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1.
J Cell Sci ; 136(9)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37162093

RESUMO

Characterizing functions of essential genes is challenging, as perturbing them is generally lethal. Conditional gene perturbation, including use of temperature-sensitive mutants, has been widely utilized to reveal functions of essential genes in the fission yeast Schizosaccharomyces pombe. However, recently we implemented a systematic and less time-consuming knockdown method, CRISPR interference (CRISPRi), in this organism using catalytically inactive Cas9 (dCas9). This technology has been expected to facilitate characterization of essential genes in S. pombe, although this still has not occurred. Here, CRISPRi was harnessed to study uncharacterized essential genes that are evolutionally conserved from yeasts to mammals. Transcription of these genes, which we call conserved essential obscure (ceo) genes, was repressed using conventional dCas9-mediated CRISPRi and by implementing technologies that enhance repression efficiency or alleviate limitations on small guide RNA (sgRNA) design. These CRISPRi methods successfully reduced transcription of target genes and allowed us to characterize resulting phenotypes. Knockdown of ceo genes inhibited cell proliferation and altered cellular morphology. Thus, dCas9-based CRISPRi methods utilized in this study enhanced accessibility of genetic analyses targeting essential genes in S. pombe.


Assuntos
Schizosaccharomyces , Animais , Schizosaccharomyces/genética , Proliferação de Células , Técnicas de Silenciamento de Genes , Fenótipo , Mamíferos
2.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34028542

RESUMO

In the fission yeast, Schizosaccharomyces pombe, the high-affinity hexose transporter, Ght5, must be transcriptionally upregulated and localized to the cell surface for cell division under limited glucose. Although cell-surface localization of Ght5 depends on Target of rapamycin complex 2 (TORC2), the molecular mechanisms by which TORC2 ensures proper localization of Ght5 remain unknown. We performed genetic screening for gene mutations that restore Ght5 localization on the cell surface in TORC2-deficient mutant cells, and identified a gene encoding an uncharacterized α-arrestin-like protein, Aly3/SPCC584.15c. α-arrestins are thought to recruit a ubiquitin ligase to membrane-associated proteins. Consistently, Ght5 is ubiquitylated in TORC2-deficient cells, and this ubiquitylation is dependent on Aly3. TORC2 supposedly enables cell-surface localization of Ght5 by preventing Aly3-dependent ubiquitylation and subsequent ubiquitylation-dependent translocation of Ght5 to vacuoles. Surprisingly, nitrogen starvation, but not glucose depletion, triggers Aly3-dependent transport of Ght5 to vacuoles in S. pombe, unlike budding yeast hexose transporters, vacuolar transport of which is initiated upon changes in hexose concentration. This study provides new insights into the molecular mechanisms controlling the subcellular localization of hexose transporters in response to extracellular stimuli.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Arrestina , Glucose , Proteínas Facilitadoras de Transporte de Glucose , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas de Transporte de Monossacarídeos/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
3.
R Soc Open Sci ; 10(10): 230404, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37859837

RESUMO

Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03-0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2-3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20-1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.

4.
G3 (Bethesda) ; 11(4)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33617628

RESUMO

Controllable and reversible transcriptional repression is an essential method to study gene functions. A systematic knock-down method using catalytically inactive Cas9 (dCas9) was originally established in bacteria. dCas9 forms a ribonucleoprotein with a small guide RNA and uses it to recognize a specific DNA sequence via Watson-Crick base-pairing. When specifically bound to a targeted DNA, dCas9 impairs RNA polymerase activity and represses transcription of that target gene. This technology, CRISPRi, has been implemented in several organisms, but not in Schizosaccharomyces pombe using dCas9. Here, we provide a plasmid that expresses dCas9 and sgRNA in fission yeast. With this plasmid, CRISPRi repressed endogenous gene transcription by as much as 87%. This transcriptional repression method is controllable, reversible, and efficient enough to alter cellular phenotypes. Here, we offer a CRISPRi method to choose proper targeting sequences for transcriptional repression in fission yeast. Implementation of CRISPRi will help to reveal gene functions and to develop tools based on dCas9 technology in S. pombe.


Assuntos
Sistemas CRISPR-Cas , Schizosaccharomyces , Sistemas CRISPR-Cas/genética , Expressão Gênica , Plasmídeos , RNA Guia de Cinetoplastídeos/genética , Schizosaccharomyces/genética
5.
Open Biol ; 11(4): 200369, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33823662

RESUMO

Mitochondria are essential for regulation of cellular respiration, energy production, small molecule metabolism, anti-oxidation and cell ageing, among other things. While the mitochondrial genome contains a small number of protein-coding genes, the great majority of mitochondrial proteins are encoded by chromosomal genes. In the fission yeast Schizosaccharomyces pombe, 770 proteins encoded by chromosomal genes are located in mitochondria. Of these, 195 proteins, many of which are implicated in translation and transport, are absolutely essential for viability. We isolated and characterized eight temperature-sensitive (ts) strains with mutations in essential mitochondrial proteins. Interestingly, they are also sensitive to limited nutrition (glucose and/or nitrogen), producing low-glucose-sensitive and 'super-housekeeping' phenotypes. They fail to produce colonies under low-glucose conditions at the permissive temperature or lose cell viability under nitrogen starvation at the restrictive temperature. The majority of these ts mitochondrial mutations may cause defects of gene expression in the mitochondrial genome. mrp4 and mrp17 are defective in mitochondrial ribosomal proteins. ppr3 is defective in rRNA expression, and trz2 and vrs2 are defective in tRNA maturation. This study promises potentially large dividends because mitochondrial quiescent functions are vital for human brain and muscle, and also for longevity.


