Current Biology, Vol. 12, 141–145, January 22, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(01)00654-6
14-3-3 Protein Interferes with the Bindingof RNA to the Phosphorylated Form ofFission Yeast Meiotic Regulator Mei2p
mutant ([11]; Figure 1A). Cdc25p is essential for bothmeiosis I and II [12], and 14-3-3 has been implicatedin downregulation of Cdc25p activity [13–15]. Thus, toexamine the possibility that Rad24p suppressed pat1-driven meiosis through Cdc25p, we analyzed a pat1-114
Masamitsu Sato,1 Yoshinori Watanabe,1,2,3
Yuji Akiyoshi,1 and Masayuki Yamamoto1
1 Department of Biophysics and BiochemistryGraduate School of ScienceUniversity of Tokyo
cdc25� cdc2-3w strain, JW94, which survives withoutHongoCdc25p [16]. As shown in Figure 1A, overexpression ofTokyo 113-0033rad24 suppressed pat1-driven meiosis effectively in this2 PRESTOstrain, i.e., in the complete absence of Cdc25p, indicat-Japan Science and Technology Corporationing that Cdc25p is not a key factor for the suppressionKawaguchiof pat1 by rad24 overexpression.Saitama 332-0012
We next examined effects of deletion of rad24 onJapanmeiosis. Diploid rad24� cells initiated meiosis on MMmedium [26] containing as much as 2.5% NH4Cl, whilestandard MM � N medium, containing 0.5% NH4Cl, in-
Summary hibited meiosis in wild-type cells, suggesting thatRad24p has a suppressive role in natural meiosis (data
The switch from mitosis to meiosis is controlled by not shown). Its negative role was also evident in pat1-the Pat1(Ran1) kinase-Mei2p system in Schizosac- driven ectopic meiosis. Whereas the pat1-114 singlecharomyces pombe [1]. Mei2p promotes both premei- mutant entered meiosis around 30�C, the pat1-114otic DNA synthesis and meiosis I, and its RNA binding rad24� mutant ceased mitotic growth and initiated mei-ability is essential for these two processes [2, 3]. Mei2p osis at 26.5�C (Figure 1B). Consistently, the temperature-forms a dot structure in the nucleus prior to meiosis sensitive mei2-33 mutant [2] recovered meiotic profi-I, aided by a specific RNA species named “meiRNA” ciency at the restrictive temperature if combined with[3–5]. Pat1 kinase phosphorylates Mei2p on two posi- deletion of rad24 (data not shown). These results sug-tions and downregulates its activity [4]. Pat1 kinase gest that Rad24p is an inhibitory factor for meiosis,undergoes inactivation under meiotic conditions, as a which closely interacts with the Pat1 kinase-Mei2presult of the production of a tethering pseudosubstrate system.Mei3p [6], and accumulation of the unphosphorylatedform of Mei2p commits cells to meiosis [4]. However, Rad24p Counteracts meiRNA in Functionthe mechanism of how phosphorylation of Mei2p sup- We investigated genetic interaction between rad24 andpresses its activity to induce meiosis remains largely sme2, the latter of which encodes meiRNA. The sme2�unknown. Here we show that S. pombe Rad24p, a 14- strain cannot generate a nuclear Mei2p dot and accom-3-3 protein, functions as a negative factor for meiosis plish meiosis I [2, 3]. We deleted rad24 from this strain.by antagonizing the function of meiRNA to promote Intriguingly, the sme2� rad24� double mutant com-the formation of a nuclear Mei2p dot. Rad24p binds pleted meiosis and produced spores efficiently (Figurepreferentially to Mei2p phosphorylated by Pat1 kinase. 2A), raising the possibility that Rad24p may counteractIt inhibits association of meiRNA to the phosphory- meiRNA in function. Recovery of the Mei2p dot in thelated form of Mei2p but not to the unphosphorylated sme2� rad24� genetic background has been observed
(data not shown).form in vitro. We speculate that Rad24p, bound tightlyOverexpression of mei2 turned out to inhibit cellto the residues phosphorylated by Pat1 kinase, may
