RESUMEN
Aberrant repair of DNA double-strand breaks can recombine distant chromosomal breakpoints. Chromosomal rearrangements compromise genome function and are a hallmark of ageing. Rearrangements are challenging to detect in non-dividing cell populations, because they reflect individually rare, heterogeneous events. The genomic distribution of de novo rearrangements in non-dividing cells, and their dynamics during ageing, remain therefore poorly characterized. Studies of genomic instability during ageing have focussed on mitochondrial DNA, small genetic variants, or proliferating cells. To characterize genome rearrangements during cellular ageing in non-dividing cells, we interrogated a single diagnostic measure, DNA breakpoint junctions, using Schizosaccharomyces pombe as a model system. Aberrant DNA junctions that accumulated with age were associated with microhomology sequences and R-loops. Global hotspots for age-associated breakpoint formation were evident near telomeric genes and linked to remote breakpoints elsewhere in the genome, including the mitochondrial chromosome. Formation of breakpoint junctions at global hotspots was inhibited by the Sir2 histone deacetylase and might be triggered by an age-dependent de-repression of chromatin silencing. An unexpected mechanism of genomic instability may cause more local hotspots: age-associated reduction in an RNA-binding protein triggering R-loops at target loci. This result suggests that biological processes other than transcription or replication can drive genome rearrangements. Notably, we detected similar signatures of genome rearrangements that accumulated in old brain cells of humans. These findings provide insights into the unique patterns and possible mechanisms of genome rearrangements in non-dividing cells, which can be promoted by ageing-related changes in gene-regulatory proteins.
Asunto(s)
Reordenamiento Génico/genética , Inestabilidad Genómica/genética , Estructuras R-Loop/genética , Envejecimiento/genética , Aberraciones Cromosómicas , Puntos de Rotura del Cromosoma , Roturas del ADN de Doble Cadena , Genómica/métodos , Modelos Genéticos , Mutación/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Telómero/genéticaRESUMEN
The relationship between DNA sequence, biochemical function, and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in noncoding regions, particularly, in eukaryote genomes. In part, this is because we lack a complete description of the essential noncoding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence, and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66-90% of the genome, including substantial portions of the noncoding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3'- and 5'-untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary, and biochemical data can provide new insights into the relationship between genome function and molecular evolution.
Asunto(s)
Aptitud Genética , Genoma Fúngico , Schizosaccharomyces/genética , Modelos Genéticos , Mutagénesis InsercionalRESUMEN
Cohesin is a highly conserved, ring-shaped protein complex found in all eukaryotes. It consists of at least two structural maintenance of chromosomes (SMC) proteins, SMC1 and SMC3 in humans (Psm1 and Psm3 in fission yeast), and the kleisin RAD21 (Rad21 in fission yeast). Mutations in its components or regulators can lead to genetic syndromes, known as cohesinopathies, and various types of cancer. Studies in several organisms have shown that only a small fraction of each subunit assembles into complexes, making it difficult to investigate dynamic chromatin loading and unloading using fluorescent fusions in vivo because of excess soluble components. In this study, we introduce bimolecular fluorescent cohesin (BiFCo), based on bimolecular fluorescent complementation in the fission yeast Schizosaccharomyces pombe BiFCo selectively excludes signals from individual proteins, enabling the monitoring of complex assembly and disassembly within a physiological context throughout the entire cell cycle in living cells. This versatile system can be expanded and adapted for various genetic backgrounds and other eukaryotic models, including human cells.
Asunto(s)
Schizosaccharomyces , Humanos , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , División Celular , CohesinasRESUMEN
During meiosis, faithful chromosome segregation requires monopolar spindle microtubule-kinetochore arrays in MI to segregate homologous chromosomes, but bipolar in MII to segregate sister chromatids. Using fission yeasts, we found that the universal Aurora B kinase localizes to kinetochores in metaphase I and in the mid-spindle during anaphase I, as in mitosis; but in the absence of an intervening S phase, the importin α Imp1 propitiates its release from the spindle midzone to re-localize at kinetochores during meiotic interkinesis. We show that "error-correction" activity of kinetochore re-localized Aurora B becomes essential to erase monopolar arrangements from anaphase I, a prerequisite to satisfy the spindle assembly checkpoint (SAC) and to generate proper bipolar arrays at the onset of MII. This microtubule-kinetochore resetting activity of Aurora B at the MI-MII transition is required to prevent chromosome missegregation in meiosis II, a type of error often associated with birth defects and infertility in humans.
