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The meiosis-specific mug28+ gene of Schizosaccharomyces pombe encodes a putative RNA-binding protein with three RNA recognition motifs (RRMs). Live observations of meiotic cells that express Mug28 tagged with green fluorescent protein (GFP) revealed that Mug28 is localized in the cytoplasm, and accumulates around the nucleus from metaphase I to anaphase II. Disruption of mug28+ generated spores with low viability, due to the aberrant formation of the forespore membrane (FSM). Visualization of the FSM in living cells expressing GFP-tagged Psy1, an FSM protein, indicated that mug28Δ cells harbored abnormal FSMs that contained buds, and had a delayed disappearance of Meu14, a leading edge protein. Electron microscopic observation revealed that FSM formation was abnormal in mug28Δ cells, showing bifurcated spore walls that were thicker than the nonbifurcated spore walls of the wild type. Analysis of Mug28 mutants revealed that RRM3, in particular phenylalanin-466, is of primary importance for the proper localization of Mug28, spore viability, and FSM formation. Together, we conclude that Mug28 is essential for the proper maturation of the FSM and the spore wall.
Meiosis, a specialized cell cycle consisting of one round of DNA synthesis followed by two successive meiotic divisions, is an important process in haploid gamete formation, which is essential for the transmission of genetic information to the next generation in sexually reproducing species. The fission yeast Schizosaccharomyces pombe is a useful model organism for studying the regulatory mechanisms during meiosis, such as recombination, chromosome behavior, and sporulation, because it synchronously enters meiosis and can easily be manipulated by a variety of genetic and cell biological techniques. The cell cycle switch from mitosis to meiosis is accompanied by a remarkable change in gene expression profiles (Mata et al., 2002 ), and meiosis-specific transcripts are selectively removed by Mmi1 if they are expressed during vegetative growth (Harigaya and Yamamoto, 2007 ). Meiosis in the fission yeast is initiated when haploid cells of opposite mating types (h+/h−) experience nitrogen starvation and conjugate (Xue-Franzén et al., 2006 ). After karyogamy (nuclear fusion), premeiotic DNA synthesis occurs. The nucleus then develops a horse-tail shape and moves back and forth about the axis of the cell. This behavior permits homologous pairing and recombination to take place in the cells (Asakawa et al., 2007 ). After the successive nuclear divisions of meiosis I (MI) and meiosis II, capsular plasma membranes form around each haploid nucleus in the cytoplasm of the mother cell. Then, spore morphogenesis occurs to generate four mature ascospores.
The most important event of sporulation is the assembly of double-layered intracellular membranes, termed forespore membranes (FSMs), which develop into the plasma membrane of spores (Shimoda, 2004 ). In meiosis II, FSMs are assembled by the fusion of membrane vesicles. After the two sequential meiotic divisions, FSMs expand and encapsulate the haploid nuclei, producing the membrane-surrounded spore precursors. Psy1, a plasma membrane protein that resembles the mammalian target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin-1A, plays an essential role in FSM formation (Nakamura et al., 2001 ). Psy1-GFP labels four cup-shaped membranes near the spindle pole body (SPB) at metaphase II, each of which surrounds one of the nuclei during anaphase II. Then, the expanding FSM encloses and completely engulfs each haploid nucleus in a synchronous manner (Nakamura et al., 2008 ). Leading edge proteins are involved in the initiation, development, and closure of FSMs during the expansion of FSMs at meiosis II. Meu14, one of the meiosis-specific coiled-coil proteins (Ohtaka et al., 2007 ), localizes at the leading edge of the FSM and regulates spore morphogenesis, in particular the determination of FSM closure during meiosis II (Okuzaki et al., 2003 ). Meu14-GFP first appears inside the nuclear region at prophase II, after which it accumulates beside the two SPBs at metaphase II, forming two ring-shaped structures that surround the nucleus at early anaphase II. Meu14-GFP disappears at post-anaphase II. The basic process of sporulation is also conserved in the budding yeast Saccharomyces cerevisiae (Neiman, 2005 ).
Large-scale cDNA sequencing projects in mammals have revealed that eukaryotic cells contain numerous polyadenylated noncoding RNAs (ncRNAs) that are expected to play physiological roles, especially in the regulation of gene expression (Okazaki et al., 2002 ; Mattick, 2004 ; Carninci et al., 2005 ; Tomilin, 2008 ). These ncRNAs are also abundant in fission yeast, particularly during meiosis (Watanabe et al., 2001 ; Kakihara et al., 2003 ). By comparing the genome-wide distribution of peaks for double-strand DNA breaks (DSBs) to that of the prl class of polyadenylated ncRNA (Watanabe et al., 2002 ), it was found that peaks for DSBs almost colocalize with prl loci, which are most often situated within intergenic regions (Wahls et al., 2008 ); this suggests that meiotic recombination hotspots of fission yeast are directed to the loci that express polyadenylated ncRNAs. Other ncRNAs may play important roles in the progression of meiosis in fission yeast, but their functions and their putative RNA-binding protein (RBP) association partners remain elusive.
