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Cell. Author manuscript; available in PMC Oct 18, 2008.
Published in final edited form as:
Cell. Apr 18, 2008; 133(2): 280–291.
doi:  10.1016/j.cell.2008.02.032
PMCID: PMC2396536
NIHMSID: NIHMS47705

Meiosis I is established through division-specific translational control of a cyclin

Summary

In budding yeast key meiotic events such as DNA replication, recombination, and the meiotic divisions are controlled by Clb cyclin-dependent kinases (Clb-CDKs). Using a novel synchronization procedure, we have characterized the activity of these Clb-CDKs and observed a surprising diversity in their regulation during the meiotic divisions. Clb1-CDK activity is restricted to meiosis I, and Clb3-CDK activity to meiosis II through 5’UTR-mediated translational control of its transcript. The analysis of cells inappropriately producing Clb3-CDKs during meiosis I furthermore defines Clb3 as an inhibitor of the meiosis I chromosome segregation program. Our results demonstrate an essential role for Clb-CDK regulation in establishing the meiotic chromosome segregation pattern.

Introduction

Meiosis is a specialized cell division used by sexually reproducing organisms to produce haploid gametes from diploid progenitor cells. Pre-meiotic DNA replication is followed by two chromosome segregation phases, meiosis I and meiosis II. During meiosis I homologous chromosomes are segregated, while during meiosis II sister chromatids are split. As during the mitotic cell cycle, cyclin-dependent kinases (CDKs) promote progression through the meiotic program. Budding yeast contains six B-type cyclins, Clb1 – Clb6 (reviewed in Bloom and Cross, 2007). Clb5 and Clb6, in conjunction with the sole CDK, Cdc28, are essential for the initiation of pre-meiotic S-phase (Dirick et al., 1998; Stuart and Wittenberg, 1998) and the initiation of homologous recombination (Henderson et al., 2006). B-type cyclins and Cdc28 are also required for the two meiotic divisions (Benjamin et al., 2003). The major mitotic cyclin CLB2 is not expressed during meiosis. Instead, CLB1, CLB3 and CLB4 promote progression through the meiotic divisions (Dahmann and Futcher, 1995). Deletion of any two of these three cyclins results in cells executing only a single meiotic division. During this single division, homologous chromosomes are segregated (Dahmann and Futcher, 1995), but meiosis II events such as loss of Sgo1, the sister chromatid cohesion control factor, from chromosomes occur (Kiburz et al., 2008).

Studies of Clb-CDKs during mitosis revealed that these kinases are regulated at multiple levels. Transcription of the CLB genes is periodic during the cell cycle, with their expression typically confined to those cell cycle stages when their activity is needed. In addition to transcriptional control, cell cycle regulated degradation of Clb cyclins is essential for restricting Clb-CDK activities to the appropriate stages of the cell cycle (reviewed in Mendenhall and Hodge, 1998). At the metaphase – anaphase transition an ubiquitin ligase known as the Anaphase Promoting Complex or Cyclosome (APC/C), together with the specificity factor Cdc20, degrades Clb5 and a fraction of Clb2. During late anaphase another APC/C specificity factor called Cdh1 degrades Clb1, Clb3 and the remaining pool of Clb2. Down-regulation of Clb-CDK activity at the end of mitosis is primarily brought about by degradation of the Clb proteins, but the Clb-CDK inhibitor Sic1, which directly binds to the cyclin-CDK complex, helps restrain Clb-CDK activity during exit from mitosis and G1 (reviewed in Bloom and Cross, 2007).

CDK activity associated with the different Clb cyclins has been characterized extensively during the mitotic cell cycle. However, the asynchrony with which sporulating cultures of S. cerevisiae proceed through the meiotic divisions has prevented the detailed characterization of CDK activity during meiosis. We developed a novel synchronization method that produces meiotic cultures that proceed through the meiotic divisions with a high degree of synchrony, comparable to that of synchronized mitotic cultures. Using this synchronization method we characterized Clb1-, Clb3-, Clb4- and Clb5-CDK activity and observed a striking diversity in their regulation during meiosis. Clb1-CDK activity, but not Clb1 protein, is restricted to meiosis I. Clb3-CDK activity, on the other hand, is meiosis II-specific because Clb3 protein is not translated during meiosis I. This meiosis I-specific translational inhibition is mediated by the 5’ untranslated region (UTR) of the RNA. Finally, we show that restricting Clb3 protein to meiosis II is essential for establishing the meiosis I chromosome segregation pattern. Our results demonstrate a high degree of specialization of Clb-CDK regulation during meiosis and demonstrate an essential role for Clb-CDK regulation in establishing the meiotic chromosome segregation pattern.