RESUMEN
In Schizosaccharomyces pombe, cytokinesis occurs by ordered recruitment of actomyosin components at the division site, followed by lateral condensation to produce a ring-like structure early in anaphase, which eventually matures and contracts at the end of mitosis. We found that in temperature-sensitive hsp90-w1 mutant cells, encoding an Hsp90 mutant protein, ring components were recruited to form a cortical network at the division site, but this network failed to condense into a compact ring, suggesting a role for Hsp90 in this particular step. hsp90-w1 mutant shows strong genetic interaction with specific mutant alleles of the fission yeast cdc2, such as cdc2-33. Interestingly, actomyosin ring defects in hsp90-w1 cdc2-33 mutant cells resembled that of hsp90-w1 single mutant at restrictive temperature. Noteworthy, similar genetic interaction was found with a mutant allele of polo-like kinase, plo1-ts4, suggesting that Hsp90 collaborates with Cdc2 and Plo1 cell cycle kinases to condense medial ring components. In vitro analyses suggested that Cdc2 and Plo1 physically interact with Hsp90. Association of Cdc2 to Hsp90 was ATP independent, while Plo1 binds to this chaperone in an ATP-dependent manner, indicating that these two kinases interact with different Hsp90 complexes. Overall, our analyses of hsp90-w1 reveal a possible role for this chaperone in medial ring condensation in association with Cdc2 and Plo1 kinases.
Asunto(s)
Actomiosina/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteína Quinasa CDC2/genética , Proteínas HSP90 de Choque Térmico/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Mitosis , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast Schizosaccharomyces pombe As opposed to many other alkylating agents, ENU only induces an S N 1-type reaction with a low s constant (s = 0.26), attacking preferentially O2 and O4 in thymine and O6 deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields.
Asunto(s)
Etilnitrosourea/farmacología , Mutágenos/farmacología , Schizosaccharomyces/efectos de los fármacos , Análisis Mutacional de ADN , Metanosulfonato de Etilo/farmacología , Mutagénesis , Mutación , Schizosaccharomyces/genéticaRESUMEN
Target of Rapamycin Complex 1 (TORC1) signaling promotes growth and aging. Inhibition of TORC1 leads to reduced protein translation, which promotes longevity. TORC1-dependent post-transcriptional regulation of protein translation has been well studied, while analogous transcriptional regulation is less understood. Here we screen fission yeast mutants for resistance to Torin1, which inhibits TORC1 and cell growth. Cells lacking the GATA factor Gaf1 (gaf1Δ) grow normally even in high doses of Torin1. The gaf1Δ mutation shortens the chronological lifespan of non-dividing cells and diminishes Torin1-mediated longevity. Expression profiling and genome-wide binding experiments show that upon TORC1 inhibition, Gaf1 directly upregulates genes for small-molecule metabolic pathways and indirectly represses genes for protein translation. Surprisingly, Gaf1 binds to and downregulates the tRNA genes, so it also functions as a transcription factor for RNA polymerase III. Thus, Gaf1 controls the transcription of both protein-coding and tRNA genes to inhibit translation and growth downstream of TORC1.
Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Transactivadores/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos , Mutación/genética , Naftiridinas/farmacología , Sistemas de Lectura Abierta/genética , Unión Proteica/efectos de los fármacos , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Transcriptoma/genéticaRESUMEN
Cdc25 phosphatase primes entry to mitosis by removing the inhibitory phosphate that is transferred to mitosis promoting factor (MPF) by Wee1 related kinases. A positive feedback loop then boosts Cdc25 and represses Wee1 activities to drive full-scale MPF activation and commitment to mitosis. Dominant mutations in the Schizosaccharomyces pombe spindle pole body (SPB) component Cut12 enable cdc25.22 mutants to overcome a G2 arrest at 36 degrees and enter mitosis. The recessive temperature-sensitive cut12.1 mutation results in the formation of monopolar spindles in which the spindle pole marker Sad1 is enriched on the nonfunctional SPB at 36 degrees . We identified mutations at five loci that suppressed the lethality of the recessive cut12.1 mutation at 36 degrees and conferred lethality at 20 degrees . Three of the five mutations led to the formation of monopolar spindles at restrictive temperatures, affected cell size at commitment to mitosis, and generated multiple Sad1 foci at nuclear periphery. The five loci, tfb2.rt1, tfb5.rt5, pla1.rt3, rpl4301.rt4, and rot2.1, and multicopy suppressors, including tfb1(+) and dbp10(+), are involved in transcription, translation, or RNA processing, prompting us to establish that elevating Cdc25 levels with the dominant cdc25.d1 allele, suppressed cut12.1. Thus, rot mutants provide a further link between protein production and cell-cycle progression.
Asunto(s)
Ciclo Celular , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Supresión Genética , Transcripción Genética , Clonación Molecular , Proteínas Mutantes/aislamiento & purificación , Fenotipo , Biosíntesis de Proteínas , Schizosaccharomyces/genética , Huso Acromático/metabolismo , Fosfatasas cdc25/genéticaRESUMEN
The synthesis, processing and function of coding and non-coding RNA molecules and their interacting proteins has been the focus of a great deal of research that has boosted our understanding of key molecular pathways that underlie higher order events such as cell cycle control, development, innate immune response and the occurrence of genetic diseases. In this study, we have found that formamide preferentially weakens RNA related processes in vivo. Using a non-essential Schizosaccharomyces pombe gene deletion collection, we identify deleted loci that make cells sensitive to formamide. Sensitive deletions are significantly enriched in genes involved in RNA metabolism. Accordingly, we find that previously known temperature-sensitive splicing mutants become lethal in the presence of the drug under permissive temperature. Furthermore, in a wild type background, splicing efficiency is decreased and R-loop formation is increased in the presence of formamide. In addition, we have also isolated 35 formamide-sensitive mutants, many of which display remarkable morphology and cell cycle defects potentially unveiling new players in the regulation of these processes. We conclude that formamide preferentially targets RNA related processes in vivo, probably by relaxing RNA secondary structures and/or RNA-protein interactions, and can be used as an effective tool to characterize these processes.
