Monoallelically expressed noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression.

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.

Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli.

Although they are organelles without a limiting membrane, nucleoli have an exclusive structure, built upon the rDNA-rich acrocentric short arms of five human chromosomes (nucleolar organizer regions or NORs). This has raised the question: what are the structural features of a chromosome required for its inclusion in a nucleolus? Previous work has suggested that sequences adjacent to the tandemly repeated rDNA repeat units (DJ, distal junction sequence) may be involved, and we have extended such studies by addressing several issues related to the requirements for the association of NORs with nucleoli. We exploited both a set of somatic cell hybrids containing individual human acrocentric chromosomes and a set of Human Artificial Chromosomes (HACs) carrying different parts of a NOR, including an rDNA unit or DJ or PJ (proximal junction) sequence. Association of NORs with nucleoli was increased when constituent rDNA was transcribed and may be also affected by the status of heterochromatin blocks formed next to the rDNA arrays. Furthermore, our data suggest that a relatively small size DJ region, highly conserved in evolution, is also involved, along with the rDNA repeats, in the localization of p-arms of acrocentric chromosomes in nucleoli. Thus, we infer a cooperative action of rDNA sequence-stimulated by its activity-and sequences distal to rDNA contributing to incorporation into nucleoli. Analysis of NOR sequences also identified LncRNA_038958 in the DJ, a candidate transcript with the region of the suggested promoter that is located close to the DJ/rDNA boundary and contains CTCF binding sites. This LncRNA may affect RNA Polymerase I and/or nucleolar activity. Our findings provide the basis for future studies to determine which RNAs and proteins interact critically with NOR sequences to organize the higher-order structure of nucleoli and their function in normal cells and pathological states.

Regulatory roles of nucleolus organizer region-derived long non-coding RNAs.

The nucleolus is the largest sub-nuclear domain, serving primarily as the place for ribosome biogenesis. A delicately regulated function of the nucleolus is vital to the cell not only for maintaining proper protein synthesis but is also tightly associated with responses to different types of cellular stresses. Recently, several long non-coding RNAs (lncRNAs) were found to be part of the regulatory network that modulate nucleolar functions. Several of these lncRNAs are encoded in the ribosomal DNA (rDNA) repeats or are transcribed from the genomic regions that are located near the nucleolus organizer regions (NORs). In this review, we first discuss the current understanding of the sequence of the NORs and variations between different NORs. We then focus on the NOR-derived lncRNAs in mammalian cells and their functions in rRNA transcription and the organization of nucleolar structure under different cellular conditions. The identification of these lncRNAs reveals great potential of the NORs in harboring novel genes involved in the regulation of nucleolar functions.

Determination of the relationship among cell proliferation, metabolic activity and stage of pregnancy by AgNORs as markers in bovine placenta / Determinación de la relación entre la proliferación celular, la actividad metabólica y la etapa de preñez por los AgNOR como marcadores en la placenta bovina

SUMMARY: Nucleolus Organizer Regions (NORs) are defined as nucleolar components containing argyrophilic proteins selectively stained by silver methods (AgNORs). Several investigations have shown the AgNOR quantity and area represent a valuable parameter of cell kinetics, since they reflect the level of activity and cellular proliferation. This article addresses an evaluation of the functional activity and relation between days of pregnancy and proliferative capacity of trophoblastic mononucleate and binucleate cells from bovine placentomes. Both the number and size of AgNORs were determined in different phases of gestation by silver nitrate staining in conventional histological slides. The results showed a significant increase (from 1 to 12 AgNORs) in the number of AgNORS per trophoblastic mononucleate cell in the 3rd trimester, with predominance of 4-6 AgNORs/cell. In the 1st and 2nd trimesters, the number ranged between 1 and 9 AgNORs/cell, with predominance of 1-3 AgNORs. No significant differences were observed between the 2nd and 3rd trimesters, but in the first, in binucleate cells (19-27 and 10-18 AgNORs/cell, respectively) - this number was higher than the one registered in trophoblastic mononucleate cells in the same period. Thus, AgNORs can be used as markers of the proliferative placental cell cycle and established a relation between number of AgNORs and days of gestation. This relation can be used for diagnoses and prognoses of several placental pathologies, including pregnancy losses from manipulated embryos.

