Isolation of a mammalian homologue of a fission yeast differentiation regulator

In the fission yeast Schizosaccharomyces pombe the nrd1(+) gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombe temperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have cloned ROD1, a functional homologue of nrd1(+), from rat and human cDNA libraries. Like nrd1(+), ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast, ROD1 behaves in a way that is functionally similar to nrd1(+), being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1 is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1(+) for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.     

The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe

A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo. Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

Rapid screen of human genes for relevance to cancer using fission yeast

A total of 437 human full-length cDNAs isolated by microarray analysis of liver and/or gastric cancer tissues were evaluated for their relevance to cancer using the fission yeast Schizosaccharomyces pombe. Overexpression of 161 human cDNAs in S. pombe caused growth inhibition and/or morphological changes, which can be considered as cancer-related phenotypes of S. pombe. Sixteen genes causing growth defects and morphological changes at the same time were chosen to validate their ostensible oncogenic properties. They were highly expressed in liver and/or gastric cancer cell lines. Also, when the mouse embryonic fibroblast cell type NIH3T3 was transfected with these genes, the proliferation rates of cells were increased by 32% to 120%. This study demonstrates that fission yeast can be used as an advantageous and powerful tool for the rapid screening of human genes relevant to cancer. Furthermore, the human genes screened can be tested further as diagnostic markers and potential therapeutic targets for liver and stomach cancers. They also can be studied further for the elucidation of mechanisms involved in carcinogenesis.     

Homologous mRNA 3'' end formation in fission and budding yeast.

Sequences resembling polyadenylation signals of higher eukaryotes are present downstream of the Schizosaccharomyces pombe ura4+ and cdc10+ coding regions and function in HeLa cells. However, these and other mammalian polyadenylation signals are inactive in S. pombe. Instead, we find that polyadenylation signals of the CYC1 gene of budding yeast Saccharomyces cerevisiae function accurately and efficiently in fission yeast. Furthermore, a 38 bp deletion which renders this RNA processing signal non-functional in S. cerevisiae has the equivalent effect in S. pombe. We demonstrate that synthetic pre-mRNAs encoding polyadenylation sites of S. pombe genes are accurately cleaved and polyadenylated in whole cell extracts of S. cerevisiae. Finally, as is the case in S. cerevisiae, DNA sequences encoding regions proximal to the S. pombe mRNA 3' ends are found to be extremely AT rich; however, no general sequence motif can be found. We conclude that although fission yeast has many genetic features in common with higher eukaryotes, mRNA 3' end formation is significantly different and appears to be formed by an RNA processing mechanism homologous to that of budding yeast. Since fission and budding yeast are evolutionarily divergent, this lower eukaryotic mechanism of mRNA 3' end formation may be generally conserved.     

Functional expression of human mitochondrial CYP11B2 in fission yeast and identification of a new internal electron transfer protein, etp1

Mitochondrial cytochrome P450 enzymes play a crucial role in the steroid biosynthesis in human adrenals, catalyzing regio- and stereospecific hydroxylations. In search of a new model system for the study of these enzymes, we expressed the human CYP11B2 (aldosterone synthase, P450(aldo)) in fission yeast Schizosaccharomyces pombe. Analysis of the subcellular localization of the P450 enzyme by Western blot analysis, fluorescence microscopy, and electron microscopy demonstrated that the mitochondrial localization signal of the human protein is functional in S. pombe. The transformed yeasts show the inducible ability to convert in vivo considerable amounts of 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone, respectively. Although in mammalian cells, mitochondrial steroid hydroxylases depend for their activity on an electron transport chain that consists of two proteins, adrenodoxin and adrenodoxin reductase, no coexpression of these proteins is needed for efficient substrate conversion by intact fission yeast cells. Searching the fission yeast genome for adrenodoxin homologues, a gene was identified that codes for a protein with an amino terminal domain homologous to COX15 of Saccharomyces cerevisiae and a carboxy terminal ferredoxin domain. It was found that overexpression of this gene significantly enhances steroid hydroxylase activity of CYP11B2 expressing fission yeast cells. Moreover, the bacterially expressed ferredoxin domain of this protein can replace adrenodoxin in a reconstituted steroid hydroxylation assay and transfer electrons from adrenodoxin reductase to a mammalian or a bacterial cytochrome P450. Therefore, we suggest to name this protein etp1 (electron-transfer protein 1).     

HIV-1 Vif protein mediates the degradation of APOBEC3G in fission yeast when over-expressed using codon optimization

Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S.pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator.

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.     

Analysis of the BiP gene and identification of an ER retention signal in Schizosaccharomyces pombe.

We have cloned the gene for the resident luminal ER protein BiP from the fission yeast, Schizosaccharomyces pombe. The predicted protein product is equally divergent from the budding yeast and mammalian homologues. Disruption of the BiP gene in S. pombe is lethal and BiP mRNA levels are regulated by a variety of stresses including heat shock. Immunofluorescence of cells expressing an epitope-tagged BiP protein show it to be localized to the nuclear envelope, around the cell periphery and in a reticular structure through the cytoplasm. Unexpectedly, we find the BiP protein contains an N-linked glycosylation site which can be utilized. The C-terminal four amino acids of BiP are Ala-Asp-Glu-Leu, a new variant of the XDEL sequence found at the C-termini of luminal endoplasmic reticulum proteins. To determine whether this sequence acts as a sorting signal in S.pombe we expressed an acid phosphatase fusion protein extended at its C-terminus with the amino acids ADEL. Analysis of the sorting of this fusion protein indicates that the ADEL sequence is sufficient to cause the retention of proteins in the endoplasmic reticulum. The sequences DDEL, HDEL and KDEL can also direct ER-retention of acid phosphatase in S.pombe.     

Complementation of the DNA repair-deficient swi10 mutant of fission yeast by the human ERCC1 gene.

In human cells DNA damage caused by UV light is mainly repaired by the nucleotide excision repair pathway. This mechanism involves dual incisions on both sides of the damage catalyzed by two nucleases. In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. The amino acid sequence of the human excision repair protein ERCC1 is homologous with the fission yeast Swi10 protein. In order to test whether these proteins are functional homologues, we overexpressed the human gene in a Schizosaccharomyces pombe swi10 mutant. A swi10 mutation has a pleiotropic effect: it reduces the frequency of mating type switching (a mitotic transposition event from a silent cassette into the expression site) and causes increased UV sensitivity. We found that the full-length ERCC1 gene only complements the transposition defect of the fission yeast mutant, while a C-terminal truncated ERCC1 protein also restores the DNA repair capacity of the yeast cells. Using the two-hybrid system of Saccharomyces cerevisiae we show that only the truncated human ERCC1 protein is able to interact with the S . pombe Rad16 protein, which is the fission yeast homologue of human XPF. This is the first example yet known that a human gene can correct a yeast mutation in nucleotide excision repair.     

Conservation of polyamine regulation by translational frameshifting from yeast to mammals

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.