RESUMEN
Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.
Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Saccharomyces cerevisiae , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cohesinas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Mitosis , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability.
Asunto(s)
ADN Ribosómico , Inestabilidad Genómica , Mutación , ARN Polimerasa I , Procesamiento Postranscripcional del ARN , ARN Ribosómico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Polimerasa I/metabolismo , ARN Polimerasa I/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1RESUMEN
Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.
Asunto(s)
Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , ARN Polimerasa I/genética , ADN Ribosómico/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Precursores del ARN/genética , ARN Ribosómico , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance.
Asunto(s)
Nucléolo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fracciones Subcelulares/metabolismoRESUMEN
DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of -0.3 to -0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in 'theta' conditions, in which they are poised for compartmentalization and phase separation.
Asunto(s)
Cromosomas Fúngicos/química , Modelos Genéticos , Cromatina/química , ADN de Hongos/química , Cinética , Movimiento (Física) , Nucleosomas/químicaRESUMEN
Ribosome biogenesis requires more than 200 trans-acting factors to achieve the correct production of the two mature ribosomal subunits. Here, we have identified Efg1 as a novel, nucleolar ribosome biogenesis factor in Saccharomyces cerevisiae that is directly linked to the surveillance of pre-40S particles. Depletion of Efg1 impairs early pre-rRNA processing, leading to a strong decrease in 18S rRNA and 40S subunit levels and an accumulation of the aberrant 23S rRNA. Using Efg1 as bait, we revealed a novel degradation pathway of the 23S rRNA. Co-immunoprecipitation experiments showed that Efg1 is a component of 90S pre-ribosomes, as it is associated with the 35S pre-rRNA and U3 snoRNA, but has stronger affinity for 23S pre-rRNA and its novel degradation intermediate 11S rRNA. 23S is cleaved at a new site, Q1, within the 18S sequence by the endonuclease Utp24, generating 11S and 17S' rRNA. Both of these cleavage products are targeted for degradation by the TRAMP/exosome complexes. Therefore, the Q1 site defines a novel endonucleolytic cleavage site of ribosomal RNA exclusively dedicated to surveillance of pre-ribosomal particles.
Asunto(s)
Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico 23S/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Nucléolo Celular/metabolismo , Endorribonucleasas/metabolismo , Exorribonucleasas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Biogénesis de Organelos , Precursores del ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Spatial organisation of chromosomes is a determinant of genome stability and is required for proper mitotic segregation. However, visualization of individual chromatids in living cells and quantification of their geometry, remains technically challenging. Here, we used live cell imaging to quantitate the three-dimensional conformation of yeast Saccharomyces cerevisiae ribosomal DNA (rDNA). rDNA is confined within the nucleolus and is composed of about 200 copies representing about 10% of the yeast genome. To fluorescently label rDNA in living cells, we generated a set of nucleolar proteins fused to GFP or made use of a tagged rDNA, in which lacO repetitions were inserted in each repeat unit. We could show that nucleolus is not modified in appearance, shape or size during interphase while rDNA is highly reorganized. Computationally tracing 3D rDNA paths allowed us to quantitatively assess rDNA size, shape and geometry. During interphase, rDNA was progressively reorganized from a zig-zag segmented line of small size (5,5⯵m) to a long, homogeneous, line-like structure of 8,7⯵m in metaphase. Most importantly, whatever the cell-cycle stage considered, rDNA fibre could be decomposed in subdomains, as previously suggested for 3D chromatin organisation. Finally, we could determine that spatial reorganisation of these subdomains and establishment of rDNA mitotic organisation is under the control of the cohesin complex.
Asunto(s)
Nucléolo Celular/metabolismo , ADN Ribosómico/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , División Celular/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Ribosómico/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30â nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope.