Results

A method to generate synchronous meiotic cultures in budding yeast

In budding yeast, the resolution with which meiotic events can be observed is limited by the relative asynchrony of meiotic cultures, much of which stems from variations in the timing of entry into the meiotic program (Nachman et al, 2007). We eliminated this source of asynchrony by developing conditions that arrest cells reversibly in prophase I, prior to the two meiotic divisions. NDT80 is a transcription factor that is required for progression out of the pachytene stage of meiosis and into meiosis I (Xu et al., 1995; Chu et al., 1998). To arrest cells reversibly in pachytene, the NDT80 open reading frame was placed under the control of the inducible GAL1-10 promoter (GAL-NDT80; Benjamin et al., 2003). In cells producing a Gal4-estrogen receptor fusion protein (Gal4.ER), transcription from the GAL1-10 promoter can be induced by the addition of estrogen to the medium (Picard et al., 1999; Benjamin et al., 2003; Figure 1A).

Figure 1
A meiotic block-release synchronization system

In the absence of β-estradiol GAL4.ER GAL-NDT80 cells failed to undergo any meiotic divisions (Figure 1B, C). However, when 1 μM β-estradiol was added 5 hours after transfer into meiosis-inducing conditions (henceforth called the GAL-NDT80 block), GAL4.ER GAL-NDT80 cells underwent both meiotic divisions synchronously (Figure 1B, C). Cells initiated meiosis I one hour post release, and approximately 80% of cells had undergone this division one hour later (Figure 1B, left panel). Meiosis II occurred with a similarly high degree of synchrony (Figure 1B, right panel). In contrast, cultures of cells that expressed NDT80 from its native promoter took approximately four hours to complete the first division and another four hours to complete meiosis II with the peaks of meiosis I and meiosis II largely overlapping (Figure 1B). The high degree of synchrony of meiotic cultures that were blocked in pachytene and then released from the block was particularly evident when the percentages of cells in metaphase I, anaphase I, metaphase II and anaphase II were examined (Figure 1C). More than 40% of cells progressed through the different cell cycle stages simultaneously, whereas only 10% of cells with NDT80 under its native promoter did (Figure 1C). This analysis also showed that cells spent longer periods of time in metaphase II and anaphase II, compared to their meiosis I counterparts (Figure 1C). Why progression through meiosis II takes longer than progression through meiosis I is at present unknown.

Tetrad analysis confirmed that the pachytene arrest caused by the depletion of NDT80 and the release from the block did not interfere with meiotic progression. Sporulation efficiency and spore viability were high (96.5% and 95.5% respectively; Figure 1D). We conclude that modulating the production of Ndt80 can be used to generate meiotic cultures that progress through the meiotic divisions with a high degree of synchrony.

Clb1-CDK activity is restricted to meiosis I, Clb3-CDK activity to meiosis II

To determine how Clb-CDK activity is controlled during the meiotic divisions we examined the expression and activity of four of these cyclins, CLB1, CLB3, CLB4 and CLB5 using the synchronization procedure described above. Consistent with the role of CLB5 in pre-meiotic DNA-replication, both Clb5 protein and associated kinase activity were present upon release from the NDT80 block, peaked during metaphase I, declined in anaphase I and peaked again as cells formed metaphase II spindles (Figure 2A, D; Supplemental Figure 1C, 2A). Comparison of Clb5 protein levels and Clb5-associated kinase activity showed that Clb5-CDK activity paralleled Clb5 protein levels (Figure 2D). We conclude that Clb5-CDK activity is regulated primarily at the level of Clb5 protein abundance and that Clb5 protein and associated kinase activity appear in two waves, one during meiosis I and one during meiosis II.

Figure 2
Cyclin expression and activity during the meiotic divisions

Clb4-CDKs displayed a more complex pattern of regulation. CLB4 RNA and protein accumulated as cells entered meiosis I and remained high throughout the two meiotic divisions (Figure 2E, F, G; Supplemental Figure 2B). Comparison of Clb4 protein levels with Clb4-CDK activity showed that the two correlated well until metaphase I. Thereafter, the decline in Clb4-CDK activity was not paralleled by a decline in Clb4 protein. This was particularly evident during exit from meiosis II (Figure 2E, H, Supplemental Figure 1A). The loss of Clb4-CDK activity was not due to a failure to immunoprecipitate Clb4 during late stages of meiosis (data not shown) indicating that during exit from meiosis I and meiosis II posttranslational mechanisms other than degradation of Clb4 protein down-regulate Clb4-CDK activity.