A polyadenylated ncRNA named meiRNA, a cofactor of an RNA-binding protein called Mei2, is expressed only during meiosis and is essential for MI (Watanabe and Yamamoto, 1994 ; Yamamoto, 1996 ). Mei2 forms a dot structure in meiotic prophase nuclei, and meiRNA is required for this localization pattern of Mei2 (Yamashita et al., 1998 ). Spo5, another meiosis-specific RNA-binding protein of S. pombe, also forms a nuclear focus at meiotic prophase I and regulates meiotic progression and homologous recombination (Kasama et al., 2006 ). Although the Spo5 focus colocalizes with the Mei2 dot in the nucleus at prophase I, Spo5 does not interact with meiRNA. Meu5/Crp79, yet another meiosis-specific RNA-binding protein of S. pombe (Watanabe et al., 2001 ), also was isolated as a novel mRNA export factor that binds to poly(A)+ RNA and regulates its export from the nucleus to the cytoplasm (Thakurta et al., 2002 ).
Although many polyadenylated ncRNAs have been isolated from S. pombe, a detailed understanding of their functions and their putative association partners (RBPs) remain elusive, partly due to the lack of information available on meiosis-specific RBPs. Except for Mei2, it remains unknown whether any polyadenylated ncRNA specifically associates and functions with Spo5 and Meu5/Crp79, the meiosis-specific RBPs described above. Indeed, we need a more detailed analysis of other uncharacterized meiosis-specific RBPs of S. pombe. As an initial step to understanding the putative physiological roles of these meiotic RBPs, we searched for meiosis-specific proteins that harbor putative RNA recognition motifs (RRMs). We report here the functional analysis of a novel meiosis-specific gene, mug28+, which encodes Mug28, a protein containing three RRMs. We show that Mug28 is required for spore maturation, particularly for the proper formation of FSMs.
The S. pombe strains and plasmids used in this study are listed in Supplemental Table S1. The media used were yeast extract-peptone-dextrose or yeast extract plus histidine (75 μg/ml) complete media, Edinburgh minimal medium 2 (EMM2) synthetic medium, and malt extract, EMM2-nitrogen (EMM2-N), or SPA sporulation media. The induction of synchronous meiosis was assessed as described previously (Shimada et al., 2002 ).
To construct the mug28+-green fluorescent protein (GFP) strain, we performed PCR as described previously (Saito et al., 2004 ) and obtained a DNA fragment carrying the open reading frame and 3′ downstream region of the mug28+ gene. For this purpose, we synthesized the following two oligonucleotides and used them as primers (Supplemental Table S1): mug28-5′Fw-AscI-SalI and mug28–3′Rv-NotI. The PCR product of the 3′ downstream region of the mug28+ gene was inserted into the pRGT41 vector via the XhoI-SacI sites. The PCR product of the mug28+ open reading frame, the 1.8-kb HindIII fragment containing the ura4+ cassette (Grimm et al., 1988 ), and the pRGT41-mug28+-3′ untranslated region vector were inserted into the pBluescriptII KS(+) vector via the EcoRI-NotI, HindIII, and NotI-SacI sites, respectively. This plasmid construct was then digested with BamHI, and the resulting construct was introduced into the TP4-1D wild-type strain (AS6). The Ura+ transformants were then screened by PCR to identify the mug28+::mug28+-gfp-ura4+ strain.
To detect Mug28-GFP in living meiotic cells, mid-log phase mug28+-GFP cells (AS108) were cultured in EMM2-N to induce meiosis as described previously (Saito et al., 2005 ). The living cells were then stained with 3.0 μg/ml Hoechst33342 for 5 min and spotted on a coverslip. Fluorescence images of these cells were observed using a fluorescence microscope (BX51; Olympus, Tokyo, Japan) with a Cool SNAP charge-coupled-device camera (Roper Scientific, San Diego, CA). Fluorescence images were acquired using Photoshop 7.0 (Adobe Systems, Mountain View, CA).
For time-lapse observations, YN68, YOD50, SOP091w, AS117, AS160, and AS161 cells, which expressed GFP-Psy1 and Meu14-GFP, were cultured in 10 ml of EMM2 with supplements adenine (75 mg/ml), histidine (75 mg/ml), leucine (250 mg/ml), and uracil (75 mg/ml) until they reached the mid-log phase at 28°C. They were then induced to enter meiosis by incubation in EMM2-N at 28°C. After 10 h of nitrogen starvation, live cells were placed on a glass-bottomed dish coated with 0.2% concanavalin A. Images were acquired with an IX71 fluorescence microscope (Olympus) every 3 min (with a 3-s exposure time), and three optical sections at 200-nm intervals were acquired for each time point. The projected images obtained were analyzed with MetaMorph software (Molecular Devices, Sunnyvale, CA).