RESUMEN: Las Regiones Organizadoras de Nucléolos (NOR) se definen como componentes nucleolares que contienen proteínas argirofílicas teñidas selectivamente por métodos de plata (AgNOR). Varias investigaciones han demostrado que la cantidad y el área de AgNOR representan un parámetro importante de la cinética celular, ya que reflejan el nivel de actividad y proliferación celular. Este trabajo analiza la actividad funcional y la relación entre los días de preñez y la capacidad proliferativa de las células trofoblásticas mononucleadas y binucleadas de placentomas bovinos. Tanto el número como el tamaño de los AgNOR se determinaron en diferentes fases de la gestación mediante tinción con nitrato de plata en portaobjetos histológicos convencionales. Los resultados mostraron un aumento significativo (de 1 a 12 AgNOR) en el número de AgNORS por célula mononucleada trofoblástica en el tercer trimestre, con predominio de 4-6 AgNOR / célula. En el primer y segundo trimestre, el número osciló entre 1 y 9 AgNOR / célula, con predominio de 1-3 AgNOR. No se observaron diferencias significativas entre el 2do y 3er trimester; en el primer trimestre, en células binucleadas (19-27 y 10-18 AgNORs / célula, respectivamente) - este número fue superior a la cantidad registrada en células mononucleadas trofoblásticas en el mismo período. Por tanto, los AgNOR se pueden utilizar como marcadores del ciclo celular placentario proliferativo y se establece una relación entre el número de AgNOR y los días de gestación. Esta relación puede ser útil en el diagnóstico y pronóstico de varias patologías placentarias, incluidas las pérdidas de preñeces de embriones manipulados.

The nucleolus from a liquid droplet perspective.

The nucleolus is a membrane-less organelle sequestered from the nucleus by liquid droplet formation through a liquid-liquid phase separation (LLPS). It plays important roles in cell homoeostasis through its internal thermodynamic changes. Reversible nucleolar transitions between coalescence and dispersion are dependent on the concentrations, conformations and interactions of its molecular liquid droplet-forming components, including DNA, RNA and protein. The liquid droplet-like properties of the nucleolus enable its diverse dynamic roles. The liquid droplet formation mechanism, by which the nucleolus is sequestered from the nucleoplasm despite the absence of a membrane, explains a number of complex nucleolar functions.

Human nucleoli comprise multiple constrained territories, tethered to individual chromosomes.

It is unknown how ribosomal gene (rDNA) arrays from multiple chromosomal nucleolar organizers (NORs) partition within human nucleoli. Exploration of this paradigm for chromosomal organization is complicated by the shared DNA sequence composition of five NOR-bearing acrocentric chromosome p-arms. Here, we devise a methodology for genetic manipulation of individual NORs. Efficient "scarless" genome editing of rDNA repeats is achieved on "poised" human NORs held within monochromosomal cell hybrids. Subsequent transfer to human cells introduces "active" NORs yielding readily discernible functional customized ribosomes. We reveal that ribosome biogenesis occurs entirely within constrained territories, tethered to individual NORs inside a larger nucleolus.

Increased numbers of nucleoli in a genome-wide RNAi screen reveal proteins that link the cell cycle to RNA polymerase I transcription.