Asunto(s)
Formamidas/metabolismo , ARN/metabolismo , Formamidas/farmacología , Sitios Genéticos , Genoma Fúngico , Mutación/genética , Fenotipo , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismoRESUMEN
During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.
Asunto(s)
Anafase/fisiología , Núcleo Celular/metabolismo , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/genética , Proteína que Contiene ValosinaRESUMEN
Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.
Asunto(s)
Ingeniería Genética/métodos , Schizosaccharomyces/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Vectores Genéticos/genética , Genoma Bacteriano/genética , Schizosaccharomyces/efectos de los fármacos , Homología de Secuencia de Ácido Nucleico , Estreptotricinas/farmacología , Transformación GenéticaRESUMEN
Activation of mitosis-promoting factor (MPF) drives mitotic commitment. In human cells active MPF appears first on centrosomes. We show that local activation of MPF on the equivalent organelle of fission yeast, the spindle pole body (SPB), promotes Polo kinase activity at the SPBs long before global MPF activation drives mitotic commitment. Artificially promoting MPF or Polo activity at various locations revealed that this local control of Plo1 activity on G2 phase SPBs dictates the timing of mitotic commitment. Cytokinesis of the rod-shaped fission yeast cell generates a naive, new, cell end. Growth is restricted to the experienced old end until a point in G2 phase called new end take off (NETO) when bipolar growth is triggered. NETO coincided with MPF activation of Plo1 on G2 phase SPBs (ref. 4). Both MPF and Polo activities were required for NETO and both induced NETO when ectopically activated at interphase SPBs. NETO promotion by MPF required polo. Thus, local MPF activation on G2 SPBs directs polo kinase to control at least two distinct and temporally separated, cell-cycle transitions at remote locations.
Asunto(s)
Factor Promotor de Maduración/metabolismo , Mitosis , Morfogénesis , Schizosaccharomyces/fisiología , Centrosoma , Activación Enzimática , Estabilidad de Enzimas , Retroalimentación Fisiológica , Fase G2 , Proteínas Fluorescentes Verdes/metabolismo , Semivida , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismo , Imagen de Lapso de TiempoRESUMEN
The fission yeast spindle pole body (SPB) comprises a cytoplasmic structure that is separated from an ill-defined nuclear component by the nuclear envelope. Upon mitotic commitment, the nuclear envelope separating these domains disperses as the two SPBs integrate into a hole that forms in the nuclear envelope. The SPB component Cut12 is linked to cell cycle control, as dominant cut12.s11 mutations suppress the mitotic commitment defect of cdc25.22 cells and elevated Cdc25 levels suppress the monopolar spindle phenotype of cut12.1 loss of function mutations. We show that the cut12.1 monopolar phenotype arises from a failure to activate and integrate the new SPB into the nuclear envelope. The activation of the old SPB was frequently delayed, and its integration into the nuclear envelope was defective, resulting in leakage of the nucleoplasm into the cytoplasm through large gaps in the nuclear envelope. We propose that these activation/integration defects arise from a local deficiency in mitosis-promoting factor activation at the new SPB.
Asunto(s)
Proteínas Asociadas a Microtúbulos/fisiología , Membrana Nuclear/metabolismo , Fosfoproteínas/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mitosis/fisiología , Modelos Biológicos , Membrana Nuclear/ultraestructura , Schizosaccharomyces/citología , Schizosaccharomyces/genéticaRESUMEN
Genetic studies in yeasts enable an in vivo analysis of gene functions required for the cell division cycle (cdc genes) in eukaryotes. In order to characterize new functions involved in cell cycle regulation, we searched for genes causing cell division defects by overexpression in the fission yeast Schizosaccharomyces pombe. By using this dominant genetic strategy, 26 independent clones were isolated from a Sz. pombe cDNA library. The cloned cDNAs were partially sequenced and identified by computer analysis. The 26 clones isolated corresponded to 21 different genes. Among them, six were genes previously characterized in Sz. pombe, 11 were homologues to genes identified and characterized in other organisms, and four represented genes with unknown functions. In addition to known cell cycle regulators encoding inhibitory protein kinases (wee1, pka1) and DNA checkpoint proteins (Pcna, rad24), we have identified genes that are involved in a number of cellular processes. This includes protein synthesis (ribosomal proteins L7, L10, L29, L41, S6, S11, S17 and the PolyA-Binding Protein PABP), protein degradation (UBI3), nucleolar rRNA expression (fib, imp1, dbp2), cell cytoskeleton (act1) and glycolysis (pfk1). The interference caused in the cell cycle by overexpression of these genes may elucidate novel mechanisms coupling different cellular processes with the control of the cell division. The effect caused by some of them is described in more detail.