RNA triphosphatase is essential in Schizosaccharomyces pombe and Candida albicans

Background

The first two steps in the capping of cellular mRNAs are catalyzed by the enzymes RNA triphosphatase and RNA guanylyltransferase. Although structural and mechanistic differences between fungal and mammalian RNA triphosphatases recommend this enzyme as a potential antifungal target, it has not been determined if RNA triphosphatase is essential for the growth of fungal species that cause human disease.

Results

We show by classical genetic methods that the triphosphatase (Pct1) and guanylyltransferase (Pce1) components of the capping apparatus in the fission yeast Schizosaccharomyces pombe are essential for growth. We were unable to disrupt both alleles of the Candida albicans RNA triphosphatase gene CaCET1, implying that the RNA triphosphatase enzyme is also essential for growth of C. albicans, a human fungal pathogen.

Conclusions

Our results provide the first genetic evidence that cap synthesis is essential for growth of an organism other than Saccharomyces cerevisiae and they validate RNA triphosphatase as a target for antifungal drug discovery.     

Conserved Orb6 phosphorylation sites are essential for polarized cell growth in Schizosaccharomyces pombe

The Ndr-related Orb6 kinase is a key regulator of polarized cell growth in fission yeast, however the mechanism of Orb6 activation is unclear. Activation of other Ndr kinases involves both autophosphorylation and phosphorylation by an upstream kinase. Previous reports suggest that the Nak1 kinase functions upstream from Orb6. Supporting this model, we show that HA-Orb6 overexpression partially restored cell polarity in nak1 ts cells. We also demonstrated by coimmunoprecipitation and in vitro binding assays that Nak1 and Orb6 physically interact, and that the Nak1 C-terminal region is required for Nak1/Orb6 complex formation in vivo. However, results from in vitro kinase assays did not show phosphorylation of recombinant Orb6 by HA-Nak1, suggesting that Orb6 activation may not involve direct phosphorylation by Nak1. To investigate the role of Orb6 phosphorylation and activity, we substituted Ala at the ATP-binding and conserved phosphorylation sites. Overexpression of kinase-dead HA-Orb6(K122A) in wild-type cells resulted in a loss of cell polarity, suggesting that it has a dominant-negative effect, and it failed to rescue the polarity defect of nak1 or orb6 ts mutants. Recombinant GST-Orb6(S291A) did not autophosphorylate in vitro suggesting that Ser291 is the primary autophosphorylation site. HA-Orb6(S291A) overexpression only partially rescued the orb6 polarity defect and failed to rescue the nak1 defect, suggesting that autophosphorylation is important for Orb6 function. GST-Orb6(T456A) autophosphorylated in vitro, indicating that the conserved phosphorylation site at Thr456 is not essential for kinase activity. However, HA-Orb6(T456A) overexpression had similar effects as overexpressing kinase-dead HA-Orb6(K122A), suggesting that Thr456 is essential for Orb6 function in vivo. Also, we found that both phosphorylation site mutations impaired the ability of Myc-Nak1 to coimmunoprecipitate with HA-Orb6. Together, our results suggest a model whereby autophosphorylation of Ser291 and phosphorylation of Thr456 by an upstream kinase promote Nak1/Orb6 complex formation and Orb6 activation.     

The unique centromeric chromatin structure of Schizosaccharomyces pombe is maintained during meiosis

In meiosis I sister centromeres are unified in their polarity on the spindle, and this unique behavior is known to require the function of meiosis-specific factors that set some intrinsic property of the centromeres. The fission yeast, Schizosaccharomyces pombe, possesses complex centromeres consisting of repetitive DNA elements, making it an excellent model in which to study the behavior of complex centromeres. In mitosis, during which sister centromeres mediate chromosome segregation by establishing bipolar chromosome attachments to the spindle, the central core of the S. pombe centromere chromatin has a unique irregular nucleosome pattern. Deletion of repeats flanking this core structure have no effect on mitotic chromosome segregation, but have profound effects during meiosis. While this demonstrates that the outer repeats are critical for normal meiotic sister centromere behavior, exactly how they function and how monopolarity is established remains unclear. In this study we provide the first analysis of the chromatin structure of a complex centromere during meiosis. We show that the nature and extent of the unique central core chromatin structure is maintained with no measurable expansion. This demonstrates that monopolarity of sister centromeres, and subsequent reversion to bipolarity, does not involve a global change to the centromeric chromatin structure.     