CLB1 mRNA and Clb1 protein levels rose during meiosis I and remained high until exit from meiosis II (Figure 2I, J, K; Supplemental Figure 2C). A slower migrating form of Clb1 was only detected during meiosis I and correlated with Clb1-CDK activity (Figure 2I, Supplemental Figure 1B, 2E) suggesting that this form of Clb1 signifies active Clb1-CDK complexes. Quantification of Clb1 protein and associated kinase activity confirmed this result and demonstrated that Clb1-CDK was only active during meiosis I (Figure 2L, Supplemental Figure 2E). The loss of Clb1-CDK activity during meiosis II was not due to an inability to immunoprecipitate Clb1 (data not shown), excluding the possibility that degradation of Clb1 in extracts or insolubility of the protein were responsible for the lack of Clb1-CDK activity during meiosis II. Our results show that during the meiotic divisions Clb1-CDK is a meiosis I-specific CDK. The finding that Clb1, which is an APC/C-Cdh1 substrate during the mitotic divisions (J. Simpson and M. Brandeis, personal communications), is not degraded during exit from meiosis I furthermore indicates that APC/C-Cdh1 is inactive during this transition. The observation that Clb1-CDK activity is nevertheless low during meiosis II demonstrates that posttranslational mechanisms other than protein degradation inhibit Clb1-CDK activity during exit from meiosis I and during meiosis II.

The CLB3 transcript accumulated somewhat later than that of CLB1 and CLB4 but nevertheless reached high levels during meiosis I and peaked during meiosis II (Figure 2N, O; Supplemental Figure 2D). Interestingly, a 3HA tagged version of Clb3 protein and its associated kinase activity did not appear until the onset of meiosis II (Figure 2M, P; Supplemental Figure 1A). Untagged Clb3 also did not accumulate until the onset of meiosis II (Supplemental Figure 1D, 2C). This data shows that during the meiotic divisions Clb3 is a meiosis II-specific cyclin, and that post-transcriptional mechanisms restrict the protein to meiosis II.

Protein degradation is not responsible for restricting Clb3 to meiosis II

Clb3 is an APC/C substrate (Zachariae et al., 1996), and in chicken cells APC/C-Cdh1 is active during G2 (Sudo et al., 2001). It was therefore possible that during the prolonged premeiotic G2 arrest induced by the NDT80 block APC/C-Cdh1 was activated leading to the degradation of Clb3, or that a novel-degradation pathway was responsible for keeping Clb3 protein levels low during meiosis I. To test this hypothesis we examined the effects of inhibiting the 26S proteasome and the APC/C on Clb3 levels during meiosis I.

Addition of MG-132 to cells inhibits the proteasome. When added to meiotic cultures, 15% of cells arrested in metaphase I (Figure 3C) and progression through the meiotic divisions was somewhat hampered (Figure 3B) indicating that the proteasome was only partially inactivated by MG-132 in these experiments. This partial inhibition was nevertheless sufficient to prevent degradation of Clb3 during exit from meiosis II (Figure 3A). However, treatment with MG-132 did not lead to accumulation of Clb3 during meiosis I (Figure 3A) suggesting that proteasome-mediated protein degradation was not responsible for keeping Clb3 protein levels low during meiosis I.

Figure 3
Clb3 does not accumulate during meiosis I in proteasome inhibited cells or in APC mutant cells

Inactivation of the APC/C by depleting the APC/C component Cdc27 or the APC/C activator Cdc20, or by employing a temperature sensitive allele of the APC/C subunit Cdc23 (cdc23-1) did not allow Clb3 to accumulate during meiosis I. Meiotic depletions of Cdc20 and Cdc27 were achieved by placing these genes under the control of the mitosis specific CLB2 promoter (Lee and Amon, 2003). Cdc20-depleted cells (cdc20-mn cells) arrested in metaphase I (Figure 3E, F), and Cdc27-depleted cells (cdc27-mn cells) were delayed in metaphase I (Figure 3E,F) indicating that Cdc27 depletion was not complete or that Cdc27 was not essential for the metaphase I – anaphase I transition. cdc23-1 cells were delayed in metaphase I, but some cells completed meiosis I before arresting in metaphase II (Figure 3H,I). In cdc20-mn, cdc27-mn and cdc23-1 cells Pds1-18Myc, an APC/C target whose degradation is required for anaphase I and anaphase II onset, was stabilized during exit from meiosis II indicating that APC/C function is impaired in these mutants (Salah and Nasmyth, 2000; Figure 3D and 3G). However, Clb3 did not accumulate during meiosis I in any of these strains (Figure 3D and 3G). In fact its accumulation was greatly delayed in the APC/C mutants that are defective in progressing through meiosis I further confirming that Clb3 is indeed a meiosis II-specific cyclin. Our results do not rule out a minor role for APC/proteasome dependent degradation in preventing Clb3 accumulation during meiosis I, but they indicate that other posttranscriptional mechanisms are primarily responsible for preventing the accumulation of Clb3 protein during meiosis I.

The 5’UTR of CLB3 is required to restrict Clb3 protein to meiosis II

Although Clb1-, Clb3-, and Clb4-CDK activity all appeared to be regulated in an interesting manner during the meiotic divisions, we chose to further study Clb3 regulation because our results raised the possibility that translational control restricts Clb3 protein to meiosis II. This form of regulation would represent a novel mechanism of controlling Clb-CDKs in yeast.