To identify a novel meiosis-specific RBP that may associate with the meiosis-specific mRNA-like ncRNAs of S. pombe that we reported previously (Watanabe et al., 2001 , 2002 ), we searched the S. pombe genome database (http://www.genedb.org/genedb/pombe/index.jsp) for uncharacterized genes that both harbor RNA recognition motifs and show enhanced expression during meiosis. We screened 278 genes in the genome database AmiGO (http://www.genedb.org/amigo-cgi/browse.cgi?speciesdb = GeneDB_Spombe) using “RNA binding” as a query word. In addition, we selected genes whose expression is enhanced at least 20-fold in the meiotic phase over that in the G1-arrested phase, based on the cDNA microarray data. This selection yielded the following 10 candidate genes: SPAC1610.02c (64), SPAC1610.03c (=meu5+/crp79+) (293), SPAC343.07 (=mrb2+/mug28+) (110), SPAC4G9.05 (=mpf1+) (93), SPBC28E12.02 (286), SPBC29A10.02 (=mrb1+/spo5+/mug12+) (214), SPBC609.01 (30), SPCC1682.08c (=mcp2+) (20.7), SPCC320.07c (= mde7+) (112), SPCC74.09 (415), with the numbers in parentheses indicating the ratio of the maximum expression to the expression in the G1-arrested phase. We initiated the functional analysis of SPAC343.07 by naming it mrb2+ after meiotic RNA-binding protein 2; however, we hereafter refer to it as mug28+ (Martín-Castellanos et al., 2005 ), which is its official name. We selected the mug28+ gene for further analysis because it resembled SPAC1610.03c (=meu5+/crp79+), which we previously isolated as one of the meu (meiotic expression upregulated) genes (Watanabe et al., 2001 ) and later identified as a regulator of RNA transport from the nucleus to the cytoplasm (Thakurta et al., 2002 ).
Mug28 (mug28+ protein) consists of 609 amino acids, harbors three RNA recognition motifs, and has no homologous genes in other species (Figure 1A). To examine whether the transcription of mug28+ is meiosis specific, we performed Northern blot and reverse transcribed (RT)-PCR analyses. For Northern blot analysis, we used RNA obtained from CD16-1 (h+/h−) and CD16-5 (h−/h−) cells harvested at various times after the induction of meiosis by nitrogen starvation; CD16-1, but not CD16-5 (negative control), cells enter meiosis. We found that mug28+ transcripts were detected exclusively from 6 to 12 h after meiotic induction (Figure 1B). We next assessed the mRNA level of mug28+ by RT-PCR using RNA obtained from JZ670 (h−/h− pat1-114) cells, which enter meiosis in a highly synchronous manner when shifted to the restrictive temperature. We found that mug28+ transcripts are observed exclusively from 2 to 6 h after meiotic induction, peaking 5 h after the temperature shift (Figure 1C), timing that is consistent with that revealed by Northern blot analysis. These results indicate that the transcription of the mug28+ gene is meiosis specific.
To accurately examine the expression of Mug28 during meiosis, we constructed a mug28+-3HA strain, which expresses Mug28 tagged with three copies of the HA epitope at its C-terminal end. To attain synchronous meiosis, we used the pat1-114 temperature-sensitive strain. We first replaced the mug28+ gene of the pat1-114 strain with the mug28+-3HA fusion gene. Then, h−/h− pat1-114 mug28+-3HA diploid cells were induced to enter synchronized meiosis by temperature shift, and their lysates were subjected to Western blot analysis using the anti-hemagglutinin (HA) antibody. This Western blot analysis detected the Mug28-3HA protein of the expected size, which was expressed only during meiosis, 4–8 h after the temperature shift, and peaking at 6–7 h (Figure 1D). As a control to monitor the timing of meiosis, we tested for tyrosine 15 phosphorylation of Cdc2, which appears around the premeiotic S phase.
To examine the subcellular localization of the Mug28 protein during meiosis, we prepared an S. pombe (h90) strain expressing both Mug28-GFP and Sad1-mCherry, a SPB marker. We induced it to enter zygotic meiosis, and observed the GFP and mCherry signals in living cells, without fixation, and determined the timing of meiotic progression with a fluorescence microscope by tracking the nucleus with Hoechst33342 staining, and the SPB by monitoring the mCherry signal. As shown in Figure 2A, no Mug28-GFP signal was detected when cells were in the vegetative growth phase and undergoing karyogamy. In contrast, after meiotic progression, Mug28-GFP faintly appeared in the cytoplasm at the horsetail phase. At metaphase I, Mug28-GFP signal was strongly detected uniformly in the cytoplasm but not in the nucleus. Then, the signal gradually accumulated around the nucleus until the cell reached anaphase I. Thereafter, Mug28-GFP signals became apparent not only around the nucleus but also in the nucleus. After late anaphase II and during sporulation, the Mug28-GFP signal disappeared. This subcellular localization of Mug28-GFP corresponds with the results of the time course Western blot analysis (Figure 1D), showing that expression of Mug28-3HA declines after peaking 6–7 h after the meiotic induction.
We also examined the detailed subcellular localization of Mug28 using an S. pombe (h90) strain that expresses both Mug28-GFP and Psy1-mCheery, an FSM marker. As shown in Figure 2, B and C, Mug28-GFP was faintly visible in the cytoplasm at the horsetail phase as described above. At metaphase I, Mug28-GFP signal was uniformly detected in the cytoplasm, but not in the nucleus. Then, the signal gradually accumulated around the nucleus until the cell reached anaphase I. After that, the Mug28-GFP signal became visible not only in the area around the nucleus, which displayed colocalization with Psy1-mCherry, but also in the nucleus at anaphase II. After late anaphase II and during sporulation, the Mug28-GFP signal disappeared. This result suggests that Mug28 protein plays a role in formation of the FSM during meiosis II.