The multiprotein exocyst complex is essential for cell separation in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. A mulitlayered division septum is assembled in concert with ring constriction. Finally, cleavage of the inner layer of the division septum results in the liberation of daughter cells. Although numerous studies have focused on actomyosin ring and division septum assembly, little information is available on the mechanism of cell separation. Here we describe a mutant, sec8-1, that is defective in cell separation but not in other aspects of cytokinesis. sec8-1 mutants accumulate about 100-nm vesicles and have reduced secretion of acid phosphatase, suggesting that they are defective in exocytosis. Sec8p is a component of the exocyst complex. Using biochemical methods, we show that Sec8p physically interacts with other members of the exocyst complex, including Sec6p, Sec10p, and Exo70p. These exocyst proteins localize to regions of active exocytosis-at the growing ends of interphase cells and in the medial region of cells undergoing cytokinesis-in an F-actin-dependent and exocytosis-independent manner. Analysis of a number of mutations in various exocyst components has established that these components are essential for cell viability. Interestingly, all exocyst mutants analyzed appear to be able to elongate and to assemble division septa but are defective for cell separation. We therefore propose that the fission yeast exocyst is involved in targeting of enzymes responsible for septum cleavage. We further propose that cell elongation and division septum assembly can continue with minimal levels of exocyst function.     

The dihydroceramide desaturase is not essential for cell viability in Schizosaccharomyces pombe

Recent studies have identified a new family of desaturase-like polypeptide sequences in many higher eukaryotes. Functional characterisation of one member of this family, from Schizosaccharomyces pombe, revealed the enzyme to be a sphingolipid desaturase. This S. pombe gene designated SDCB3b8.07c was identified as the dihydroceramide Delta(4)-desaturase, responsible for the synthesis of sphingosine. Homologous recombination was used to disrupt the endogenous S. pombe dihydroceramide Delta(4)-desaturase. Surprisingly, this had no effect on cell viability, indicating that sphingosine may not be crucial for normal S. pombe functions. This observation has implications for our understanding of the role of sphingosine and its phosphorylated metabolite sphingosine-1-phosphate in lower eukaryotes.     

Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe.

Topoisomerases catalyse changes in the topological state of DNA and are required for many aspects of DNA metabolism. While the functions of topoisomerases I and II in eukaryotes are well established, the role of topoisomerase III remains poorly defined. We have identified a gene in the fission yeast Schizosaccharomyces pombe, designated top3 (+), which shows significant sequence similarity to genes encoding topoisomerase III enzymes in other eukaryotic species. In common with murine TOP3 alpha, but in contrast to Saccharomyces cerevisiae TOP3, the S.pombe top3 (+)gene is essential for long-term cell viability. Fission yeast haploid spores containing a disrupted top3 (+)gene germinate successfully, but then undergo only a limited number of cell divisions. Analysis of these top3 mutants revealed evidence of aberrant mitotic chromosome segregation, including the 'cut' phenotype, where septation is completed prior to nuclear division. Consistent with the existence of an intimate association (originally identified in S.cerevisiae ) between topoisomerase III and DNA helicases of the RecQ family, deletion of the rqh1 (+)gene encoding the only known RecQ helicase in S.pombe suppresses lethality in top3 mutants. This conservation of genetic interaction between two widely diverged yeasts suggests that the RecQ family helicases encoded by the Bloom's and Werner's syndrome genes are likely to act in concert with topoisomerase III isozymes in human cells. Our data are consistent with a model in which the association of a RecQ helicase and topoisomerase III is important for facilitating decatenation of late stage replicons to permit faithful chromosome segregation during anaphase.     

