Showing papers on "Nucleolus published in 2023"
TL;DR: In this paper , de novo frameshift variants in HMGB1 were found to cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome.
Abstract: Abstract Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions 1–3 . Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus 4,5 . This suggests that mutations in disordered proteins may alter condensate properties and function 6–8 . Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.
8 citations
TL;DR: In this paper , a review of the ribosomal RNA (rRNA) gene copy number and RNA polymerase I (RNA polymerase II) is presented, where the crucial maintenance of rRNA gene copy numbers through control of gene amplification and rRNA production is discussed.
Abstract: One of the first biological machineries to be created seems to have been the ribosome. Since then, organisms have dedicated great efforts to optimize this apparatus. The ribosomal RNA (rRNA) contained within ribosomes is crucial for protein synthesis and maintenance of cellular function in all known organisms. In eukaryotic cells, rRNA is produced from ribosomal DNA clusters of tandem rRNA genes, whose organization in the nucleolus, maintenance and transcription are strictly regulated to satisfy the substantial demand for rRNA required for ribosome biogenesis. Recent studies have elucidated mechanisms underlying the integrity of ribosomal DNA and regulation of its transcription, including epigenetic mechanisms and a unique recombination and copy-number control system to stably maintain high rRNA gene copy number. In this Review, we disucss how the crucial maintenance of rRNA gene copy number through control of gene amplification and of rRNA production by RNA polymerase I are orchestrated. We also discuss how liquid-liquid phase separation controls the architecture and function of the nucleolus and the relationship between rRNA production, cell senescence and disease.
7 citations
TL;DR: In this article , a nucleolus Hi-C (nHi-C) experimental technique was developed to enrich the nucleoli-associated chromatin interactions, and the authors identified 264 high-confidence NADs (hNADs) that formed strong heterochromatin interactions associated with the nucleus and formed 24% of the whole genome in HeLa cells.
Abstract: As the largest substructures in the nucleus, nucleoli are the sites of ribosome biogenesis. Increasing evidence indicates that nucleoli play a key role in the organization of 3D genome architecture, but systematic studies of nucleolus-associated chromatin interactions are lacking. Here, we developed a nucleolus Hi-C (nHi-C) experimental technique to enrich nucleolus-associated chromatin interactions. Using the nHi-C experiment, we identify 264 high-confidence nucleolus-associated domains (hNADs) that form strong heterochromatin interactions associated with the nucleolus and consist of 24% of the whole genome in HeLa cells. Based on the global hNAD inter-chromosomal interactions, we find five nucleolar organizer region (NOR)-bearing chromosomes formed into two clusters that show different interaction patterns, which is concordant with their epigenetic states and gene expression levels. hNADs can be divided into three groups that display distinct cis/trans interaction signals, interaction frequencies associated with nucleoli, distance from the centromeres, and overlap percentage with lamina-associated domains (LADs). Nucleolus disassembly caused by Actinomycin D (ActD) significantly decreases the strength of hNADs and affects compartment/TAD strength genome-wide. In summary, our results provide a global view of heterochromatin interactions organized around nucleoli and demonstrate that nucleoli act as an inactive inter-chromosomal hub to shape both compartments and TADs.
4 citations
3 citations
TL;DR: In this article , the authors provide distinct perspectives on how nucleolar DNA checkpoint pathways are activated by different stresses or by liquid-liquid phase separation (LLPS) in the nucleolus.
3 citations
TL;DR: In this paper , a 1.5 mega-base rDNA locus was repositioned within the megachromosome in a fused-karyotype strain of Saccharomyces cerevisiae.
Abstract: The nucleolus is the most prominent membraneless compartment within the nucleus-dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations-where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location-the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This "megablock" rDNA engineering was performed in a fused-karyotype strain of Saccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to the CEN, in that the locus that is CEN-distal undergoes size reduction at a higher frequency compared to the CEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogous cis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.
2 citations
TL;DR: In this article , the authors report changes in nucleolar morphology and function during myogenic differentiation, which are consistent with changes to nucleolar phase separation properties, and support an mTOR dependent re-organization of nucleolar structure during myogenesis, enhancing our understanding of myogenesis and possibly facilitating new approaches to therapeutic interventions in muscle pathologies.