Asunto(s)
Ciclo Celular , Forma del Núcleo Celular , Microscopía Confocal/métodos , Saccharomycetales/citología , Carbono/farmacología , Ciclo Celular/efectos de los fármacos , Forma del Núcleo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Imagenología Tridimensional , Interfase/efectos de los fármacos , Membrana Nuclear/efectos de los fármacos , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Saccharomycetales/efectos de los fármacos , Saccharomycetales/metabolismoRESUMEN
Several DNA cis-elements and trans-acting factors were described to be involved in transcription termination and to release the elongating RNA polymerases from their templates. Different models for the molecular mechanism of transcription termination have been suggested for eukaryotic RNA polymerase I (Pol I) from results of in vitro and in vivo experiments. To analyse the molecular requirements for yeast RNA Pol I termination, an in vivo approach was used in which efficient termination resulted in growth inhibition. This led to the identification of a Myb-like protein, Ydr026c, as bona fide termination factor, now designated Nsi1 (NTS1 silencing protein 1), since it was very recently described as silencing factor of ribosomal DNA. Possible Nsi1 functions in regard to the mechanism of transcription termination are discussed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ARN Polimerasa I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Transcripción Genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de AminoácidoRESUMEN
Chromosome dynamics are recognized to be intimately linked to genomic transactions, yet the physical principles governing spatial fluctuations of chromatin are still a matter of debate. Using high-throughput single-particle tracking, we recorded the movements of nine fluorescently labeled chromosome loci located on chromosomes III, IV, XII, and XIV of Saccharomyces cerevisiae over an extended temporal range spanning more than four orders of magnitude (10(-2)-10(3) sec). Spatial fluctuations appear to be characterized by an anomalous diffusive behavior, which is homogeneous in the time domain, for all sites analyzed. We show that this response is consistent with the Rouse polymer model, and we confirm the relevance of the model with Brownian dynamics simulations and the analysis of the statistical properties of the trajectories. Moreover, the analysis of the amplitude of fluctuations by the Rouse model shows that yeast chromatin is highly flexible, its persistence length being qualitatively estimated to <30 nm. Finally, we show that the Rouse model is also relevant to analyze chromosome motion in mutant cells depleted of proteins that bind to or assemble chromatin, and suggest that it provides a consistent framework to study chromatin dynamics. We discuss the implications of our findings for yeast genome architecture and for target search mechanisms in the nucleus.
Asunto(s)
Cromatina/metabolismo , Cromosomas Fúngicos , Ensayos Analíticos de Alto Rendimiento , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/genética , Sitios Genéticos , Genoma Fúngico , Modelos Moleculares , Simulación de Dinámica Molecular , Saccharomyces cerevisiae/genética , Telómero/genéticaRESUMEN
The basal transcription/repair factor II H (TFIIH), found mutated in cancer-prone or premature aging diseases, plays a still unclear role in RNA polymerase I transcription. Furthermore, the impact of this function on TFIIH-related diseases, such as trichothiodystrophy (TTD), remains to be explored. Here, we studied the involvement of TFIIH during the whole process of ribosome biogenesis, from RNAP1 transcription to maturation steps of the ribosomal RNAs. Our results show that TFIIH is recruited to the ribosomal DNA in an active transcription-dependent manner and functions in RNAP1 transcription elongation through ATP hydrolysis of the XPB subunit. Remarkably, we found a TFIIH allele-specific effect, affecting RNAP1 transcription and/or the pre-rRNA maturation process. Interestingly, this effect was observed in mutant TFIIH-TTD cells and also in the brains of TFIIH-TTD mice. Our findings provide evidence that defective ribosome synthesis represents a new faulty mechanism involved in the pathophysiology of TFIIH-related diseases.
Asunto(s)
Mutación , ARN Ribosómico/genética , Factor de Transcripción TFIIH/genética , Síndromes de Tricotiodistrofia/genética , Animales , Humanos , Ratones , Ratones Noqueados , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Factor de Transcripción TFIIH/metabolismo , Transcripción Genética , Síndromes de Tricotiodistrofia/metabolismoRESUMEN
Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of â¼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.
Asunto(s)
ADN Ribosómico/metabolismo , Proteínas HMGB/química , Proteínas HMGB/metabolismo , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Nucléolo Celular/metabolismo , Secuencia Conservada , Proteínas HMGB/genética , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Proteínas del Complejo de Iniciación de Transcripción Pol1/química , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa I/metabolismo , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Relación Estructura-ActividadRESUMEN
Transcription by RNA polymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC(50) in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity. We have shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis.