To investigate the possibility that translational control confines Clb3 protein to meiosis II, we tested whether the UTRs of the mRNA mediate such regulation. Because the Clb3 protein employed in this study carries a tag at its C-terminus resulting in the disruption of the native 3’UTR of CLB3 (Longtine et al., 1998) we examined the role of the 5’UTR in preventing Clb3 accumulation during meiosis I by replacing the promoter and 5’UTR of CLB3 with that of the GAL1-10 gene. Thus cells carrying the GAL4.ER fusion express CLB3 upon addition of β–estradiol. Upon release from the GAL-NDT80 block GAL-CLB3 was expressed within 30 minutes (Figure 4B). The amount of CLB3 RNA generated from the GAL1-10 promoter was significantly lower than that produced by the native CLB3 promoter. This was best seen when the amounts of CLB3 RNA at the 7.5, 7.75 and 8 hour time points of the wild-type were compared to that of the 6.5, 6.75 and 7 hour time points of the GAL-CLB3 carrying strain (Figure 4B). Clb3 protein however was undetectable in wild-type cells at the 7.5, 7.75 and 8 hour time points but the protein was highly produced at the 6.5, 6.75 and 7 hour time points of the GAL-CLB3 carrying strain (Figure 4A).

Figure 4
The CLB3 promoter and 5’UTR are required to prevent Clb3 accumulation during meiosis I

Quantification of the amount of Clb3 protein produced by CLB3 mRNA demonstrated that the promoter and 5’UTR of CLB3 affected Clb3 protein levels in at least two ways. First, it functions to restrict Clb3 protein to meiosis II. The ratio of Clb3 protein/CLB3 mRNA was low during meiosis I but high during meiosis II (Figure 4E, left panel). Second, the 5’UTR prevents efficient translation. This was evident when we compared the amount of Clb3 protein generated per CLB3 mRNA in wild-type cells with the amount of Clb3 protein generated from CLB3 mRNA carrying the GAL1-10 5’UTR (Figure 4E, right panel). The amount of Clb3 protein produced per RNA was much higher in the GAL-CLB3 expressing strains than in wild-type cells. Similar results were obtained when CLB2 was controlled by the CLB3 promoter and 5’UTR (Figure 6A). We conclude that the promoter and 5’UTR of CLB3 are required for restricting translation of Clb3 protein to meiosis II.

Figure 6
The CLB3 5’UTR is sufficient to prevent protein accumulation during meiosis I

The 5’UTR of CLB3 is sufficient to prevent accumulation of proteins during meiosis I

To determine whether the CLB3 promoter and 5’UTR were sufficient to restrict accumulation of proteins to meiosis II we replaced the open reading frame and 3’UTR of CLB3 with that of CLB2 and ADH1, respectively (see Experimental Procedures). As a control, we also placed CLB2 fused to the ADH1 3’UTR under the control of the CLB4 promoter. Expression of CLB2 from the CLB3 or CLB4 promoters did not significantly interfere with progression through meiosis, although exit from meiosis II appeared slightly delayed in PCLB4-CLB2 strains (Figure 5C–D).

Figure 5
The CLB3 promoter and 5’UTR are sufficient to prevent protein accumulation during meiosis I

As expected, in both PCLB3-CLB2 and PCLB4-CLB2 strains CLB2 transcript levels mirrored those of CLB3 and CLB4, respectively (Figure 5B). However, despite the presence of CLB2 transcript during meiosis I in PCLB3-CLB2 cells, Clb2 protein did not accumulate during meiosis I, but was, as Clb3, present only during meiosis II (Figure 5A). In PCLB4-CLB2 strains Clb2 appeared during meiosis I, indicating that Clb2 protein can accumulate during the first division (Figure 5A). Our results indicate that the promoter and 5’UTR of CLB3 are sufficient to confine translation of proteins to meiosis II.

To further define the sequences that bring about translational control to CLB3 we determined the length of the CLB3 5’UTR. This analysis revealed a start site at 130 bp upstream of the ATG and additional transcriptional initiation between 130 bp and 156 bp upstream (Supplemental Figure 3). Thus, the 5’UTR has a maximal length of 156 bases, which is in good agreement with a recent genome-wide analysis of the yeast transcriptome (David et al., 2006). Importantly, the length of the 5’UTR was the same during vegetative growth, meiosis I and meiosis II (Supplemental Figure 3) indicating that changes in 5’UTR length are not likely to be responsible for the differences in translation observed between meiosis I and meiosis II.