Assuntos
Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Fenótipo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Metabolismo Energético , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Essenciais , Humanos , Estresse Fisiológico
6.
Mol Biol Cell ; 26(2): 373-86, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25411338

RESUMO

Hexose transporters are required for cellular glucose uptake; thus they play a pivotal role in glucose homeostasis in multicellular organisms. Using fission yeast, we explored hexose transporter regulation in response to extracellular glucose concentrations. The high-affinity transporter Ght5 is regulated with regard to transcription and localization, much like the human GLUT transporters, which are implicated in diabetes. When restricted to a glucose concentration equivalent to that of human blood, the fission yeast transcriptional regulator Scr1, which represses Ght5 transcription in the presence of high glucose, is displaced from the nucleus. Its displacement is dependent on Ca(2+)/calmodulin-dependent kinase kinase, Ssp1, and Sds23 inhibition of PP2A/PP6-like protein phosphatases. Newly synthesized Ght5 locates preferentially at the cell tips with the aid of the target of rapamycin (TOR) complex 2 signaling. These results clarify the evolutionarily conserved molecular mechanisms underlying glucose homeostasis, which are essential for preventing hyperglycemia in humans.


Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacocinética , Glucose/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Immunoblotting , Alvo Mecanístico do Complexo 2 de Rapamicina , Microscopia de Fluorescência , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Fosfoproteínas Fosfatases/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Serina-Treonina Quinases TOR/genética , Imagem com Lapso de Tempo/métodos
7.
Science ; 321(5892): 1088-91, 2008 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-18719285

RESUMO

The centromere is essential for the inheritance of genetic information on eukaryotic chromosomes. Epigenetic regulation of centromere identity has been implicated in genome stability, karyotype evolution, and speciation. However, little is known regarding the manner in which centromere dysfunction affects the chromosomal architectures. Here we show that in the fission yeast Schizosaccharomyces pombe, the conditional deletion of the centromere produces survivors that carry either a neocentromere-acquired chromosome at the subtelomeric region or an acentric chromosome rescued by intertelomere fusion with either of the remaining chromosomes. The ratio of neocentromere formation to telomere fusion is considerably decreased by the inactivation of genes involved in RNA interference-dependent heterochromatin formation. By affecting the modes of chromosomal reorganization, the genomic distribution of heterochromatin may influence the fate of karyotype evolution.


Assuntos
Centrômero/fisiologia , Cromossomos Fúngicos/fisiologia , Heterocromatina/metabolismo , Schizosaccharomyces/genética , Telômero/fisiologia , Imunoprecipitação da Cromatina , Segregação de Cromossomos , Replicação do DNA , Expressão Gênica , Genes Fúngicos , Histonas/metabolismo , Cariotipagem , Cinetocoros/metabolismo , Metilação , Mitose , Mutação , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo
8.
Genes Cells ; 12(3): 311-28, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17352737

RESUMO

Spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance system for chromosome missegregation. We isolated fission yeast Hos2, a component of the Dam1/DASH complex, as a multicopy suppressor of temperature-sensitive (ts) growth of nnf1-495 mutant that exhibits the minichromosome instability (mis) phenotype, producing lethal aneuploids without prominent mitotic delay. It remains elusive why SAC is satisfied in mis mutants despite the occurrence of missegregation. We found that Hos2 binds to the inner-kinetochore regions in both prometaphase and metaphase. Hos2 is essential for kinetochore localization of Dis1, a microtubule (MT) associated Dis1/XMAP215/TOG family protein that is required for proper MT dynamics. Cells lacking DASH exhibit cold-sensitive (cs) growth with the defective in sister-chromatid disjoining (dis) phenotype, which is characterized by hyper-condensed sister-chromatid pairs and elongated spindle MTs. Although DASH-deficient cells are viable at high temperatures, DASH-deletion transforms all the inner-kinetochore mis mutants so far tested into a constitutively active state of SAC, leading to the dis phenotype. We also discovered that Hos2 over-expression commonly suppresses growth retardation in a variety of inner-kinetochore mutants. These genetic interactions highlight the DASH-action(s) in satisfying SAC when aneuploids are formed during mitosis in the inner-kinetochore-defective mis mutants.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , DNA Fúngico/genética , Genes Fúngicos , Cinetocoros/metabolismo , Metáfase , Proteínas Associadas aos Microtúbulos/genética , Mitose , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fuso Acromático/metabolismo
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