growth in a rad24� strain but not in the isogenic rad24�mask the RNA recognition motifs on Mei2p. This modelstrain (Figure 2B), suggesting that accumulation ofwill explain, at least partly, why phosphorylation byMei2p in the absence of Rad24p may switch off thePat1 kinase inhibits the meiosis-inducing activity ofmitotic cell cycle. GFP-tagged Mei2p was localizedMei2p.mainly in the cytoplasm, and nuclear dots were rarelyseen in vegetative rad24� cells ([3]; Figure 2C). However,Results and Discussionvegetative rad24� cells expressing GFP-Mei2p dis-played a single fluorescent dot in a spherical interphaseRad24p Is an Inhibitory Factor for Meiosisnucleus (Figure 2C). We have shown previously that
The 14-3-3 proteins bind preferentially to proteins car-ectopic overexpression of meiRNA in vegetative cells
rying phosphoserine [7] and modulate the function ofresults in the generation of nuclear Mei2p dots [5]. Alto-
them in a variety of ways [8]. The rad24 and rad25 genes gether, we conclude that meiRNA and Rad24p executeencode the major and the minor 14-3-3 isoforms of S. opposing functions in meiosis: meiRNA advances for-pombe, respectively, which participate in DNA-damage mation of the Mei2p dot, whereas Rad24p retards it.checkpoint control by regulating the M phase activatorCdc25p [9, 10]. Overexpression of rad24 could inhibit Phosphorylation of Mei2p Strengthensectopic meiosis in the temperature-sensitive pat1-114 Its Interaction with Rad24p
Mei2p could interact with Rad24p and Rad25p in a con-ventional yeast two-hybrid assay (data not shown; see3 Correspondence: [email protected]
Current Biology142
Figure 1. Genetic Interaction of rad24 withpat1
(A) Suppression of ectopic meiosis in pat1-114 mutant cells by overexpression of rad24.Cells of the h� pat1-114 (JZ409) and h� pat1-114 cdc25� cdc2-3w (JW94) strains weretransformed with pREP41-Rad24-FLAG (de-noted as rad24 o.e.) or pREP41-FLAG (vec-tor). (Top panel) Transformants were grownon MM � N plate at 25�C for 24 hr, then shiftedto 32�C and incubated for 4 days. (Bottompanel) Cell morphology of each strain wasexamined by Nomarski and DAPI-stained flu-orescence microscopy. Merged images areshown. Scale bar, 10 �m. The cdc2-3w muta-tion was introduced into the tester strain torender activation of Cdc2 kinase independentof Cdc25p and hence to circumvent the le-thality caused by deletion of cdc25 [16].(B) Stimulation of meiosis in pat1-114 rad24�
cells. (Left panel) The h� rad24� (JW41), h�
pat1-114 (JZ409), and h� pat1-114 rad24�
(JW10) strains, grown at 25�C, were streakedon YE � adenine plates and were incubatedat 26.5�C and 34�C for 3 days. (Right panel)Cell morphology of the pat1-114 and pat1-114 rad24� strains incubated at 26.5�C, ex-amined as in (A).
below). The 14-3-3 protein is known to recognize phos- phothreonine appears to play a more important role thanphosphoserine in the interaction of Mei2p with Rad24p.phoserine [7] and, less frequently, phosphothreonine in
target proteins [17]. The two Pat1 kinase target sites on The high affinity of Rad24p for phosphorylated Mei2pwas confirmed by immunoprecipitation assay. We ex-Mei2p (Ser438 and Thr527) [4] are enclosed by se-
quences similar to the consensus recognition motif for amined association of FLAG-tagged Rad24p to threetypes of HA-tagged Mei2p variants, namely, wild-type14-3-3 [7] (Figure 3A). Therefore, we examined whether
phosphorylation of Mei2p by Pat1 kinase might enhance Mei2p, Mei2-FAp, which has no affinity for meiRNA andno ability to induce meiosis [4], and Mei2-FA-SATAp,its interaction with Rad24p. The two-hybrid interaction
between Mei2p and Rad24p was indeed strengthened which carries the SATA mutation in addition to the FAmutation. The SATA mutation was combined with theby simultaneous expression of active Pat1 kinase in
the S. cerevisiae host (Figure 3B). The weak interaction FA mutation because production of Mei2-SATAp with-out the FA mutation would result in unconditional meio-between Rad24p and unphosphorylated Mei2p was not
detectable under the stringent assay conditions used sis and change the cell physiology extensively. Mei2-FA-SATAp, which mimicked the unphosphorylated form,here.