Isolation of an essential Schizosaccharomyces pombe gene, prp31(+), that links splicing and meiosis

We carried out a screen for mutants that arrest prior to premeiotic S phase. One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31⁺ gene. The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression. It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature. We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype. Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation.     

Nuclear import of Spo12p, a protein essential for meiosis

In Saccharomyces cerevisiae, Spo12p is involved in mitosis and is essential for meiosis. We found that Spo12p is imported into the nucleus by the karyopherin Kap121p. A complex containing Spo12p and Kap121p was isolated from cytosol and was also reconstituted with recombinant proteins, indicating that this interaction is direct. Spo12p was mislocalized to the cytosol in pse1-1, a temperature-sensitive strain harboring a mutation of Kap121p, at the permissive temperature, confirming an essential role for Kap121p in Spo12p import. Spo12p was also mislocalized in a pse1-1/pse1-1 homozygous strain, suggesting it is imported via the same pathway in diploid cells. Furthermore, we found that pse1-1/pse1-1 shows a sporulation defect similar to that of spo12Delta/spo12Delta. In addition, we have characterized the Spo12p nuclear localization signal, mapped it to residues 76-130, and identified residues within this region that are important for nuclear localization signal function.     

Reconstruction of the kinetochore during meiosis in fission yeast Schizosaccharomyces pombe

During the transition from mitosis to meiosis, the kinetochore undergoes significant reorganization, switching from a bipolar to a monopolar orientation. To examine the centromere proteins that are involved in fundamental reorganization in meiosis, we observed the localization of 22 mitotic and 2 meiotic protein components of the kinetochore during meiosis in living cells of the fission yeast. We found that the 22 mitotic proteins can be classified into three groups: the Mis6-like group, the NMS (Ndc80-Mis12-Spc7) group, and the DASH group, based on their meiotic behavior. Mis6-like group proteins remain at the centromere throughout meiosis. NMS group proteins disappear from the centromere at the onset of meiosis and reappear at the centromere in two steps in late prophase. DASH group proteins appear shortly before metaphase of meiosis I. These observations suggest that Mis6-like group proteins constitute the structural basis of the centromere and that the NMS and DASH group proteins reassemble to establish the functional metaphase kinetochore. On the other hand, the meiosis-specific protein Moa1, which plays an important role in forming the meiotic monopolar kinetochore, is loaded onto the centromere significantly earlier than the NMS group, whereas another meiosis-specific protein, Sgo1, is loaded at times similar to the NMS group.     

Nonrandom homolog segregation at meiosis I in Schizosaccharomyces pombe mutants lacking recombination

Physical connection between homologous chromosomes is normally required for their proper segregation to opposite poles at the first meiotic division (MI). This connection is generally provided by the combination of reciprocal recombination and sister-chromatid cohesion. In the absence of meiotic recombination, homologs are predicted to segregate randomly at MI. Here we demonstrate that in rec12 mutants of the fission yeast Schizosaccharomyces pombe, which are devoid of meiosis-induced recombination, homologs segregate to opposite poles at MI 63% of the time. Residual, Rec12-independent recombination appears insufficient to account for the observed nonrandom homolog segregation. Dyad asci are frequently produced by rec12 mutants. More than half of these dyad asci contain two viable homozygous-diploid spores, the products of a single reductional division. This set of phenotypes is shared by other S. pombe mutants that lack meiotic recombination, suggesting that nonrandom MI segregation and dyad formation are a general feature of meiosis in the absence of recombination and are not peculiar to rec12 mutants. Rec8, a meiosis-specific sister-chromatid cohesin, is required for the segregation phenotypes displayed by rec12 mutants. We propose that S. pombe possesses a system independent of recombination that promotes homolog segregation and discuss possible mechanisms.     

Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.     

Schizosaccharomyces pombe histone acetyltransferase Mst1 (KAT5) is an essential protein required for damage response and chromosome segregation

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature-sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1), and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.