To assess the physiological role of mug28+, a mug28+ disrupted strain (mug28Δ) was constructed by one-step gene replacement. Diploid cells in which one of the mug28+ genes had been replaced by ura4+ were sporulated and germinated. The generated spores were viable, indicating that the mug28+ gene is not essential for vegetative growth. However, many of the diploid h90 mug28Δ cells failed to form proper spores 18 h after meiotic induction, and the spore viability of the mug28Δ mutant was only half that of the mug28+ cells (Figure 3A). We confirmed that the ectopic expression of Mug28-GFP suppressed this abnormal sporulation phenotype of mug28Δ cells (Supplemental Figure S1). To investigate the cause of abnormal spore formation in mug28Δ cells, the morphologies of FSMs in these mutant cells were analyzed by fluorescence microscopy using an FSM marker, GFP-Psy1 (Nakamura et al., 2001 ). We found that about half of the mug28Δ spores showed the abnormal snowman-like morphology, whereas most of the mug28+ spores were normal (Figure 3B). After incubation on sporulation plates for 18 h, many of the wild-type cells displayed GFP-Psy1 fluorescence as circles of uniform brightness, representing FSMs with a haploid nucleus already enclosed. In contrast, many mug28Δ cells displayed buds of various sizes, or spores with large buds forming a shape resembling a snowman (Figure 3C). When we classified these abnormal morphologies into six types (i~vi), we noticed that many mug28Δ cells displayed more than five GFP-Psy1 circles, which is due to the budded shape of the FSMs. Interestingly, some spores had FSMs extending from the FSM-encapsulated nucleus (Figure 3, D-v and vi). Enlarged images of such spores are surrounded by squares and presented in Figure 3D-v (bottom row) and Figure 3D-vi (top row) (Figure 3E). Frequencies of each abnormal spore type are presented as bar graphs in Figure 3F. These results indicate that Mug28 is required for normal spore formation.
Because the snowman-like phenotype of mug28Δ cells, shown in Figure 3, is strikingly similar to that of meu10Δ cells (Tougan et al., 2002 ), we investigated whether Mug 28 regulates the protein expression or subcellular distribution of Meu10, which functions in normal spore wall assembly as a glycosylphosphatidylinositol (GPI)-anchored cell surface protein. We first examined meu10+ mRNA expression in mug28Δ cells during meiosis by microarray analysis and found no apparent difference from wild-type cells (Supplemental Figure S2iv). We next determined the subcellular localization of Meu10 by constructing a Meu10-GFP-expressing strain in the h90 mug28Δ genetic background and inducing it to undergo meiosis by nitrogen starvation. Fluorescence microscopy during meiosis II showed that Meu10-GFP signals colocalized to the snowman-like FSM visualized by mCherry-tagged Psy1 (arrowheads in Supplemental Figure S3A). However, Meu10-GFP appeared in the spore cytoplasm and near the spore wall when wild-type or mug28Δ cells entered meiosis II (right panels in Supplemental Figure S3A, i and ii). Furthermore, Meu10-GFP showed similar signal intensity in wild-type and mug28Δ cells. Thus, although the abnormal spore wall of mug28Δ cells is similar to that of Meu10 mutants (Tougan et al., 2002 ), our results indicate that Meu10 is not a regulatory target of Mug28.
Meu14 is known to regulate the proper formation of FSMs by localizing at the leading edge of the FSM during meiosis II in fission yeast; the Meu14 protein then disappears as FSMs enclose the nucleus (Okuzaki et al., 2003 ). To explore the cause of abnormal FSM formation in mug28Δ cells, we monitored the subcellular localization of Meu14-GFP in mug28Δ and mug28+ cells by fluorescence microscopy (Figure 4A). We found that the disappearance of Meu14-GFP was incomplete in most (85.8% in Figure 4B) of the mug28Δ cells (panels surrounded by red squares in Figure 4A) at a time when most (80.0% in Figure 4B) of the mug28+ cells no longer carried Meu14-GFP signals (Figure 4Aiv). This also suggests that the sporulation process is delayed in mug28Δ cells, because more than five times as many mug28Δ cells as mug28+ cells (20.0% in Figure 4B) had a residual Meu14-GFP signal at the closing aperture of the FSM. The residual Meu14-GFP signal was observed even after completion of FSM formation at the end of sporulation in mug28Δ cells (Figure 4Aviii).
To analyze this abnormality in more detail, we categorized the cells into three classes (Figure 4C). Class I cells contain satellite dots (white arrowheads) in the vicinity of the major Meu14-GFP rings. In class II cells, Meu14-GFP signals are detected at the leading edge of the FSM, even after the completion of FSM formation. These signals usually disappeared at the end of anaphase II in mug28+ cells. Class III cells show abnormally localized dots at uncharacterized locations in the cytoplasm, adjacent to the FSM. The phenotypes marked with squares in Figure 4C are enlarged in Figure 4D. The fact that class II and class III cells are frequently produced from mug28Δ cells suggests that Mug28 is required for the degradation of Meu14 and/or the evacuation of Meu14 from the FSM at the last phase of the sporulation process. These results suggest that Meu14 aberrantly remaining at the leading edge of FSM induced the formation of bud-like extra FSM in mug28Δ cells.