To assess the degree of contribution of phosphory- showed only a weak affinity for Rad24p, compared towild-type Mei2p and Mei2-FAp, which were supposedlylated Ser438 and Thr527 to the interaction between
Mei2p and Rad24p, we analyzed three mutant forms of phosphorylated by Pat1 kinase in the host cell and ac-quired a stronger affinity for Rad24p (Figure 3C).Mei2p in the above assay. The first mutant carried ala-
nine instead of Ser438 (mei2-SA), the second carriedalanine instead of Thr527 (mei2-TA), and the third carried Rad24p Inhibits Binding of meiRNA
to Phosphorylated Mei2pthese two substitutions (mei2-SATA) [4]. The TA muta-tion greatly reduced the interaction between Mei2p and We have shown previously that bacterially produced
Mei2p can bind to meiRNA in vitro, whether it is phos-Rad24p in the two-hybrid assay system with Pat1 kinaseexpressed, whereas the SA mutation gave significant phorylated by Pat1 kinase or not [4]. Given that Rad24p
preferentially binds to phosphorylated Mei2p and thatbut much less influence (Figure 3B). Thus, phos-
Brief Communication143
Rad24p inhibits formation of a nuclear Mei2p dot innonmeiotic cells, by suppressing association of meiRNAto Mei2p phosphorylated by Pat1 kinase.
ConclusionsThe initial observations of this study, i.e., that S. pombeRad24p is a negative factor for meiosis and that Rad24pphysically interacts with Mei2p, have been noticed alsoby others [18]. Our analysis has further demonstratedthat Rad24p suppresses the assembly of Mei2p into anuclear dot, antagonizing the function of meiRNA. Mei2pshuttles between the cytoplasm and the nucleus [5],and artificial acceleration of nuclear import of Mei2pstimulates formation of a Mei2p dot in the absence ofmeiRNA [3]. However, deletion of rad24 does not en-hance the nuclear accumulation rate of Mei2p in a semi-quantitative assay (our unpublished data), as is true withoverexpression of meiRNA [5], implying that Rad24pand meiRNA may affect the Mei2p dot formation in thesame context but oppositely. The affinity of Rad24p forMei2p increases if Mei2p is phosphorylated by Pat1kinase, and Rad24p interferes with the binding ofmeiRNA to the phosphorylated form of Mei2p in vitro.These observations suggest that Rad24p, bound to theThr527 and Ser438 residues phosphorylated by Pat1kinase, may mask the RRMs on Mei2p and hamper theaccess of meiRNA to them, as schematized in Figure4B. This scheme will explain neatly why Mei2p does not
Figure 2. Genetic Interaction of rad24 with sme2 and mei2 form a nuclear dot in proliferating cells, which containactive Pat1 kinase.(A) Deletion of rad24 suppresses meiotic deficiency of meiRNA-
missing cells. Cells of a homothallic sme2� strain JZ463 and a A possible question to be raised on the proposedhomothallic sme2� rad24� strain JW204 were incubated on sporula- scheme will be why deletion of rad24 can suppresstion-inducing medium SPA at 20�C. After 2 days, cell morphology complete loss of meiRNA, if Rad24p inhibits the functionwas examined under the Nomarski microscope (top left, JZ463; top
of Mei2p by blocking the association of meiRNA. Al-right, JW204; Scale bar, 10 �m). JW204 generated roundish asci duethough we do not have a definite answer to this question,to the rad24� mutation. The bottom panel shows patches of thesethe following explanation appears to be the most rea-strains cultured on SPA and exposed to I2 vapor. I2 stains cells that
have undergone meiosis and sporulation in dark brown [25]. sonable at present. We have shown previously that(B) Overexpression of mei2 inhibits mitotic growth in rad24� cells. Mei2p can form a nuclear dot in the complete absenceA rad24� strain carrying the nmt1-mei2 fusion gene at the leu1 locus of meiRNA, when this protein is enriched in the nucleus(JX381), an isogenic rad24� strain (JW98), and a control rad24�
[3]. The dot formation without the aid of meiRNA yetstrain (JW39) were cultured at 30�C for 5 days, either on the mediumappears to depend on one or more RNA species, be-MM � N � Thiamine, repressing the nmt1 promoter (left), or on MMcause the RNA binding ability of Mei2p is indispensable� N, derepressing it (right).