Nucleoli are dynamic nuclear condensates in eukaryotic cells that originate through ribosome biogenesis at loci that harbor the ribosomal DNA. These loci are known as nucleolar organizer regions (NORs), and there are 10 in a human diploid genome. While there are 10 NORs, however, the number of nucleoli observed in cells is variable. Furthermore, changes in number are associated with disease, with increased numbers and size common in aggressive cancers. In the near-diploid human breast epithelial cell line, MCF10A, the most frequently observed number of nucleoli is two to three per cell. Here, to identify novel regulators of ribosome biogenesis we used high-throughput quantitative imaging of MCF10A cells to identify proteins that, when depleted, increase the percentage of nuclei with ≥5 nucleoli. Unexpectedly, this unique screening approach led to identification of proteins associated with the cell cycle. Functional analysis on a subset of hits further revealed not only proteins required for progression through the S and G2/M phase, but also proteins required explicitly for the regulation of RNA polymerase I transcription and protein synthesis. Thus, results from this screen for increased nucleolar number highlight the significance of the nucleolus in human cell cycle regulation, linking RNA polymerase I transcription to cell cycle progression.

Reduced ribosomal DNA transcription in the prefrontal cortex of suicide victims: consistence of new molecular RT-qPCR findings with previous morphometric data from AgNOR-stained pyramidal neurons.

Prefrontal cortical regions play a key role in behavioural regulation, which is profoundly disturbed in suicide. The study was carried out on frozen cortical samples from the anterior cingulate cortex (dorsal and ventral parts, ACd and ACv), the orbitofrontal cortex (OFC), and the dorsolateral cortex (DLC) obtained from 20 suicide completers (predominantly violent) with unknown psychiatric diagnosis and 21 non-suicidal controls. The relative level of ribosomal RNA (rRNA) as a marker of the transcriptional activity of ribosomal DNA (rDNA) was evaluated bilaterally in prefrontal regions mentioned above (i.e. in eight regions of interest, ROIs) by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The overall statistical analysis revealed a decrease in rDNA activity in suicide victims versus controls, particularly in male subjects. Further ROI-specific post hoc analyses revealed a significant decrease in this activity in suicides compared to non-suicides in five ROIs. This effect was accentuated in the ACv, where it was observed bilaterally. Our findings suggest that decreased rDNA transcription in the prefrontal cortex plays an important role in suicide pathogenesis and corresponds with our previous morphometric analyses of AgNOR-stained neurons.

Argentic staining reveals changes in cerebellar tissue organisation by prenatal glucocorticoid administration in rats.

It was almost 150 years ago that Golgi revolutionised histology with silver-based stains. Major advances in knowledge of the nervous system became possible because of silver impregnations. Silver staining combined with classical histological staining, cytochemistry methods, and electron microscopy is useful for studying mechanisms and components at subcellular, cellular, and tissue levels. Despite the advantages of silver staining, its use has decreased over time. The aim of this work was to use argentic staining to study the cerebellar effects of controversial prenatal glucocorticoid (GC) therapy. At postnatal day 12 (P12), the cerebellum of corticosterone (CC)-treated rats impregnated with AgNOR staining exhibited diminished thickness of the external granule layer (EGL) and irregular Purkinje cell arrangement. There was a greater number of nucleoli and nucleolar organiser regions (NORs) in 24% of Purkinje cells. Cerebellar granule neuron progenitor (CGNP) cells of the EGL showed a decrease in cellular density (confirmed by proliferating cell nuclear antigen [PCNA] immunolocalization) and NORs. At postnatal day 6 (P6), the Golgi-Kopsch technique allowed us to observe disturbances in the distribution pattern of CGNP cells (during proliferation, migration, and differentiation) and premature growth of the Bergmann glia. Our findings reveal disturbances in the cerebellar development program with early cellular and tissue changes.

Nucleophosmin, a multifunctional nucleolar organizer with a role in DNA repair.

Nucleophosmin (NPM1) is a mostly nucleolar protein with crucial functions in cell growth and homeostasis, including regulation of ribosome biogenesis and stress response. Such multiple activities rely on its ability to interact with nucleic acids and with hundreds of proteins, as well as on a dynamic subcellular distribution. NPM1 is thus regulated by a complex interplay between localization and interactions, further modulated by post-translational modifications. NPM1 is a homopentamer, with globular domains connected by long, intrinsically disordered linkers. This configuration allows NPM1 to engage in liquid-liquid phase separation phenomena, which could underlie a key role in nucleolar organization. Here, we will discuss NPM1 conformational and functional versatility, emphasizing its emerging, and still largely unexplored, role in DNA damage repair. Since NPM1 is altered in a subtype of acute myeloid leukaemia (AML), we will also present ongoing research on the molecular mechanisms underlying its pathogenic role and potential NPM1-targeting therapeutic strategies.

Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin.

The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.

Does capsaicin have therapeutic benefits in human colon adenocarcinoma? Selection of the most reliable dose via AgNOR

Background/aim: To determine the effect of different doses of capsaicin on AgNOR protein synthesis in human colon adenocarcinoma derivate from colon cancer (Caco-2 cell). Materials and methods: In this experimental study, after the cultured of Caco-2 cell line, the cells are divided into 4 groups as control and different capsaicin exposed doses (25uµ, 50uµ, and 75uµ). Mean AgNOR number and total AgNOR area/nuclear area (TAA/NA) were calculated. Results: A significant differences were detected between control and capsaicin (50uµ) (P = 0.001), between control and capsaicin (75uµ) (P = 0.000), between capsaicin (25uµ) and capsaicin (50uµ) (P = 0.001) and between capsaicin (25uµ) and capsaicin (75uµ) (P = 0.000) for TAA/NA. Also, there were significant differences between control and capsaicin (50uµ) (P = 0.001), between control and capsaicin (75uµ) (P = 0.000), between capsaicin (25uµ) and capsaicin (50uµ) (P = 0.000) and between capsaicin (25uµ) and capsaicin (75uµ) (P = 0.000) for mean AgNOR number. Conclusion: A certain amount of capsaicin has a protective effect against colon adenocarcinoma and the dose concentrations are important for the most reliable treatment.

NORs on human acrocentric chromosome p-arms are active by default and can associate with nucleoli independently of rDNA.

Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.

Common Features of the Pericentromere and Nucleolus.

Both the pericentromere and the nucleolus have unique characteristics that distinguish them amongst the rest of genome. Looping of pericentromeric DNA, due to structural maintenance of chromosome (SMC) proteins condensin and cohesin, drives its ability to maintain tension during metaphase. Similar loops are formed via condensin and cohesin in nucleolar ribosomal DNA (rDNA). Condensin and cohesin are also concentrated in transfer RNA (tRNA) genes, genes which may be located within the pericentromere as well as tethered to the nucleolus. Replication fork stalling, as well as downstream consequences such as genomic recombination, are characteristic of both the pericentromere and rDNA. Furthermore, emerging evidence suggests that the pericentromere may function as a liquid-liquid phase separated domain, similar to the nucleolus. We therefore propose that the pericentromere and nucleolus, in part due to their enrichment of SMC proteins and others, contain similar domains that drive important cellular activities such as segregation, stability, and repair.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus.

Nucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1-100 carbon ions per point (about 0.3-30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.

Disruption of nuclear speckles reduces chromatin interactions in active compartments.

BACKGROUND: Nuclei of eukaryotes contain various higher-order chromatin architectures and nuclear bodies (NBs), which are critical for proper nuclear functions. Recent studies showed that active chromatin regions are associated with nuclear speckles (NSs), a type of NBs involved in RNA processing. However, the functional roles of NSs in 3D genome organization remain unclear. RESULTS: Using mouse hepatocytes as the model, we knocked down SRRM2, a core protein component scaffolding NSs, and performed Hi-C experiments to examine genome-wide chromatin interactions. We found that Srrm2 depletion disrupted the NSs and changed the expression of 1282 genes. The intra-chromosomal interactions were decreased in type A (active) compartments and increased in type B (repressive) compartments. Furthermore, upon Srrm2 knockdown, the insulation of TADs was decreased specifically in active compartments, and the most significant reduction occurred in A1 sub-compartments. Interestingly, the change of intra-TAD chromatin interactions upon Srrm2 depletion was not associated with the alteration of gene expression. CONCLUSIONS: We show that disruption of NSs by Srrm2 knockdown causes a global decrease in chromatin interactions in active compartments, indicating critical functions of NSs in the organization of the 3D genome.