Asunto(s)
Elipticinas/farmacología , Inhibidores Enzimáticos/farmacología , ARN Polimerasa I/metabolismo , Transcripción Genética , Bromodesoxiuridina/farmacología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Química Farmacéutica/métodos , Diseño de Fármacos , Humanos , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Ribosómico/metabolismoRESUMEN
The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPK.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Transporte de ARN , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Viabilidad Microbiana , Datos de Secuencia Molecular , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tolerancia a la Sal , Estrés FisiológicoRESUMEN
Over the past decade, tremendous progress has been made in understanding the spatial organization of genes and chromosomes. Nuclear organization can be thought of as information that is not encoded in DNA, but which nevertheless impacts gene expression. Nuclear organizational influences can be cell-specific and are potentially heritable. Thus, nuclear organization fulfills all the criteria necessary for it to be considered an authentic level of epigenetic information. Chromosomal nuclear organization is primarily dictated by the biophysical properties of chromatin. Diffusion models of polymers confined in the crowded nuclear space accurately recapitulate experimental observation. Diffusion is a Brownian process, which implies that the positions of chromosomes and genes are not defined deterministically but are likely to be dictated by the laws of probability. Despite the small size of their nuclei, budding yeast have been instrumental in discovering how epigenetic information is encoded in the spatial organization of the genome. The relatively simple organization of the yeast nucleus and the very high number of genetically identical cells that can be observed under fluorescent microscopy allow statistically robust definitions of the gene and chromosome positions in the nuclear space to be constructed. In this review, we will focus on how the spatial organization of the chromatin in the yeast nucleus might impact transcription. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
Asunto(s)
Fenómenos Biofísicos , Núcleo Celular/genética , Cromatina/genética , Saccharomyces cerevisiae/genética , Ensamble y Desensamble de Cromatina/genética , Cromosomas/genética , Difusión , Regulación Fúngica de la Expresión Génica , Genoma , Modelos TeóricosRESUMEN
Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Membrana Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Transactivadores/metabolismo , Transcripción Genética/genética , Difusión , Genes Reporteros/genética , Modelos Genéticos , Mutación/genética , Membrana Nuclear/genética , Unión Proteica , ARN de Hongos/biosíntesis , ARN de Hongos/genética , Proteínas de Saccharomyces cerevisiae/genética , Transactivadores/genéticaRESUMEN
Cohesin is a DNA translocase that is instrumental in the folding of the genome into chromatin loops, with functional consequences on DNA-related processes. Chromatin loop length and organization likely depend on cohesin processivity, translocation rate and stability on DNA. Here, we investigate and provide a comprehensive overview of the roles of various cohesin regulators in tuning chromatin loop expansion in budding yeast Saccharomyces cerevisiae. We demonstrate that Scc2, which stimulates cohesin ATPase activity, is also essential for cohesin translocation, driving loop expansion in vivo. Smc3 acetylation during the S phase counteracts this activity through the stabilization of Pds5, which finely tunes the size and stability of loops in G2.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Acetilación , Proteínas de Ciclo Celular/metabolismo , Cromatina , Proteínas Cromosómicas no Histona/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
The nonrandom positioning of genes inside eukaryotic cell nuclei is implicated in central nuclear functions. However, the spatial organization of the genome remains largely uncharted, owing to limited resolution of optical microscopy, paucity of nuclear landmarks and moderate cell sampling. We developed a computational imaging approach that creates high-resolution probabilistic maps of subnuclear domains occupied by individual loci in budding yeast through automated analysis of thousands of living cells. After validation, we applied the technique to genes involved in galactose metabolism and ribosome biogenesis. We found that genomic loci are confined to 'gene territories' much smaller than the nucleus, which can be remodeled during transcriptional activation, and that the nucleolus is an important landmark for gene positioning. The technique can be used to visualize and quantify territory positions relative to each other and to nuclear landmarks, and should advance studies of nuclear architecture and function.
Asunto(s)
Núcleo Celular/fisiología , Núcleo Celular/ultraestructura , Cromosomas/genética , Cromosomas/ultraestructura , Interpretación Estadística de Datos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Compartimento Celular , Sensibilidad y EspecificidadRESUMEN
In eukaryotes, ribosome biogenesis is a process of major interest that requires more than 200 factors acting coordinately in time and space. Using genetic and proteomic studies, most of the components have now been identified. Based on its nucleolar localization, we characterized the protein encoded by the open reading frame YGR251W, we renamed Nop19p as playing an essential role in ribosome biogenesis. Depletion of the Nop19p in yeast impairs pre-rRNA processing at sites A0, A1 and A2, leading to a strong decrease in 18S rRNA and 40S subunit levels. Nop19p is a component of 90S preribosomes which assembly is believed to result from stepwise incorporation of UTP modules. We show that Nop19p depletion does not impair the incorporation of UTP subcomplexes on preribosomes and conversely that depletion of UTP subcomplexes does not affect Nop19p recruitment on 90S preribosomes. TAP experiments under stringent conditions revealed that Nop19p interacts preferentially with the DEAH-box RNA helicase Dhr2p and Utp25p, both required for A 0, A 1 and A 2 cleavages. Nop19p appeared essential for the incorporation of Utp25p in preribosomes. In addition, our results suggest that in absence of Nop19p, Dhr2p remains trapped within aberrant preribosomes.