(C) Overproduced Mei2p forms a nuclear dot in rad24� cells. A for it. It is hence presumable that such RNA speciesrad24� strain carrying the nmt1-GFP-mei2 fusion gene at the chro- may also support the Mei2p dot formation in sme2�mosomal mei2 locus (JW224, left) and an isogenic rad24� strain rad24� cells, where no Rad24p exists and the RRMs on(JW225, right) were grown in liquid MM � N for 16 hr at 30�C to the
Mei2p are presumably exposed to any RNA species.concentration of 5 � 106 cells/ml. Cells were examined for GFPWhereas meiRNA is required only for the promotionfluorescence without fixation. Nuclei were stained with Hoechst
of meiosis I, Mei2p is essential for both premeiotic DNA33342. Scale bar, 10 �m.
synthesis and meiosis I [2, 4]. Because the FA mutationin the C-terminal RRM renders Mei2p nonfunctional inboth of the two steps, Mei2p is supposed to cooperateRad24p counteracts meiRNA with respect to the Mei2p
dot formation, we suspected that the presence of with at least one more RNA species other than meiRNAto complete natural meiosis, though its identity is un-Rad24p might interfere with the binding of meiRNA to
phosphorylated Mei2p. To examine this hypothesis, we known [2, 3]. Phosphorylation of Mei2p by Pat1 kinaseblocks the onset of meiosis, including the initiation ofadded Rad24p to the in vitro binding assay system we
employed previously [4]. As shown in Figure 4A, the premeiotic DNA synthesis [2, 4], and overexpression ofRad24p impedes not only meiosis I but also the onsetaddition of GST-tagged Rad24p suppressed the binding
of meiRNA to phosphorylated Mei2p but did not signifi- of meiosis. Therefore, a highly likely scheme will be thatRad24p hampers the access of RNA in general to thecantly affect its binding to unphosphorylated Mei2p. The
control GST caused no effect on either type of Mei2p. phosphorylated form of Mei2p. Consistent with thisscheme, deletion of rad24 cannot suppress the mei2-Pat1 kinase did not phosphorylate Rad24p (data not
shown). Altogether, these observations suggest that FA mutation (our unpublished data).
Current Biology144
Figure 3. Mei2p Phosphorylated by Pat1 Ki-nase Has a Higher Affinity for Rad24p
(A) Schematic illustration of the phosphoryla-tion sites on Mei2p by Pat1 kinase, namely,Ser438 and Thr527. RRM, RNA recognitionmotif. The truncated C-terminal region (427–733), which is the shortest sequence knownto be functional as Mei2p [4], was used intwo-hybrid analysis. A comparison of theconsensus 14-3-3 binding motif and thephosphorylation sites of Mei2p is given onthe right. pS and pT represent phosphoserineand phosphothreonine, respectively.(B) Two-hybrid analysis with Pat1 kinase ex-pressed. Two-hybrid interaction of Rad24pwith either Mei2p or its mutant form as indi-cated was examined in the presence of intactPat1 kinase or its kinase-dead derivative. Foreach combination of bait and prey, four inde-pendent transformants were assayed for ex-pression of the reporter HIS3. HIS3 expres-sion was monitored on histidine-free SC platesupplemented with either 5, 10, 20, or 30 mM3-amino-1,2,4-triazole to modify the strin-gency of the assay conditions. More detailedprocedures are given in the SupplementaryMaterial available with this article online.(C) Immunoprecipitation proving the associa-tion of Rad24p with Mei2p in vivo. Extractswere prepared from JY741 cells expressingRad24-FLAG and one type of Mei2-3HA, asindicated, and treated with anti-FLAG mono-clonal antibody M2. For each sample, the to-tal extract (denoted as “ext.”) and the immu-noprecipitate (IP) were analyzed by Westernblotting with either anti-HA monoclonal anti-body 12CA5 (upper panel) or anti-FLAG M2(lower panel). The extract used for immuno-precipitation was approximately six times asmuch as that loaded on the gel.
