RESUMEN
Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.
Asunto(s)
ADN Ribosómico , Ribosomas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , ADN Ribosómico/genética , Ribosomas/metabolismo , Ribosomas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Regulación Fúngica de la Expresión Génica , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Transducción de Señal/genética , Ciclo Celular/genética , Eliminación de GenRESUMEN
Nucleoli form interchromosomal contacts with genes controlling differentiation and carcinogenesis. DUX4 genes specify transcription factor possessing two homeodomains. Previously, using Circular Chromosome Conformation Capture (4С) approach on population of cells, it was demonstrated that DUX4 gene clusters form frequent contacts with nucleoli. It was found also that these contacts are almost completely abolished after heat shock treatment. 4C approach as all ligation-mediated methods is capable to detect rather close interactions between chromatin loops in nuclei. In order to independently confirm the formation and the frequency of the contacts in single cells we used FISH approach. Here, we show that DUX genes in single cells form stable contacts in all tested HEK293T cells. During heat shock, DUX4 genes reversibly move 1-3 µm away from the nuclei. We conclude that interchromosomal contacts formed by nucleoli are strong, dynamic, and reversible, providing both the initiation and maintenance of a differentiated state.
Asunto(s)
Nucléolo Celular , Proteínas de Homeodominio , Humanos , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células HEK293 , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Hibridación Fluorescente in SituRESUMEN
The nucleolus functions as a multi-layered regulatory hub for ribosomal RNA (rRNA) biogenesis and ribosome assembly. Long noncoding RNAs (lncRNAs) in the nucleolus, originated from transcription by different RNA polymerases, have emerged as critical players in not only fine-tuning rRNA transcription and processing, but also shaping the organization of the multi-phase nucleolar condensate. Here, we review the diverse molecular mechanisms by which functional lncRNAs operate in the nucleolus, as well as their profound implications in a variety of biological processes. We also highlight the development of emerging molecular tools for characterizing and manipulating RNA function in living cells, and how application of such tools in the nucleolus might enable the discovery of additional insights and potential therapeutic strategies.
Asunto(s)
Nucléolo Celular , ARN Largo no Codificante , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Humanos , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , Regulación de la Expresión Génica , Animales , Transcripción Genética , Ribosomas/metabolismoRESUMEN
Different developmental genes shape frequent dynamic inter-chromosomal contacts with rDNA units in human and Drosophila cells. In the course of differentiation, changes in these contacts occur, coupled with changes in the expression of hundreds of rDNA-contacting genes. The data suggest a possible role of nucleoli in the global regulation of gene expression. However, the mechanism behind the specificity of these inter-chromosomal contacts, which are rebuilt in every cell cycle, is not yet known. Here, we describe the strong association of rDNA-contacting genes with numerous long intergenic non-coding RNAs (lincRNAs) in HEK293T cells and in initial and differentiated K562 cells. We observed that up to 600 different lincRNAs were preferentially co-expressed with multiple overlapping sets of rDNA-contacting developmental genes, and there was a strong correlation between the genomic positions of rDNA-contacting genes and lincRNA mappings. These two findings suggest that lincRNAs might guide the corresponding developmental genes toward rDNA clusters. We conclude that the inter-chromosomal interactions of rDNA-contacting genes with nucleoli might be guided by lincRNAs, which might physically link particular genomic regions with rDNA clusters.
Asunto(s)
Nucléolo Celular , ADN Ribosómico , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Células HEK293 , Células K562RESUMEN
RNA-binding proteins (RBPs) are pivotal in acute myeloid leukaemia (AML), a lethal disease. Although specific phase separation-competent RBPs are recognized in AML, the effect of their condensate formation on AML leukaemogenesis, and the therapeutic potential of inhibition of phase separation are underexplored. In our in vivo CRISPR RBP screen, fibrillarin (FBL) emerges as a crucial nucleolar protein that regulates AML cell survival, primarily through its phase separation domains rather than methyltransferase or acetylation domains. These phase separation domains, with specific features, coordinately drive nucleoli formation and early processing of pre-rRNA (including efflux, cleavage and methylation), eventually enhancing the translation of oncogenes such as MYC. Targeting the phase separation capability of FBL with CGX-635 leads to elimination of AML cells, suggesting an additional mechanism of action for CGX-635 that complements its established therapeutic effects. We highlight the potential of PS modulation of critical proteins as a possible therapeutic strategy for AML.
Asunto(s)
Proteínas Cromosómicas no Histona , Leucemia Mieloide Aguda , Precursores del ARN , Procesamiento Postranscripcional del ARN , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Precursores del ARN/metabolismo , Precursores del ARN/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Animales , Línea Celular Tumoral , Biosíntesis de Proteínas , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Ratones , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Regulación Leucémica de la Expresión Génica , Separación de FasesRESUMEN
With increasingly used assisted reproductive technology (ART), the acquisition of high-quality oocytes and early embryos has become the focus of much attention. Studies in mice have found that the transition of chromatin conformation from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) is essential for oocyte maturation and early embryo development, and similar chromatin transition also exists in human oocytes. In this study, we collected human NSN and SN oocytes and investigated their transcriptome. The analysis of differentially expressed genes showed that epigenetic functions, cyclin-dependent kinases and transposable elements may play important roles in chromatin transition during human oocyte maturation. Our findings provide new insights into the molecular mechanism of NSN-to-SN transition of human oocyte and obtained new clues for improvement of oocyte in vitro maturation technique.