To determine whether the CLB3 5’UTR is sufficient to restrict translation of proteins to meiosis II, we placed CLB2 under the control of a fusion between the GAL1-10 promoter and 153 bases of the 5’UTR of CLB3 (GAL-5’UTRCLB3-CLB2). Cells that carry CLB2 under the control of the GAL1-10 promoter and GAL1 5’UTR (GAL-CLB2) were used as a control. Expression of CLB2 RNA was similar in the two strains (Figure 6B). In contrast, Clb2 protein accumulated during meiosis I in GAL-CLB2 cells, but did not accumulate until meiosis II in GAL-5’UTRCLB3-CLB2 strains (Figure 6A). Furthermore, consistent with our analysis of Clb3 translation, Clb2 translation was significantly less efficient when the 5’ UTR of CLB3 was employed to drive CLB2 expression (Figure 6A). Our results show that the CLB3 5’UTR is sufficient to prevent protein accumulation during meiosis I.

Consequences of translating Clb3 during meiosis I

To determine whether preventing Clb3 accumulation during meiosis I was important for the successful execution of this division we produced Clb3 during meiosis I by placing the gene under the control of the GAL1-10 promoter. Importantly, the amount of Clb3 produced from this promoter during meiosis I was less than the amount of Clb3 that accumulates during meiosis II in wild-type cells (Figure 4A) indicating that the protein was not significantly overproduced when expressed from the GAL1-10 promoter. To follow the fate of chromosomes during the meiotic divisions we integrated a tandem array of tetO sequences at LEU2 on both copies of chromosome III (homozygous LEU2-GFP dots). These cells also expressed a tetR-GFP fusion, which binds to tetO, to visualize the repeats (Michaelis et al., 1997).

Wild-type and GAL-CLB3 cells carrying NDT80 under its native promoter were induced to enter meiosis. Three hours thereafter, when most cells had completed DNA replication, β-estradiol was added to induce expression of CLB3 in the GAL-CLB3 cells. In wild-type cells, homologous chromosomes segregated during meiosis I giving rise to binucleate cells with a LEU2-GFP dot in each nucleus (Figure 7A, left panel). In contrast, 17% of GAL-CLB3 cells mis-segregated homologs to the same pole during meiosis I leading to binucleate cells with a LEU2-GFP dot in only one of the two nuclei (Figure 7A, left panel). The distribution of LEU2-GFP dots was also abnormal in cells that had completed meiosis II (Figure 7A, right panel). Despite these segregation abnormalities, both meiotic nuclear divisions occurred in GAL-CLB3 cells to the same extent as in wild-type cells (65% tetranuclate cells in GAL-CLB3 cultures compared to 57% in wild-type cultures; n=200). Tetrad formation was however reduced (30.5% tetrads in GAL-CLB3 cells compared to 59% in wild-type cells; n=200). Analysis of the spore viability revealed that in cells that were able to form spores chromosome segregation was little affected. 66% of spores of GAL-CLB3 strains were viable compared to 93% of the wild-type (n=160).

Figure 7
Production of Clb3 or Clb2 during meiosis I causes premature sister chromatid separation

Next we examined whether production of Clb3 during meiosis I affected sister chromatid segregation by analyzing the behavior of strains in which only one of the two homologs carried LEU2-GFP dots (heterozygous LEU2-GFP dots). In this situation, a wild-type meiosis I chromosome segregation pattern gives rise to binucleate cells with a LEU2-GFP dot in one of the two nuclei. In GAL-CLB3 cells, 30% of binucleate cells contained a GFP dot in both nuclei (Figure 7B; left panel). This outcome was not due to increased recombination between the centromere and the LEU2 locus because GFP dots at the centromere of chromosome V (CENV-GFP dots) behaved similarly (Figure 7B, right panel). Our results show that expression of Clb3 prior to and during meiosis I interferes with homolog disjunction and causes cells to segregate sister chromatids during the first nuclear division.

Are the effects of expressing Clb3 on meiosis I chromosome segregation specific to this cyclin or does any high Clb-CDK activity interfere with meiotic chromosome segregation? To address this question we examined the consequences of producing Clb2, another cyclin that is normally not expressed during meiosis I. Sister chromatid separation during the first meiotic division also occurred in cells expressing CLB2 from the GAL1-10 promoter but at a lower frequency (12% in GAL-CLB2 cells compared to 51% in GAL-CLB3 cells; Figure 7B, C). In contrast, replacing the promoter of a cyclin that is normally expressed during meiosis I (CLB4) with the GAL1-10 promoter did not interfere with meiotic chromosome segregation (Figure 7C), sporulation efficiency and spore viability (94.5%; n=160). Our results show that expression of a cyclin that is normally expressed during meiosis I from the GAL1-10 promoter (Clb4) does not affect meiosis I chromosome segregation. In contrast, expression of a cyclin that is not normally expressed during meiosis I such as Clb2 or Clb3, which likely results in an increase in overall Clb-CDK activity during meiosis I, interferes with meiotic chromosome segregation. Furthermore, it appears that Clb3 is a more potent inhibitor of meiosis I chromosome segregation than Clb2.