Abstract: Myogenesis, the process of muscle differentiation, requires an extensive remodeling of the cellular transcriptome and proteome. While the transcriptional program underpinning myogenesis is well characterized, the required adaptation in protein synthesis is incompletely understood. Enhanced protein synthesis necessitates ribosome biogenesis at the nucleolus. Nucleolar size and activity are inextricably linked with altered gene expression. Here, we report changes in nucleolar morphology and function during myogenic differentiation. Immunofluorescence analysis revealed alterations in nucleolar morphology that were dependent on the cellular state: proliferative or quiescent myogenic progenitors (myoblasts or reserve cells) contain multiple small nucleoli with a characteristic spherical shape, whereas multinucleated myotubes typically contain one large, often irregularly shaped nucleolus. These morphological alterations are consistent with changes to nucleolar phase separation properties. Re-organization of the nucleolar structure was correlated with enhanced rRNA production and protein translation. Inhibition of mTOR signaling with Rapamycin perturbed nucleolar re-organization. Conversely, hyperactivated mTOR enhanced alterations in nucleolar morphology. These findings support an mTOR dependent re-organization of nucleolar structure during myogenesis, enhancing our understanding of myogenesis and possibly facilitating new approaches to therapeutic interventions in muscle pathologies.
2 citations
TL;DR: In this article , a pH-triggered charge reversible fluorescent probe (LD-Nu) was constructed based on a cyclization-ring-opening mechanism, and the in vitro pH titration experiment and 1H NMR showed that LD-nu gradually transferred from the charged form to the electroneutral form with the increase of pH, and thus, the conjugate plane was reduced and its fluorescence blue-shifted.
Abstract: Cooperation between organelles is essential to maintain the normal functions of cells. Lipid droplets (LDs) and nucleoli, as important organelles, play an important role in the normal activities of cells. However, due to the lack of appropriate tools, in situ observation of the interaction between them has been rarely reported. In this work, taking into full consideration the pH and charge differences between LDs and nucleoli, a pH-triggered charge reversible fluorescent probe (LD-Nu) was constructed based on a cyclization-ring-opening mechanism. The in vitro pH titration experiment and 1H NMR showed that LD-Nu gradually transferred from the charged form to the electroneutral form with the increase of pH, and thus, the conjugate plane was reduced and its fluorescence blue-shifted. Most importantly, the physical contact between LDs and nucleoli was visualized for the first time. Meanwhile, the relationship between LDs and nucleoli was also further investigated, and the results showed that their interaction was more liable to be affected by the abnormality of LDs than those of nucleoli. Moreover, the cell imaging results displayed that the LDs both in the cytoplasm and nucleus were observed using the probe LD-Nu, and interestingly, the LDs in the cytoplasm were more susceptible to external stimuli than those in the nucleus. In a word, the probe LD-Nu can serve as a powerful tool for further exploration of the interaction mechanism between LDs and nucleoli in living cells.
2 citations
TL;DR: In this article , a small non-structural protein (NS5) was detected in infected mammalian or insect cells and was identified in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids.
Abstract: Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses.
2 citations
TL;DR: In this paper , the authors examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells.
Abstract: Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, we examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.
2 citations
TL;DR: A 25 kDa human protein, RIEP (Ribosomal IGS Encoded Protein) as discussed by the authors , is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS).
Abstract: Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar "non-coding" RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.
TL;DR: In this article , the differences between the cytological features of GEA and usual-type endocervical adenocarcinoma (UEA) were examined, and the authors concluded that GEA can be identified based on the presence of flat, honeycomb-like sheets of tumor cells possessing vesicular nuclei, prominent nucleoli, and abundant vacuolated cytoplasm.
Abstract: Background/Aim: Gastric-type endocervical adenocarcinoma (GEA) is a rare but distinct histological type of gynecological malignancy. This study aimed to conduct a comprehensive analysis of the cytological features of GEA. Patients and Methods: We reviewed 18 cytological samples obtained from 14 patients with GEA. All cytology slides were prepared using conventional smear and liquid-based preparations. We examined the differences between the cytological features of GEA and usual-type endocervical adenocarcinoma (UEA). Results: The cytological samples of GEA exhibited flat, honeycomb-like cellular sheets (p=0.035), vesicular nuclei (p=0.037) with prominent nucleoli (p=0.037), and vacuolated cytoplasm (p<0.001) more frequently than those of UEA, irrespective of the sampling site and preparation method. UEA showed three-dimensional cellular clusters (p<0.001), peripheral nuclear feathering (p<0.001), and nuclear hyperchromasia (p=0.014) more frequently than GEA. Conclusion: GEA can be identified cytologically based on the presence of flat, honeycomb-like sheets of tumor cells possessing vesicular nuclei, prominent nucleoli, and abundant vacuolated cytoplasm.