Western blot analysis indicated that an ineligible amount of Meu14 remained even at the end of sporulation in mug28Δ cells (Figure 4E). This was not due to remaining meu14+ transcription, because microarray analysis showed no apparent change in the meu14+ mRNA level in mug28Δ cells (Supplemental Figure S2). These data indicate that Mug28 regulates the stability of Meu14 protein but is not involved in transcriptional regulation of the meu14+ gene.
We next examined the behavior of FSM (depicted by GFP-Psy1) and Meu14-GFP simultaneously in mug28+ and mug28Δ cells. Because the mCherry signal, which is weak and short lived, is not suitable for monitoring the behavior of Psy1 and Meu14 over a long period, such as sporulation, we observed the GFP signal alone using a mug28Δ strain (AS161) that expresses both GFP-Psy1 and Meu14-GFP. This is by taking advantage of the fact that the subcellular localizations of GFP-Psy1 and Meu14-GFP are distinct, and we compared the behavior of Psy1 and Meu14 simultaneously using the same fluorescent tag. We first confirmed that the frequency of abnormal cells (83.1%) was similar to that presented in Figure 4B (85.8%), in which the disappearance of the Meu14-GFP signal at the end of the FSM formation process was incomplete (Figure 5, A and B).
To further analyze this abnormality, we classified the abnormal cells into three types, and representative images of each type are shown in Figure 5C. Enlarged images show that type I mug28Δ cells emit a strong signal of residual Meu14-GFP at the junction between mother and daughter FSMs, which strongly suggests that budding of extra FSM emerges from the closing FSM aperture due to residual Meu14. Type II mug28Δ cells harbor FSM (or GFP-Psy1) with four or more snowman-like abnormal spores structures, and without Meu14-GFP dots, which suggests that Meu14 is no longer required for the maintenance of the budded spore morphology. Type III mug28Δ cells harbor an extraordinarily abnormal spore morphology, which is probably not due to Mug28 abolishment, because even a small percentage of mug28+ spores exhibit this abnormality (Figure 5C). Enlarged views of the squared portion of Figure 5C are presented in Figure 5D. These results suggest that aberrantly retained Meu14 at the leading edge of FSM induced the formation of additional FSM in mug28Δ cells.
We also examined the effects of overexpression of Meu14 protein, which was directed from the nmt promoter during meiosis. However, we found none of the abnormal phenotypes observed in mug28Δ cells (Supplemental Figure S3B). Thus, although the aberrant amount of remaining Meu14 is an indication of abnormal spores of mug28Δ cells, it alone does not explain its abnormality (see Discussion). We also examined whether the meu14 mutation was genetically epistatic to the mug28 mutation by constructing a mug28Δ meu14Δ double mutant strain. However, we found no genetic interactions between these mutations either with respect to the abnormal spores of mug28Δ cells or the abnormal nuclear fragmentation of meu14Δ cells (Supplemental Figure S4).
To investigate the live movement of FSM formation and the movement of leading edge proteins of mug28Δ cells in detail, we performed time-lapse observations of GFP-Psy1 and/or Meu14-GFP signals, either separately or simultaneously, in both mug28+ and mug28Δ cells. When we performed a time-lapse analysis of GFP-Psy1, we found that new FSM initiated budding from already enclosed FSM only in mug28Δ cells (Figure 6A and Supplemental Movie 1) but not in mug28+ cells, which resulted in the formation of a snowman-like morphology of the spore after the FSM was completely enclosed (Supplemental Figure S5A and Supplemental Movie 2). When we similarly observed Meu14-GFP, we continually detected the residual Meu14-GFP signal at the leading edge of the FSM only in mug28Δ cells (Figure 6B and Supplemental Movie 3), even when all Meu14-GFP signals disappeared in mug28+ cells (Supplemental Figure S5B and Supplemental Movie 4). Simultaneous time-lapse analysis of both GFP-Psy1 and Meu14-GFP signals confirmed the above-mentioned observations, which are tightly related to the residual Meu14-GFP signals at the leading edge of the FSM in mug28Δ cells (Figure 6C and Supplemental Movie 5), but not in mug28+ cells (Supplemental Figure S5C and Supplemental Movie 6). Together, these results indicate that Meu14 is not properly degraded in mug28Δ cells.
Ectopic expression of Meu14DD-GFP (Meu14DD-R148AL151A or -R161AL164A), in which Arg and Leu in the two putative destruction boxes (RxxL) are replaced by Ala, caused no apparent abnormalities in FSM formation as visualized by Psy1-GFP (Supplemental Figure S6). Thus, the ubiquitin-proteasome pathway is apparently not involved in the dysregulation of timely degradation of Meu14 at the end of meiosis.
To understand the detailed spore structures of mug28Δ cells, we examined their morphologies by thin-section electron microscopy (TEM). We treated sporulating mug28+ and mug28Δ cells with rapid freezing to minimize the artifacts that can arise during the prefixation process (Kasama et al., 2006 ). The substituted cells were then fixed and observed by electron microscopy. As shown in Figure 7A, TEM observation in mug28+ cells revealed four normal spores that possess nuclei, mitochondria, rough endoplasmic reticulum, and Golgi apparatuses. In contrast, many of the single mug28Δ spores possess a spore wall that is thicker than that of mug28+ spores (Figure 7, Bii and Dii). We also found that some of the budded mug28Δ spores retained a canal-like passage of cytoplasm between the mother and daughter spores (Figure 7, Cii, Div, and Eiv), whereas in snowman-like spores, which may represent mature spores, the cytoplasm of the mother and daughter spores was separated by a common spore wall (Figure 7, Diii and Eii). Notably, many of the budded mug28Δ spores displayed ruffled and seemingly fragile spore walls (Figure 7, Ciii, Dii, and Eiii and iv). In contrast, the snowman-like spores typically seemed to have a solid spore wall (Figure 7, Bii, Diii, and Eii). For quantitative analysis, we selected four typical TEM images of both mug28+ and mug28Δ spores and measured the thickness of their spore walls (Figure 7F and Supplemental Figure S7). Indeed, the spore walls of the mug28Δ strain were ~1.5 times thicker than those of the mug28+ strain (Figure 7G). These results indicate that Mug 28 is required for proper spore wall formation.