Production of Clb3 during meiosis I causes premature sister chromatid separation

Production of Clb3 prior to and during meiosis I caused the appearance of bi-nucleate cells that had separated their sister chromatids. To determine whether this was due to premature sister chromatid separation during meiosis I we examined the behavior of heterozygous CENV-GFP dots in cells that were arrested in metaphase I, due to the depletion of Cdc20. During metaphase I kinetochores of sister chromatid pairs attach to microtubules emanating from the same pole (co-orientation). Thus the sister kinetochores are not under tension and only one CENV-GFP dot is visible (Figure 7D, Lee and Amon, 2003). In contrast, when sister chromatids are bi-oriented, their kinetochores attach to microtubules from opposite poles. The pulling force of the spindle then leads to the appearance of two CENV-GFP dots (Lee and Amon, 2003).

Production of Clb3 in Cdc20-depleted cells led to separation of sister chromatids as judged by the appearance of two GFP dots (Figure 7D). Interestingly, expression of Clb3 also suppressed the metaphase I arrest caused by the depletion of Cdc20. GFP-dots integrated at URA3 also separated in GAL-CLB3 cells and spindle elongation and the formation of binucleate cells occurred (Figure 7D-F). Our results show that production of Clb3 suppresses the need for high levels of Cdc20 in promoting entry into anaphase I and causes premature sister chromatid separation during meiosis I.

Discussion

Clb-CDK control during meiosis

We developed a method to produce budding yeast cultures that proceed through the meiotic divisions with a high degree of synchrony. The examination of Clb-CDKs using this system revealed a surprising diversity in the regulation of the different cyclin-CDK complexes that was not appreciated previously because of the poor synchrony of meiotic cultures generated by standard conditions. During vegetative growth, transcription and ubiquitin-dependent protein degradation are primarily responsible for controlling Clb-CDK activity (reviewed in Bloom and Cross, 2007). Clb6 is a substrate of the SCF ubiquitin ligase, and Clb5 and a fraction of Clb2 are degraded by APC/C-Cdc20 at the metaphase – anaphase transition. Clb1, Clb2 and Clb3 are degraded later during mitosis by APC/C-Cdh1. The ubiquitin ligase responsible for degrading Clb4 has not been identified. During meiosis only Clb5-CDK activity is regulated at the level of Clb5 protein abundance. Clb3-CDKs are regulated at the level of Clb3 translation. Clb1-CDKs and Clb4-CDKs are restricted to specific meiotic stages by posttranslational mechanisms other than protein degradation.

Which post-translational mechanisms could be responsible for down-regulating Clb4-CDKs after metaphase II and Clb1-CDKs during all of meiosis II? CDK inhibition by tyrosine 19 phosphorylation on Cdc28 or binding of the CDK inhibitor Sic1 could inhibit Clb4-CDKs during exit from meiosis II. One of the two pathways or both could also be responsible for preventing Clb1-CDKs from being active throughout meiosis II. This, however, would require a high degree of Clb-CDK specificity for these regulatory mechanisms that has not been observed previously. We therefore believe it to be more likely that a novel Clb1-CDK specific mechanism exists that prevents this particular kinase from being active during meiosis II. Such a mechanism could involve a selective inhibitor of Clb1-CDKs or preventing the association of Clb1 with Cdc28. In this regard it is interesting to note that Clb1 protein is not degraded during exit from meiosis I but exported out of the nucleus (Buonomo et al., 2003; Marston et al., 2003). Perhaps this nuclear export of Clb1 is important for maintaining Clb1-CDKs inactive during meiosis II.

Translational control in meiosis

Clb3 expression is restricted to meiosis II through translational control. In higher eukaryotes, translational control of B-type cyclins plays a key role in controlling meiotic progression. In Xenopus, progesterone mediated oocyte maturation relieves the translational inhibition of a number of mRNAs including that of cyclin B1 by promoting their polyadenylation (Stebbins-Boaz et al., 1996). Translational control of cyclin B mRNA is also observed in Drosophila. There, the PAN GU kinase promotes translation of the cyclin B message during exit from meiosis II by antagonizing the translational repressor Pumilio (Vardy and Orr-Weaver, 2007). Translational control of Clb3 appears to be meiosis I specific. CLB3 mRNA is translated during meiosis II and also during mitosis, because Clb3 protein accumulates as soon as cells enter the cell cycle (as judged by bud formation) and APC/C-Cdh1 activity is turned off (Supplemental Figure 4). The simplest interpretation of this data is that CLB3 translation is controlled by a meiosis I-specific translational repressor.