TL;DR: In this paper , the authors describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks.
Abstract: The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.
TL;DR: In this paper , the authors summarize recent findings on the molecular mechanisms of nucleolar stress and the human ribosomal diseases and cancers that arise in its wake, including DNA damage response and oncogenic stress.
Abstract: Ribosome biogenesis in the nucleolus is an important process that consumes 80% of a cell's intracellular energy supply. Disruption of this process results in nucleolar stress, triggering the activation of molecular systems that respond to this stress to maintain homeostasis. Although nucleolar stress was originally thought to be caused solely by abnormalities of ribosomal RNA (rRNA) and ribosomal proteins (RPs), an accumulating body of more current evidence suggests that many other factors, including the DNA damage response and oncogenic stress, are also involved in nucleolar stress response signaling. Cells reacting to nucleolar stress undergo cell cycle arrest or programmed death, mainly driven by activation of the tumor suppressor p53. This observation has nominated nucleolar stress as a promising target for cancer therapy. However, paradoxically, some RP mutations have also been implicated in cancer initiation and progression, necessitating caution. In this article, we summarize recent findings on the molecular mechanisms of nucleolar stress and the human ribosomal diseases and cancers that arise in its wake.
TL;DR: In this paper , the authors provided ultrastructural and molecular evidence for the presence of putative nucleolus-like subcellular domains in the TACK crenarchaeon Saccharolobus solfataricus (formerly known as Sulfolobalus solfish).
Abstract: Nucleoli are subcellular compartments where transcription and maturation of pre-ribosomal RNAs occur. While the transcription of ribosomal RNAs is common to all living cells, the presence and ultrastructure of nucleoli has been only documented in eukaryotes. Asgard-Archaea, the closest prokaryotic relatives of eukaryotes, and their near relatives TACK-Archaea have homologs of nucleolar proteins and RNAs in their genome, but the cellular organization of both is largely unexplored. Here we provide ultrastructural and molecular evidence for the presence of putative nucleolus-like subcellular domains in the TACK crenarchaeon Saccharolobus solfataricus (formerly known as Sulfolobus solfataricus). Transmission electron microscopy (TEM) revealed consistent electron-dense fibro-granular compartments, also positive to the specific silver staining for nucleolar organizer regions (AgNOR). TEM also confirmed that ribosomal DNA (rDNA) is spatially distributed in non-random, clustered arrays underlying fine structures, as observed by ultrastructural in situ hybridization (UISH). To further explore these observations, proteomic sequencing of isolated bands from AgNOR-stained protein gels was conducted and compared against a compiled inventory of putative nucleolar homologs from the S. solfataricus P1 genome. Sequenced AgNOR-sensitive peptides encoded homologs of eukaryotic nucleoli proteins, enriched for nucleolus-related functions. Our results provide first evidence that subcellular domains of nucleolar-like nature are not exclusive to eukaryotes. Based on our data, we propose a model for a putative nucleolus in S. solfataricus. Whereas technical limitations and further aspects remain a matter for future functional studies, our data supports the origin of nucleoli within the common ancestor of Eukarya and TACK-Archaea, based on a two-domain tree of life.
TL;DR: In this paper , chromatin-binding ribonucleic acid polymerase II (RNAP II) activity, newly synthesized RNA, and chromatin accessibility in mouse germinal vesicle (GV) oocytes were examined.
Abstract: Mouse germinal vesicle (GV) oocytes are divided into surrounded nucleolus (SN) and nonsurrounded nucleolus (NSN) oocytes based on chromatin morphology. NSN oocytes spontaneously transform into SN oocytes after accumulating enough maternal transcripts. SN oocytes show transcriptional silencing. When oocyte maturation is abnormal or takes place in vitro, NSN oocytes do not go through SN stage before proceeding to MII. Nontransitive oocytes show developmental retardation, a low fertilization rate, and arrest at the two‐cell embryo stage in mice. Here, chromatin‐binding ribonucleic acid polymerase II (RNAP II) activity, newly synthesized RNA, and chromatin accessibility in GV oocytes were examined. In SN oocytes, RNAP II did not bind to DNA, neo‐RNA was not generated in nuclei, and the phosphorylation state of RNAP II did not affect the chromatin‐binding activity. The number of accessible genes in SN oocytes was remarkably lower than that in NSN oocytes. The accessibility of different functional genes was also different between the two types of oocytes. Thus, low chromatin accessibility leads to transcriptional silencing in SN oocytes.