To analyze the role of each RRM domain in Mug28 function, we prepared Mug28 deletion mutants that express GFP fused to truncated Mug28 proteins (Figure 8A, left). We induced these mutant cells to enter meiosis by nitrogen starvation and observed the GFP signals of living cells without fixation, tracking the nucleus by staining with Hoechst33342. Apart from the Mug28-RRM2Δ-GFP mutant, the proper perinuclear localization of Mug28-GFP was disturbed at meiosis I in all mutants, and the Mug28-GFP signal was also observed in the nucleus (arrowheads in middle panels of Figure 8A). This result indicates that RRM2 does not contribute to the proper subcellular localization of Mug28. In contrast, at meiosis II, Mug28-GFP localization was almost normal in all mutants except for the Mug28-RRM1ΔRRM3Δ mutant, in which the nuclear accumulation of GFP signal was abolished (rightmost panel of Figure 8A). This result suggests that the RRM1 and RRM3 domains are required for the proper subcellular localization of Mug28.
The spore viabilities of the Mug28-RRM1Δ and Mug28-RRM2Δ mutants were almost normal, whereas the Mug28-RRM3Δ mutant displayed a spore viability that was reduced to the level found in the Mug28-RRM1ΔRRM2Δ double mutant (Figure 8B). Mug28-RRM1ΔRRM3Δ and Mug28-RRM2ΔRRM3Δ double mutants exhibited a further reduction in spore viability, whereas Mug28Δ, which corresponds to the triple mutant (RRM1ΔRRM2ΔRRM3Δ), showed the most remarkable reduction of spore viability. These results indicate that the RRM3 domain is most important for viable spore formation, and that the RRM1 and RRM2 domains play subsidiary roles in spore viability.
To further assess the requirements of each RRM domain for proper FSM formation, we prepared mutants expressing YFP-Psy1 and the truncated form of Mug28-3HA, and we observed the YFP signals in living cells after meiotic induction. We used YFP-Psy1 instead of GFP-Psy1 here to exclude the possibility that the above-mentioned observations were due to artifacts linked to the fluorescent signals of GFP. Indeed, we could confirm that YFP-Psy1 behaved similarly to GFP-Psy1 during fluorescence microscopy (our unpublished data). To visualize the YFP-Psy1 signals alone and to examine the influence of the GFP tag on the phenotype, we replaced the GFP tag fused to the deletion mutants with the 3HA tag. We first confirmed that Mug28-RRM3Δ-3HA cells had a reduced spore viability at a similar level of the Mug28-RRM3Δ-GFP mutant (Supplemental Figure S8). This indicates that GFP-tagged and HA-tagged deletion mutants show similar results. We also confirmed that abnormal YFP-Psy1 localization occurred at a similar frequency (bar graph of mug28Δ in Figure 8C) as abnormal GFP-Psy1 localization and that YFP-Psy1 and GFP-Psy1 labeled similar abnormal morphologies (panels indicated as abnormal FSM of mug28Δ cells in Figure 8D) (Figure 3, B and D). As for the other deletion mutants, nearly 90% of Mug28-RRM2Δ (AS199) cells displayed normal YFP-Psy1 localization, whereas only 33% of Mug28-RRM3Δ (AS200) and 27% of Mug28-RRM1ΔRRM3Δ (AS202) cells exhibited normal YFP-Psy1 localization. Percentages of normal cell populations in Mug28-RRM1Δ (AS198), Mug28-RRM2ΔRRM3Δ (AS201), and Mug28-RRM1ΔRRM2Δ (AS203) cells ranged between 53 and 92%. Notably, these percentages are similar to those of spore viability of these mutants. Together, we conclude that the RRM3 domain is pivotal for the full activity of Mug28, whereas the RRM1 and RRM2 domains have supplementary roles.
To examine whether the abnormal phenotype of Mug28-RRM3Δ cells was due to defective Mug28 RNA binding ability rather than due to conformational changes, we replaced Phe-466 (F466) with alanine in the conserved RNA-binding motif RRM3 to generate mutant cells that expressed GFP-Mug28F466A (Figure 9A). Then, we compared the GFP localization of Mug28F466A-GFP (NP252) with that of Mug28-GFP (AS164), Mug28RRM3Δ-GFP (AS176), and in mug28Δ (AS99) cells after meiotic induction by nitrogen starvation. We found that the perinuclear localization of Mug28F466A-GFP and Mug28RRM3Δ-GFP was disrupted to a similar degree during meiosis I (arrowheads in the middle panels of Figure 9A, ii and iii). In contrast, all Mug28-GFP signals were observed in the peri-nucleus (arrowhead in middle panels of Figure 9Ai). This result indicates that F466, a putative RNA binding site, is required for proper subcellular localization of Mug28-GFP. In contrast, Mug28F466A-GFP localized normally during meiosis II, as did Mug28-GFP and Mug28RRM3Δ-GFP (rightmost panel of Figure 9A).