Asunto(s)
Cromatina , Oocitos , Transcriptoma , Humanos , Oocitos/metabolismo , Cromatina/metabolismo , Cromatina/genética , Femenino , Perfilación de la Expresión Génica , Nucléolo Celular/metabolismo , Nucléolo Celular/genéticaRESUMEN
Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.
Asunto(s)
ADN Helicasas , Enzimas Multifuncionales , ARN Helicasas , ARN no Traducido , Humanos , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Daño del ADN , ADN Helicasas/metabolismo , ADN Helicasas/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Enzimas Multifuncionales/metabolismo , Enzimas Multifuncionales/genética , Agregado de Proteínas , Proteostasis , Estructuras R-Loop/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
Although DNA-PK inhibitors (DNA-PK-i) have been applied in clinical trials for cancer treatment, the biomarkers and mechanism of action of DNA-PK-i in tumor cell suppression remain unclear. Here, we observed that a low dose of DNA-PK-i and PARP inhibitor (PARP-i) synthetically suppresses BRCA-deficient tumor cells without inducing DNA double-strand breaks (DSBs). Instead, we found that a fraction of DNA-PK localized inside of nucleoli, where we did not observe obvious DSBs. Moreover, the Ku proteins recognize pre-rRNA that facilitates DNA-PKcs autophosphorylation independent of DNA damage. Ribosomal proteins are also phosphorylated by DNA-PK, which regulates pre-rRNA biogenesis. In addition, DNA-PK-i acts together with PARP-i to suppress pre-rRNA biogenesis and tumor cell growth. Collectively, our studies reveal a DNA damage repair-independent role of DNA-PK-i in tumor suppression.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína Quinasa Activada por ADN , Autoantígeno Ku , Precursores del ARN , Proteína Quinasa Activada por ADN/metabolismo , Proteína Quinasa Activada por ADN/genética , Humanos , Precursores del ARN/metabolismo , Precursores del ARN/genética , Línea Celular Tumoral , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Fosforilación , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , Animales , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
Nucleolar stress has been consistently linked to age-related diseases. In this issue, Sirozh et al.1 find that the common molecular signature of nucleolar stress is the accumulation of free ribosomal proteins, which leads to premature aging in mice; however, it can be reversed by mTOR inhibition.
Asunto(s)
Nucléolo Celular , Proteínas Ribosómicas , Ratones , Animales , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , ARN Ribosómico/metabolismoRESUMEN
DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.
Asunto(s)
ARN Helicasas DEAD-box , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/química , Humanos , Animales , ARN/metabolismo , ARN/genética , ARN/química , Empalme del ARN , Orgánulos/metabolismo , Orgánulos/genética , Ribosomas/metabolismo , Ribosomas/genética , Pliegue del ARN , Procesamiento Postranscripcional del ARN , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Histone H1 participates in chromatin condensation and regulates nuclear processes. Human somatic cells may contain up to seven histone H1 variants, although their functional heterogeneity is not fully understood. Here, we have profiled the differential nuclear distribution of the somatic H1 repertoire in human cells through imaging techniques including super-resolution microscopy. H1 variants exhibit characteristic distribution patterns in both interphase and mitosis. H1.2, H1.3, and H1.5 are universally enriched at the nuclear periphery in all cell lines analyzed and co-localize with compacted DNA. H1.0 shows a less pronounced peripheral localization, with apparent variability among different cell lines. On the other hand, H1.4 and H1X are distributed throughout the nucleus, being H1X universally enriched in high-GC regions and abundant in the nucleoli. Interestingly, H1.4 and H1.0 show a more peripheral distribution in cell lines lacking H1.3 and H1.5. The differential distribution patterns of H1 suggest specific functionalities in organizing lamina-associated domains or nucleolar activity, which is further supported by a distinct response of H1X or phosphorylated H1.4 to the inhibition of ribosomal DNA transcription. Moreover, H1 variants depletion affects chromatin structure in a variant-specific manner. Concretely, H1.2 knock-down, either alone or combined, triggers a global chromatin decompaction. Overall, imaging has allowed us to distinguish H1 variants distribution beyond the segregation in two groups denoted by previous ChIP-Seq determinations. Our results support H1 variants heterogeneity and suggest that variant-specific functionality can be shared between different cell types.