TL;DR: In this article , the nucleolar ubiquitin-specific protease USP36 was shown to act as a SUMO ligase to promote EXOSC10 SUMOylation critical for ribosome biogenesis.
Abstract: Abstract The RNA exosome is an essential 3′ to 5′ exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3′ to 5′ exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.
TL;DR: In this paper , the authors identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen.
Abstract: The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen.
TL;DR: In this article , bright red-emissive carbon dots (termed CPCDs) with excitationindependent/polarity-dependent fluorescence emission are synthesized by a one-step hydrothermal reaction between congo red and p-phenylenediamine.
Abstract: Nucleolus, which participates in many crucial cellular activities, is an ideal target for evaluating the state of a cell or an organism. Here, bright red-emissive carbon dots (termed CPCDs) with excitation-independent/polarity-dependent fluorescence emission are synthesized by a one-step hydrothermal reaction between congo red and p-phenylenediamine. The CPCDs can achieve wash-free, real-time, long-term, and high-quality nucleolus imaging in live cells, as well as in vivo imaging of two common model animals-zebrafish and Caenorhabditis elegans (C. elegans). Strikingly, CPCDs realize the nucleolus imaging of organs/flowing blood cells in zebrafish at a cellular level for the first time, and the superb nucleolus imaging of C. elegans suggests that the germ cells in the spermatheca probably have no intact nuclei. These previously unachieved imaging results of the cells/tissues/organs may guide the zebrafish-related studies and benefit the research of C. elegans development. More importantly, a novel strategy based on CPCDs for in vivo toxicity evaluation of materials/drugs (e.g., Ag+ ), which can visualize the otherwise unseen injuries in zebrafish, is developed. In conclusion, the CPCDs represent a robust tool for visualizing the structures and dynamic behaviors of live zebrafish and C. elegans, and may find important applications in cell biology and toxicology.
TL;DR: TransitID as mentioned in this paper is a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells using two proximity labeling (PL) enzymes, TurboID and APEX, targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates.
TL;DR: The role of biomolecular condensates in phase separation in viral replication was discussed in this paper . But, this work focused on the hierarchical coassembly of ribosomal RNAs and proteins in the nucleolus.
Abstract: Phase separation of viral biopolymers is a key factor in the formation of cytoplasmic viral inclusions, known as sites of virus replication and assembly. This review describes the mechanisms and factors that affect phase separation in viral replication and identifies potential areas for future research. Drawing inspiration from studies on ribosome biogenesis, we compare the hierarchical coassembly of ribosomal RNAs and proteins in the nucleolus to the coordinated coassembly of viral RNAs and proteins taking place within viral factories formed by RNA viruses containing segmented genomes. We highlight the evidence supporting the role of biomolecular condensates in viral replication and how this new understanding is reshaping our views of virus assembly mechanisms. Future research of biomolecular condensates has the potential to uncover unexplored antiviral strategies targeting these phase-separated states. Expected final online publication date for the Annual Review of Virology, Volume 10 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
TL;DR: In this article , the mass density of several condensates, including nucleoli, heterochromatin, nuclear speckles, and stress granules, was quantified using refractive index imaging.
Abstract: Biomolecular condensates play a key role in organizing cellular reactions by concentrating a specific set of biomolecules. However, whether condensate formation is accompanied by an increase in the total mass concentration within condensates or by the demixing of already highly crowded intracellular components remains elusive. Here, using refractive index imaging, we quantify the mass density of several condensates, including nucleoli, heterochromatin, nuclear speckles, and stress granules. Surprisingly, the latter two condensates exhibit low densities with a total mass concentration similar to the surrounding cyto- or nucleoplasm. Low-density condensates display higher permeability to cellular protein probes. We find that RNA tunes the biomolecular density of condensates. Moreover, intracellular structures such as mitochondria heavily influence the way phase separation proceeds, impacting the localization, morphology, and growth of condensates. These findings favor a model where segregative phase separation driven by non-associative or repulsive molecular interactions together with RNA-mediated selective association of specific components can give rise to low-density condensates in the crowded cellular environment.