We also examined spore formation and found that the frequency of abnormal spores from F466A mutant cells (20.6%) was greater than that from WT cells (4.6%) but slightly less than that from mug28Δ (36.6%) and RRM3Δ (34.8%) cells (Figure 9B). Spore viability of F466A mutant cells (77.5%) was also intermediate between WT cells (91.1%) and mug28Δ (29.4%)/RRM3Δ (47.9%) cells (Figure 9C). These results indicate that the F466 residue in RRM3 of Mug28 F466 is required for full function of Mug28.
In meiosis, the spatiotemporal coordination of meiotic nuclear divisions and FSM formation is essential for accurate distribution of the genome into four haploid spores. Here, we showed that expression of meiosis-specific Mug28, which has three RRM domains, is detected in the cytoplasm from metaphase I to anaphase II, and accumulates particularly at the periphery of the nucleus during meiosis I (Figures 1 and and2).2). Disruption of the mug28+ gene caused abnormal FSM formation, which may be one of the causes of the reduced spore viability (Figure 3A) and the formation of abnormal spores with the snowman-like morphology, as revealed by an FSM marker, GFP-Psy1 (Figure 3, B–F). Visualization of the FSM in living cells by GFP-Psy1 indicated that mug28Δ cells harbored abnormal FSMs with buds, and displayed the retarded disappearance of Meu14 (Figures 44–6).
The abnormal FSM phenotype of mug28Δ cells, displaying the snowman-like morphology, resembles that of sst4/vps27Δ cells (Onishi et al., 2007 ), in which the expression of the FSM is inefficient and bubble-like structures emerge from the leading edge of the FSM. However, whereas sst4/vps27Δ cells generating FSMs with bubbles produce oval-shaped spores with fuzzy rims, mug28Δ cells generating FSMs with buds produce spores with an abnormal morphology, due to their abnormal FSM formation. Micorarray analysis showed no alteration in the mRNA level of sst4 in mug28Δ cells (Supplemental Figure S2vii). Moreover, we found no genetic interaction between the genes mug28+ and sst4+ (Supplemental Figure S9). Thus, Mug28 and Sst4 seem to function independently during meiosis. Sst4/Vps27 is a downstream factor of the phosphatidylinositol 3-kinase pathway and functions in FSM formation. Vps27p, the Sst4/Vps27 homologue of the budding yeast S. cerevisiae, is a component of the endosomal sorting complex required for transport during protein sorting at the multi vesicular body (Bowers and Stevens, 2005 ). Because most of the genes required for vacuolar biogenesis and protein transport are conserved between S. pombe and S. cerevisiae (Takegawa et al., 2003 ), it is possible that Sst4/Vps27 is also a component of endosomal sorting complexes in S. pombe.
Spo20, a phosphatidylinositol/phosphatidylcholine transfer protein of S. pombe, is also notable because some of the abnormal FSM morphologies resembled those of mug28Δ cells in spo20-KC104 mutant cells (Nakase et al., 2001 ). However, DNA micorarray analysis showed no change in the mRNA level of spo20 in mug28Δ cells (Supplemental Figure S2vii). The result suggests that not only the phosphatidylinositol 3-kinase pathway but also the meiosis-specific RNA-binding protein Mug28 is independently involved in the proper formation of the FSM via regulation of the sorting factors of FSMs. Because the expression of an uncharacterized gene termed meu6+ (Watanabe et al., 2001 ) was higher in mug28Δ cells at the sporulation stage (Supplemental Figure S2), its significance as a downstream target of Mug28 will be analyzed in a future study.
Electron microscopy revealed that mug28Δ cells displayed not only abnormal snowman-like FSMs but also thicker spore walls than those of mug28+ cells (Figure 7). After formation of FSMs, spore wall assembly begins in the lumen between the two membranes derived from the FSM, where there is no preexisting wall structure to serve as a template. Because bgs2+ and chs1+, which encode a β-glucan synthase and a chitin synthase, respectively, are required for formation of viable spores, β-glucan and chitin must be necessary components of spore walls (Arellano et al., 2000 ; Liu et al., 2000 ; Martin et al., 2000 ). Thus, although the mRNA levels of these genes were not regulated by Mug28 (Supplemental Figure S2vii), Mug28 could still be involved in spore wall assembly.