Asunto(s)
Núcleo Celular , Histonas , Humanos , Histonas/genética , Nucléolo Celular/genética , Cromatina , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Nucleolar stress (NS) has been associated with age-related diseases such as cancer or neurodegeneration. To investigate how NS triggers toxicity, we used (PR)n arginine-rich peptides present in some neurodegenerative diseases as inducers of this perturbation. We here reveal that whereas (PR)n expression leads to a decrease in translation, this occurs concomitant with an accumulation of free ribosomal (r) proteins. Conversely, (PR)n-resistant cells have lower rates of r-protein synthesis, and targeting ribosome biogenesis by mTOR inhibition or MYC depletion alleviates (PR)n toxicity in vitro. In mice, systemic expression of (PR)97 drives widespread NS and accelerated aging, which is alleviated by rapamycin. Notably, the generalized accumulation of orphan r-proteins is a common outcome of chemical or genetic perturbations that induce NS. Together, our study presents a general model to explain how NS induces cellular toxicity and provides in vivo evidence supporting a role for NS as a driver of aging in mammals.
Asunto(s)
Neoplasias , Ribosomas , Ratones , Animales , Ribosomas/metabolismo , Envejecimiento/genética , Péptidos/metabolismo , Sirolimus/farmacología , Neoplasias/metabolismo , Nucléolo Celular/genética , MamíferosRESUMEN
The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.
Asunto(s)
Nucléolo Celular , Proteínas de Unión al ADN , Nucleofosmina , Factores de Transcripción , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Ribosómico/metabolismo , Humanos , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
Out of the several hundred copies of rRNA genes arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, ~50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to the differential expression of rRNAs. However, the mechanism(s) controlling the dosage of active versus inactive rRNA genes within each NOR in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influence rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosomes. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs influencing rRNA expression by forming constrained nucleolar territories on individual NORs.
Asunto(s)
Región Organizadora del Nucléolo , Precursores del ARN , Humanos , Animales , Región Organizadora del Nucléolo/genética , Región Organizadora del Nucléolo/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Cromosomas Humanos/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Mamíferos/genéticaRESUMEN
BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains and consequently is prone to modification by chromosomal rearrangements. We have shown that nuclear architecture is modified in spermatocytes of Robertsonian (Rb) homozygotes of Mus domesticus. In this study we analyse the synaptic configuration of the quadrivalents formed in the meiotic pro- phase of spermatocytes of mice double heterozygotes for the dependent Rb chromosomes: Rbs 11.16 and 16.17. RESULTS: Electron microscope spreads of 60 pachytene spermatocytes from four animals of Mus domesticus 2n = 38 were studied and their respective quadrivalents analysed in detail. Normal synaptonemal complex was found between arms 16 of the Rb metacentric chromosomes, telocentrics 11 and 17 and homologous arms of the Rb metacentric chromosomes. About 43% of the quadrivalents formed a synaptonemal complex between the heterologous short arms of chromosomes 11 and 17. This synaptonemal complex is bound to the nuclear envelope through a fourth synapsed telomere, thus dragging the entire quadrivalent to the nuclear envelope. About 57% of quadrivalents showed unsynapsed single axes in the short arms of the telocentric chromosomes. About 90% of these unsynapsed quadrivalents also showed a telomere-to-telomere association between one of the single axes of the telocentric chromosome 11 or 17 and the X chromosome single axis, which was otherwise normally paired with the Y chromosome. Nucleolar material was associated with two bivalents and with the quadrivalent. CONCLUSIONS: The spermatocytes of heterozygotes for dependent Rb chromosomes formed a quadrivalent where four chromosomes are synapsed together and bound to the nuclear envelope through four telomeres. The nuclear configuration is determined by the fourth shortest telomere, which drags the centromere regions and heterochromatin of all the chromosomes towards the nuclear envelope, favouring the reiterated encounter and eventual rearrangement between the heterologous chromosomes. The unsynapsed regions of quadrivalents are frequently bound to the single axis of the X chromosome, possibly perturbing chromatin condensation and gene expression.
Asunto(s)
Animales , Masculino , Ratones , Espermatocitos/fisiología , Espermatocitos/ultraestructura , Cromosoma X/fisiología , Cromosoma Y/fisiología , Complejo Sinaptonémico/fisiología , Nucléolo Celular/fisiología , Translocación Genética , Cromosoma X/genética , Cromosoma Y/genética , Complejo Sinaptonémico/genética , Heterocromatina/fisiología , Heterocromatina/genética , Nucléolo Celular/genética , Telómero/fisiología , Telómero/genética , Profase Meiótica I/fisiología , Profase Meiótica I/genética , HeterocigotoRESUMEN
Drosophila mulleri (MU) and D. arizonae (AR) are cryptic species of the mulleri complex, mulleri subgroup, repleta group. Earlier cytogenetic studies revealed that these species have different regulatory mechanisms of nucleolar organizing activity. In these species, nucleolar organizing regions are found in both the X chromosome and the microchromosome. In the salivary glands of hybrids between MU females and AR males, there is an interspecific dominance of the regulatory system of the D. arizonae nucleolar organizer involving, in males, amplification and activation of the nucleolar organizer from the microchromosome. The authors who reported these findings obtained hybrids only in that cross-direction. More recently, hybrids in the opposite direction, i.e., between MU males and AR females, have been obtained. The purpose of the present study was to evaluate, in these hybrids, the association of the nucleoli with the chromosomes inherited from parental species in order to cytogenetically confirm the dominance patterns previously described. Our results support the proposed dominance of the AR nucleolar organizer activity over that of MU, regardless of cross-direction.