The 5’UTR affects translation of Clb3 in at least two ways. The 5’UTR decreases translational efficiency and restricts translation to meiosis II. Whether these two effects are linked or are brought about by separate mechanisms remains to be determined. Translational control occurs in budding yeast, but to date has not been observed during meiosis. Micro open reading frames (μORFs) regulate the translation of the GCN4 mRNA. These μORFs are thought to serve as a measure of the translational capacity of the cell, preventing translation of GCN4 mRNA when the translational capacity is high (Hinnebusch, 2005). The 156 bp 5’UTR of CLB3 does not contain μORFs (T. M. C. unpublished observations). It is therefore not likely that translation of CLB3 is governed by such a mechanism. Processing bodies (P-bodies) are cytoplasmic foci that are sites of mRNA degradation and of storage of non-translating mRNAs, which can later reenter translation (reviewed in Parker and Sheth, 2007). PAT1 and DHH1 have been implicated in regulating mRNA stability and translation in P-bodies (Coller and Parker, 2005). Deletion of neither gene allowed Clb3 protein to accumulate during meiosis I (T. M. C., unpublished observations) indicating that this pathway was not responsible for controlling Clb3 translation. Identifying the mechanisms that prevent translation of Clb3 during meiosis I and determining whether other RNAs are regulated in this manner will be an important question in the future. Furthermore we note that the 156 bp CLB3 5’UTR sequence will prove useful in studying the effects of expressing genes specifically during meiosis II.

Importance of translational control of Clb3

During meiosis I, homologous chromosomes rather than sister chromatids segregate from each other. For this unusual chromosome segregation to occur several meiosis specific events must take place (reviewed in Marston and Amon, 2004). First, reciprocal recombination between homologs generates linkages between them, which ensure that homologs are accurately aligned on the metaphase I spindle. Second, mediated by the monopolin complex, sister kinetochores attach to microtubules emanating from the same pole (co-orientation) to facilitate sister chromatid co-segregation during anaphase I. Lastly, cohesin complexes that hold sister chromatids together are lost in a step-wise manner. Loss of arm cohesion allows for the segregation of homologs during meiosis I. Retention of centromeric cohesion ensures that sister chromatids properly align on the meiosis II spindle. Inhibition of Clb3 production during meiosis I is critical for establishing the meiosis I chromosome segregation pattern. In cells producing Clb3 during meiosis I, sister kinetochore co-orientation and the step-wise loss of cohesion appear to be disrupted. Clb3 could interfere with sister kinetochore co-orientation by preventing the association of the monopolin complex with kinetochores. How could Clb3 interfere with the step-wise loss of cohesion? During meiosis I, Sgo1 associates with kinetochores where it prevents loss of cohesins (reviewed in Ishiguro and Watanabe, 2007). Clb3 could inhibit the association of Sgo1 with kinetochores or prevent the protein from recruiting the protein phosphatase PP2A, which renders cohesins resistant to removal.

The phenotype of cells expressing CLB3 during meiosis I is reminiscent to that of cells lacking the meiosis I specific gene SPO13. In both, GAL-CLB3 cells and spo13Δ cells, sister chromatids separate during meiosis I. In both strains, the metaphase I arrest brought about by Cdc20 depletion is suppressed (Shonn et al., 2002; Katis et al., 2004) and delaying progression through early meiosis suppresses premature sister chromatid separation (McCarroll and Esposito, 1994; Supplemental Figure 5). SPO13 is, however, not required for regulating CLB3 translation. In cells lacking SPO13 translation of CLB3 did not occur prematurely (Supplemental Figure 6). Whether Clb3-CDK activity inhibits Spo13 function remains to be determined.

Clb-CDK specificity during meiosis

Is Clb3 unique amongst the Clb cyclins in repressing meiosis I chromosome segregation? Consistent with this idea is the observation that Clb2 is not as effective in suppressing the meiosis I chromosome segregation pattern as Clb3. Several other observations, however argue against this notion. First, to date no CDK substrate has been identified that is phosphorylated exclusively by one Clb-CDK subtype (Loog and Morgan, 2005). Second, deletion of individual CLB genes does not interfere with progression through meiosis (Dahmann and Futcher, 1995). Finally, expression of CLB4 from the GAL1-10 promoter instead of its native promoter, which is not expected to substantially increase overall Clb-CDK activity, does not interfere with meiotic chromosome segregation. On the other hand, moderate expression of Clb2 during meiosis I, which is expected to elevate overall Clb-CDK activity, causes some premature sister chromatid separation. Increasing Clb-CDK activity levels even further through overexpression of a stabilized version of Clb2, leads to more than half of sister chromatids segregating during the first nuclear division, which could not solely be explained by meiosis II events occurring on the anaphase I spindle (Marston et al., 2003). We therefore favor the idea that high Clb-CDK levels interfere with the meiosis I chromosome segregation pattern but will not exclude the possibility that some substrate-specificity exists among Clb1, Clb2, Clb3 and Clb4 causing Clb3 to be better at inhibiting meiosis I than other Clbs.