In sporulation, leading edge proteins are involved in the initiation and development of the FSM. In fission yeast, a leading edge protein, Meu14, functions in the expansion and closure of four FSMs in a synchronous manner; closure of the FSM must be coordinated with the completion of meiosis II, to ensure proper cell division (Okuzaki et al., 2003 ). In mug28Δ cells, abnormal behavior of Meu14 was observed during sporulation, compared with mug28+ cells (Figure 4). Although most Meu14 formed a ring-shape during early anaphase II in the mug28+ strain, both Meu14 dots and Meu14 rings were detected in mug28Δ cells (Figures 4 and and5).5). Moreover, Meu14 could not properly disappear in mug28Δ cells, even after the guidance of leading FSM formation, and was thus aberrantly retained at the edge of the FSM (Figures 5 and and6).6). This abnormal behavior of Meu14 in mug28Δ cells suggests that Mug28 might somehow regulate the proper disappearance of Meu14 from the leading edge of the FSM during sporulation. The aberrant disappearance of Meu14 is considered to cause the formation of additional FSMs in mug28Δ cells. Notably, Cam2 (type I myosin light chain calmodulin), Myo1 (type I myosin heavy chain), F-actin, and Meu14 colocalize at the leading edge of FSM (Itadani et al., 2006 ); this suggests that the actin-mediated endocytosis machinery may regulate the closure of the leading edge. Thus, Mug28 also may control the actin-mediated endocytosis machinery by controlling the proper disappearance of Meu14 at sporulation.
Interestingly, this abnormal timing of Meu14 disappearance in mug28Δ cells resembles that of Ssp1p in the ama1Δ strain of S. cerevisiae (Diamond et al., 2009 ). Three leading edge proteins have been identified in budding yeast, namely, Don1p, Ssp1p, and Ady3p (Moreno-Borchart et al., 2001 ). Among these three proteins, Ssp1p is essential for sporulation in S. cerevisiae (Nickas and Neiman, 2002 ). Ssp1p is required for localization of both Ady3p and Don1p to the leading edge of the prospore membrane (PSM), an analogue of FSM in budding yeast, whereas PSM growth is abnormal and spore formation is blocked in ssp1Δ cells. Notably, PSM closure is defective in the absence of ama1, a meiosis-specific activator of the anaphase promoting complex/cyclosome (Diamond et al., 2009 ). Moreover, the leading edge protein Ssp1p is stabilized in ama1Δ mutants, suggesting that an essential function of Ama1p is to remove Ssp1p from the PSM by degradation. Because ectopic expression of Meu14DD-GFP caused no detectable abnormality in FSM formation (Supplemental Figure S6), the ubiquitin-proteasome pathway may not be involved in the untimely degradation of Meu14.
We also showed here that Mug28 localizes to the cytoplasm, particularly to the area around the nucleus, between metaphase I and anaphase II, and also to the nucleus during meiosis II (Figure 2). Considering that mug28Δ cells have both abnormal FSM formation and perinuclear localization of Mug28-GFP during meiosis, it is possible that the FSM and the nuclear membrane are coordinately regulated by Mug28 in the perinuclear region, which controls the disappearance of the leading edge protein Meu14 at sporulation. Nonetheless, because overexpression of Meu14 (Supplemental Figure S3B) and a putatively stabilized Meu14DD (Supplemental Figure S6) in wild-type cells did not reverse the morphological defects of mug28Δ cells, and because these morphological defects are also observed in mug28Δ meu14Δ double mutant cells (Supplemental Figure S4), we cannot exclude the possibility that the effects of Mug28 on Meu14 and on FSM morphology are independent.
Analysis of Mug28 mutants indicated that the RRM3 domain that harbors F466 is pivotal for the full functioning of Mug28, which is essential for proper maturation of the FSM and the spore wall (Figure 8). Indeed, abnormal Mug28 localization, spore formation, and spore viability were observed in F466A mutant cells (Figure 9). Homology between the RRMs of Mug28 and Meu5/Crp79, another meiosis-specific RNA-binding protein (Watanabe et al., 2001 ), may be noteworthy. Meu5/Crp79 also was isolated as a novel mRNA export factor from the synthetic lethal screen of a mutation in rae1+, an essential gene required for mRNA export, during vegetative growth in S. pombe; indeed, Meu5/Crp79 binds to and exports mRNA from the nucleus to the cytoplasm (Thakurta et al., 2002 ). Although the RRM1 and RRM3 domains of Meu5/Crp79 also have an essential role in poly(A)+ RNA binding (Thakurta et al., 2002 ), it remains unclear whether Mug28 also can bind to and export any RNA during meiosis, because there is no clear homology between Mug28 and Meu5/Crp79 beyond that seen in the RRMs.
A similar common association partner may exist between Mug28 and Meu5/Crp79. The present study indicates that meiosis-specific proteins harboring RRMs are involved not only in meiotic progression but also in the proper formation or maturation of FSMs and spore walls. It is expected that Mug28 binds to some target RNAs, interaction partners, or both via its RRM domains and thus functions in the proper development and formation of the FSM during meiosis. Identifying the target RNAs and interaction partners of Mug28 will provide important information about the novel function of RBPs in the proper development and formation of FSMs during meiosis. We plan to address these issues in future studies.
We thank Drs. C. Shimoda, M. Yamamoto, T. Nakamura, W. Z. Cande, A. Ohtaka, and R. Tsien and the National BioResource Project (http://yeast.lab.nig.ac.jp/nig/) (Osaka City University) for S. pombe strains and plasmids. We are also indebted to Dr. P. Hughes for critically reading the manuscript. This work was supported in part by grants-in-aid for Scientific Research on Priority Areas “Applied Genomics,” Scientific Research (S), Exploratory Research, and the Science and Technology Incubation Program in Advanced Regions from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H. N.).
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-12-0997) on April 21, 2010.