Nucleolar DNA Double-Strand Break Responses Underpinning rDNA Genomic Stability.

Nucleoli, the sites of ribosome biogenesis, form around ribosomal gene (rDNA) arrays termed nucleolar organiser regions (NORs). These are the most transcriptionally active regions of the human genome and specialised responses have evolved to ensure their genomic stability. This review focuses on nucleolar responses to DNA double-strand breaks (DSBs) introduced into rDNA arrays using sequence-specific endonucleases, including CRISPR/Cas9. Repair of rDNA DSBs is predominantly carried out by the homology-directed repair (HDR) pathway that is facilitated by inhibition of transcription by RNA polymerase-I (Pol-I) and ensuing dramatic nucleolar reorganisation. Additionally, we review evidence that nucleoli can sense and respond to DSBs elsewhere in the genome.

Evaluation of cell proliferation in cystic lesions associated with impacted third molars.

The purpose of this study was to evaluate the metabolism and epithelial cell proliferation of odontogenic keratocyst (OKC), dentigerous cyst (DC), and unicystic ameloblastoma (UA) by quantifying the nucleolar organizing regions (AgNORs) and Ki-67 protein immunoexpression. Forty-eight cases (16 OKC, 16 DC, and 16 UA) were evaluated retrospectively. The metabolism and epithelial cell proliferation was measured by the Ki-67 positive cell percentage index and by the mean AgNOR count in each group. The Ki-67 and AgNOR counts were significantly higher in OKC comparing to the DC and UA (p < .001). Ki-67 positive cells were observed higher in suprabasal cell layers of OKC with uniform distribution, a few of them were predominantly observed in basal cell layer in DC and UA. The AgNOR count was significantly higher in the OKC basal cell layers and observed throughout the lining epithelium of DC and UA. Ki-67 and AgNOR reinforced the aggressive character of OKC, presenting high metabolism and cellular proliferation compared to DC and UA, possibly due to its more aggressive clinical behavior and high recurrence rate. RESEARCH HIGHLIGHTS: We evidence higher metabolism and epithelial cell proliferation in OKC when compared to UA and DC, supporting its aggressive aspect and its high rate of recurrence. OKC had intense and predominant labeling of Ki-67 on the suprabasal layer unlike UA and DC.

Structural features of two major nucleolar organizer regions (NORs), Nor-B1 and Nor-B2, and chromosome-specific rRNA gene expression in wheat.

The reference genome sequence of wheat 'Chinese Spring' (CS) is now available (IWGSC RefSeq v1.0), but the core sequences defining the nucleolar organizer regions (NORs) have not been characterized. We estimated that the total copy number of the rDNA units in the wheat genome is 11 160, of which 30.5%, 60.9% and 8.6% are located on Nor-B1 (1B), Nor-B2 (6B) and other NORs, respectively. The total length of the NORs is estimated to be 100 Mb, corresponding to approximately 10% of the unassembled portion of the genome not represented in RefSeq v1.0. Four subtypes (S1-S4) of the rDNA units were identified based on differences within the 3' external transcribed spacer regions in Nor-B1 and Nor-B2, and quantitative PCR indicated locus-specific variation in rDNA subtype contents. Expression analyses of rDNA subtypes revealed that S1 was predominantly expressed and S2 weakly expressed, in contrast to the relative abundance of rDNA subtypes in the wheat genome. These results suggest a regulation mechanism of differential rDNA expression based on sequence differences. S3 expression increased in the ditelosomic lines Dt1BL and Dt6BL, suggesting that S3 is subjected to chromosome-mediated silencing. Structural differences were detected in the regions surrounding the NOR among homoeologous chromosomes of groups 1 and 6. The adjacent regions distal to the major NORs were expanded compared with their homoeologous counterparts, and the gene density of these expanded regions was relatively low. We provide evidence that these regions are likely to be important for autoregulation of the associated major NORs as well as silencing of minor NORs.