Aside from the issue of Clb-CDK specificity our studies raise the following question. Why do cells restrict different Clb-CDKs to different stages of meiosis and employ such diverse strategies to accomplish this? We propose that the answer may lie in the unique feature of meiosis that is the occurrence of two consecutive chromosome divisions. One division immediately following another one requires a balancing act between the necessity to down-regulate Clb-CDKs to bring about exit from meiosis I and ensuring that a sufficient amount of Clb-CDKs are present to execute the second meiotic division. Employing APC/C-mediated protein degradation to bring about Clb-CDK down-regulation during exit from meiosis I as is done during mitosis would necessitate the re-synthesis of Clb cyclins prior to entry into meiosis II, which may be a lengthy undertaking under the extreme nutrient-limiting conditions of meiosis. It may thus be advantageous for the cell to inhibit APC/C-Cdh1 during exit from meiosis I. Indeed, our data and that of others on Clb1 protein abundance (Marston et al., 2003; Buonomo et al., 2003) indicate that this is the case. Instead, other mechanisms have evolved that down-regulate Clb1-CDKs to promote exit from meiosis I. Following exit from meiosis I, maintaining a pool of CLB3 RNA ready for translation may ensure quick entry into meiosis II. We suggest that the strategies governing progression through two consecutive meiotic divisions required novel ways of regulating Clb-CDKs.

Experimental Procedures

Strains and Plasmids

All strains are SK1 derivatives and are described in Supplemental Table 1. GAL-NDT80 and GAL4.ER constructs are described in Benjamin et al. (2003). CLB3-3HA, CLB4-3HA, CLB5-3HA, pdr5 , PCLB2-CDC20, PCLB2-3HA-CDC27, GAL-CLB3-3HA and GAL-CLB4-3HA were constructed using the PCR-based method described in Longtine et al. (1998). Endogenous CLB3 and CLB4 were replaced with CLB2 using the PCR-based method described in Longtine et al. (1998). The cdc23-1 allele was integrated at the CDC23 locus in SK1. GAL-5’UTRCLB3-CLB2 was created by cloning 153 base pairs of the CLB3 5’UTR into a GAL-CLB2 plasmid, and integrating at the CLB2 locus.

Sporulation Conditions

Strains were grown to saturation in YPD, diluted in YPA (1% yeast extract, 2% bactopeptone, 1% potassium acetate) to OD600 = 0.3, and grown overnight. Cells were resuspended in sporulation medium (0.3% potassium acetate [pH 7], 0.02% raffinose) to OD600 = 1.9 and sporulated at 30°C. A more detailed description can be found in the Supplemental Online Materials. GAL-NDT80 GAL4.ER strains were released from the arrest by the addition of 1 μM β-estradiol (5mM stock in ethanol, Sigma E2758-1G) at 6 hours unless otherwise indicated. In NDT80 GAL4.ER strains, transcription was induced from the GAL promoter with 1 μM β-estradiol at 3 hours.

Western Blot Analysis

Samples were prepared as described in Moll et al. (1991), immunoblots as in Cohen-Fix et al. (1996). Antibody concentrations are described in Monje-Casas et al. (2007), except anti-Pgk1 (Molecular Probes) was used at 1:5000. Rabbit anti-Clb2 was used at a concentration of 1:2000, rabbit anti-Cdc28 at 1:1000 and rabbit anti-Clb3 (Santa Cruz, sc-7167) at 1:500.

Other Techniques

Total RNA was isolated as described in Cross and Tinkelenberg (1991). Northern blots were performed as described in Hochwagen et al. (2005). Indirect immunofluorescence was performed as described in Visintin et al. (1999). GFP-tagged chromosomes were fixed for visualization as described in Monje-Casas et al. (2007). 200 cells were counted for each time point unless otherwise noted. Criteria for classifications of spindle morphologies are described in the Supplemental Online Material. Histone H1 kinase assays were performed as described in Hochwagen et al. (2005) and are described in detail in the Supplemental Online Materials. Quantification of immunoblots, Northern blots and kinase assays were performed using NIH ImageQuant. To synchronize mitotic cultures in G1, α-factor was added to a concentration of 5 μg/ml. When fully arrested, cells were washed and resuspended in pheromone free media. Total RNA prepared for 5’RACE analysis as described in Collart and Oliviero (1993). 5’RACE was performed using the 5’RACE System Version 2.0 (Invitrogen, 18374-058).

Supplementary Material

01

Acknowledgments

We are grateful to Kirsten Benjamin for strains and Fred Cross for the anti-Clb2 antibody. We thank Roy Parker for helpful discussions and Steve Bell, Frank Solomon, Andreas Hochwagen and members of the Amon lab for their critical reading of this manuscript. This research was supported by National Institutes of Health grant GM62207 to A.A. A.A. is also an Investigator of the Howard Hughes Medical Institute.

Footnotes

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