suggests that the point of action of Msa1 is within the target of Rad24.

Several negative regulators that control sexual differentiation have been reported. Pat1 is the most essential regulator of sexual differentiation and works at several points during conjugation and meiosis (NIELSEN and EGEL 1990 ; WATANABE and YAMAMOTO 1994 ). Cig2/Cyc17, a B-type cyclin, promotes the cell cycle start and negatively regulates differentiation through cell cycle control (OBARA-ISHIHARA and OKAYAMA 1994 ; MONDESERT et al. 1996 ). The 14-3-3 proteins are thought to play important roles in conjugation and meiosis through different acting points (KITAMURA et al. 2001 ; OZOE et al. 2002). Pac1 and Pac2 also regulate sexual differentiation by repressing ste11 expression using unknown mechanisms (IINO et al. 1991 ; KUNITOMO et al. 1995 ). An RNA-binding protein, Nrd1/Msa2, which we independently showed worked with Msa1, is a negative regulator of sexual differentiation, but its role is also not yet clear (TSUKAHARA et al. 1998 ). We described the new factor, Msa1, whose function is mainly as a negative regulator of sexual differentiation, possibly through the regulation of the pheromone-signaling-mediated pathway. The former three factors are relatively well characterized, but the functional points of the latter four are still obscure. Because sexual differentiation is undoubtedly a complicated process in cell events, it will be necessary to characterize each unknown factor one by one.

	ACKNOWLEDGMENTS

We thank D. Beach, M. Yamamoto, Y. Watanabe, T. Kato, and K. Kitamura for strains and plasmids and E. Uchida and K. Nakasato for technical assistance. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Manuscript received November 7, 2003; Accepted for publication January 22, 2004.

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