TL;DR: In this paper , the role and downstream mechanisms of Prostaglandins regulate the nucleolus of Drosophila oogenesis were investigated. And they showed that PGs carefully balance the level and forms of nuclear actin to control the level of nucleolar activity required for producing fertilization competent oocytes.
Abstract: Prostaglandins (PGs), locally acting lipid signals, regulate female reproduction, including oocyte development. However, the cellular mechanisms of PG action remain largely unknown. One cellular target of PG signaling is the nucleolus. Indeed, across organisms, loss of PGs results in misshapen nucleoli, and changes in nucleolar morphology are indicative of altered nucleolar function. A key role of the nucleolus is to transcribe ribosomal RNA (rRNA) to drive ribosomal biogenesis. Here we take advantage of the robust, in vivo system of Drosophila oogenesis to define the roles and downstream mechanisms whereby PGs regulate the nucleolus. We find that the altered nucleolar morphology due to PG loss is not due to reduced rRNA transcription. Instead, loss of PGs results in increased rRNA transcription and overall protein translation. PGs modulate these nucleolar functions by tightly regulating nuclear actin, which is enriched in the nucleolus. Specifically, we find that loss of PGs results in both increased nucleolar actin and changes in its form. Increasing nuclear actin, by either genetic loss of PG signaling or overexpression of nuclear targeted actin (NLS-actin), results in a round nucleolar morphology. Further, loss of PGs, overexpression of NLS-actin or loss of Exportin 6, all manipulations that increase nuclear actin levels, results in increased RNAPI-dependent transcription. Together these data reveal PGs carefully balance the level and forms of nuclear actin to control the level of nucleolar activity required for producing fertilization competent oocytes.
TL;DR: The nucleolus organizer regions (NORs) are eukaryotic chromosomal loci where ribosomal RNA (rRNA) genes are clustered, typically in hundreds, to thousands, of copies as discussed by the authors .
Abstract: Nucleolus organizer regions (NORs) are eukaryotic chromosomal loci where ribosomal RNA (rRNA) genes are clustered, typically in hundreds, to thousands, of copies. Transcription of these rRNA genes by RNA Polymerase I and processing of their transcripts results in the formation of the nucleolus, the sub-nuclear domain in which ribosomes are assembled. Approximately 90 years ago, cytogenetic observations revealed that NORs inherited from the different parents of an interspecific hybrid sometimes differ in morphology at metaphase. Fifty years ago, those chromosomal differences were found to correlate with differences in rRNA gene transcription and the phenomenon became known as nucleolar dominance. Studies of the past 30 years have revealed that nucleolar dominance results from selective rRNA gene silencing, involving repressive chromatin modifications, and occurs in pure species as well as hybrids. Recent evidence also indicates that silencing depends on the NOR in which a rRNA gene is located, and not on the gene's sequence. In this perspective, we discuss how our thinking about nucleolar dominance has shifted over time from the kilobase scale of individual genes to the megabase scale of NORs and chromosomes, and questions that remain unanswered in the search for a genetic and biochemical understanding of the off switch.
TL;DR: In this article , the role of nucleolus-targeted effectors in pathogenesis of P. infestans infection of Nicotiana benthamiana has been investigated, showing that nucleolar inflation occurs during the transition from the biotrophic to the necrotrophic phase.
Abstract: Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system. Although the nucleolus has emerged as a stress marker and several effectors are localized in the nucleolus, the roles of nucleolar-targeted effectors remain elusive. In this study, we showed that Phytophthora infestans infection of Nicotiana benthamiana results in nucleolar inflation during the transition from the biotrophic to the necrotrophic phase. Multiple P. infestans effectors were localized in the nucleolus: Pi23226 induced cell death in N. benthamiana and nucleolar inflation similar to that observed in the necrotrophic stage of infection, whereas its homolog Pi23015 and a deletion mutant (Pi23226ΔC) did not induce cell death or affect nucleolar size. RNA immunoprecipitation and individual-nucleotide-resolution UV crosslinking and immunoprecipitation sequencing analysis indicated that Pi23226 bound to the 3' end of 25S rRNA precursors, resulting in accumulation of unprocessed 27S pre-rRNAs. The nucleolar stress marker NAC082 was strongly upregulated under Pi23226-expressing conditions. Pi23226 subsequently inhibited global protein translation in host cells by interacting with ribosomes. Pi23226 enhanced P. infestans pathogenicity, indicating that Pi23226-induced ribosome malfunction and cell death were beneficial for pathogenesis in the host. Our results provide evidence for the molecular mechanism underlying RNA-binding effector activity in host ribosome biogenesis and lead to new insights into the nucleolar action of effectors in pathogenesis.