Even the above “universal” scheme, however, does required for sexual development [24]. Phosphorylationof Ste11p by Pat1 kinase reduces its transcription-acti-not account for the whole mechanism for Pat1 kinase to
block the Mei2p function. Compared to the conspicuous vating activity, apparently recruiting 14-3-3 to the phos-phorylated sites [23]. Although the molecular functionmeiosis-inducing phenotype of the pat1 mutant [19–22]
or the mei2-SATA mutant [4], the phenotype of the of 14-3-3 here is not clarified yet [23], this observationillustrates that 14-3-3 may execute its negative role onrad24� mutant is considerably milder. Unlike pat1-114
cells, haploid rad24� cells never initiate meiosis in rich meiosis not only through Mei2p. Because 14-3-3 poten-tially interacts with a large number of phosphoproteins,medium. This may be attributed to Rad25p, the minor
14-3-3 isoform, which possibly blocks Mei2p to be fully we presume that the activity of some other factors facili-tating the progression of meiosis may also be regulatedactivated in the rad24� mutant. However, even rad24�
haploid cells displaying a clear nuclear Mei2p dot, as negatively by 14-3-3.shown in Figure 2C, do not complete the meiotic pro-cess, despite that their mitotic cell cycle is apparently Supplementary Materialblocked. Thus, Mei2p free from phosphorylation by Pat1 Supplementary Material including Experimental Procedures and a
supplementary table can be found with this article online at http://kinase appears to acquire some additional power toimages.cellpress.com/supmat/supmatin.htm.facilitate progression of meiosis, besides the ability to
associate with meiRNA and other hypothetical RNA mol-Acknowledgmentsecules. Indeed, we have seen that Mei2p becomes more
susceptible to proteasome-dependent proteolysis in-We thank A. Carr for the rad24� strain, and A. Yamashita for advice.volving E2 Ubc2p and E3 Ubr1p, if phosphorylated byThis work was supported by a Grant-in-Aid for Specially Promoted
Pat1 kinase [23]. This will also partly account for why Research from the Ministry of Education, Science, Sports and Cul-phosphorylation of Mei2p by Pat1 kinase downregulates ture of Japan and by the Mitsubishi Foundation.its function. Whether the phosphorylation modifies anyadditional feature of Mei2p remains to be seen.
Received: October 1, 2001Ste11p is the other substrate of Pat1 kinase identified Revised: November 9, 2001
unequivocally to date [6]. It is an HMG family protein that Accepted: November 16, 2001Published: January 22, 2002activates transcription of mei2 and many other genes
Brief Communication145
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man, C., Larsson, C., Oecking, C., and Sommarin, M. (1999).Mei2p
Phosphorylation of Thr-948 at the C terminus of the plasma(A) Measurement of Mei2p-meiRNA binding by in vitro UV crosslink- membrane H(�)-ATPase creates a binding site for the regulatorying assay. Purified GST-tagged C-terminal half of Mei2p (GST- 14-3-3 protein. Plant Cell 11, 2379–2392.Mei2C) was either treated or untreated with GST-Pat1 kinase and 18. Tanaka, Y., Okuzaki, D., Yabuta, N., Yoneki, T., and Nojima, H.assayed for binding to 32P-labeled meiRNA, as described in Supple- (2000). Rad24 is essential for proliferation of diploid cells inmentary Material. Effects of the addition of GST-Rad24p to this fission yeast. FEBS Lett. 472, 254–258.system are shown. Lanes 1 and 2, no addition; lanes 3 and 4, GST- 19. Iino, Y., and Yamamoto, M. (1985). Mutants of Schizosaccharo-Rad24p; and lanes 5 and 6, GST. Products of UV crosslinking were myces pombe which sporulate in the haploid state. Mol. Gen.analyzed by SDS-PAGE and were detected by staining with Coo- Genet. 198, 416–421.massie brilliant blue (upper and middle panels) and autoradiography 20. Iino, Y., and Yamamoto, M. (1985). Negative control for the(lower panel). Note that phosphorylated Mei2C migrated slightly initiation of meiosis in Schizosaccharomyces pombe. Proc. Natl.slower than unphosphorylated Mei2C. Acad. Sci. USA 82, 2447–2451.(B) A model for the nuclear Mei2p dot formation regulated by 21. Beach, D., Rodgers, L., and Gould, J. (1985). RAN1� controlsmeiRNA and 14-3-3. the transition from mitotic division to meiosis in fission yeast.
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