TL;DR: In this article , the authors characterized Arabidopsis thaliana plants with disrupted NAS genes to modify accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression.
Abstract: In plant cells, a large pool of iron (Fe) is contained in the nucleolus, together with that in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by nicotianamine synthase (NAS). Here, we characterized Arabidopsis thaliana plants with disrupted NAS genes to modify accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contain lower quantities of the iron ligand NA, also contain less iron in the nucleolus. This is concurrent with the expression of normally silenced rRNA genes from Nucleolar Organizer Regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contain lower quantities of NA, nucleolar iron and rDNA expression are not affected. In contrast, in both nas124 and nas234, specific RNA modifications are differentially regulated in a genotype dependent manner. Taken together the data point out the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.
TL;DR: In this article , the authors show that nuclear ribopuromycylation is enhanced in malignant cancer cells and is associated with rapid cell growth, which suggests that cancer cells might have adapted a mechanism of nuclear translation to support their need for rapid growth and might also open up new possibilities for therapeutic targeting of cancerspecific cellular functions.
Abstract: Abstract Recent technological advances have re‐invigorated the interest in nuclear translation (NT), but the underlying mechanisms and functional implications of NT remain unknown. Here we show that NT is enhanced in malignant cancer cells and is associated with rapid cell growth. Nuclear ribopuromycylation analyses in a panel of diverse cell lines revealed that NT is scarce in normal immortalized cells, but is ubiquitous and robust in malignant cancer cells. Moreover, NT occurs in the nucleolus and requires normal nucleolar function. Intriguingly, NT is reduced by cellular stresses and anti‐tumor agents and positively correlates with cancer cell proliferation and growth. By using a modified puromycin‐associated nascent chain proteomics, we further identified numerous oncoproteins that are preferentially translated in the nucleus, such as transforming growth factor‐beta 2 (TGFB2) and nucleophosmin 1 (NMP1). Specific overexpression of TGFB2 and NMP1 messenger RNAs in the nucleus can increase their protein levels and promote tumorigenesis. These findings establish a previously unknown link between NT and malignancy and suggest that cancer cells might have adapted a mechanism of NT to support their need for rapid growth, which highlight the potential of NT in tumorigenesis and might also open up new possibilities for therapeutic targeting of cancer‐specific cellular functions.
30 Mar 2023
TL;DR: In this paper , the role of ARF in restraining nucleolar ribosome production is poorly understood, and the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of deficient mouse cells is reported.
Abstract: <div>Abstract<p>The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. Although its activation of p53 has been well characterized, the role of ARF in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of <i>Arf</i>-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5, which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, <i>Arf</i>-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, because reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis. <i>Cancer Res; 71(21); 6708–17. ©2011 AACR</i>.</p></div>
TL;DR: In this article , the authors demonstrate that oxaliplatin-induced ribosomal RNA (rRNA) transcriptional silencing and nucleolar stress occur downstream of DNA damage signaling involving ATM and ATR.
Abstract: Platinum (Pt) compounds are an important class of anti-cancer therapeutics, but outstanding questions remain regarding their mode of action. In particular, emerging evidence indicates that oxaliplatin, a Pt drug used to treat colorectal cancer, kills cells by inducing ribosome biogenesis stress rather than through DNA damage generation, but the underlying mechanism is unknown. Here, we demonstrate that oxaliplatin-induced ribosomal RNA (rRNA) transcriptional silencing and nucleolar stress occur downstream of DNA damage signaling involving ATM and ATR. We show that NBS1 and TOPBP1, two proteins involved in the nucleolar DNA damage response (n-DDR), are recruited to nucleoli upon oxaliplatin treatment. However, we find that rRNA transcriptional inhibition by oxaliplatin does not depend upon NBS1 or TOPBP1, nor does oxaliplatin induce substantial amounts of nucleolar DNA damage, distinguishing it from previously characterized n-DDR pathways. Taken together, our work indicates that oxaliplatin induces a distinct DDR signaling pathway that functions in trans to inhibit Pol I transcription in the nucleolus, demonstrating how nucleolar stress can be linked to DNA damage signaling and highlighting an important mechanism of Pt drug cytotoxicity.