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.

Transitions in the framework of condensate biology.

As phase separation is found in an increasing variety of biological contexts, additional challenges have arisen in understanding the underlying principles of condensate formation and function. We spoke with researchers across disciplines about their views on the ever-changing landscape of biomolecular condensates.

The p-Arms of Human Acrocentric Chromosomes Play by a Different Set of Rules.

The p-arms of the five human acrocentric chromosomes bear nucleolar organizer regions (NORs) comprising ribosomal gene (rDNA) repeats that are organized in a homogeneous tandem array and transcribed in a telomere-to-centromere direction. Precursor ribosomal RNA transcripts are processed and assembled into ribosomal subunits, the nucleolus being the physical manifestation of this process. I review current understanding of nucleolar chromosome biology and describe current exploration into a role for the NOR chromosomal context. Full DNA sequences for acrocentric p-arms are now emerging, aided by the current revolution in long-read sequencing and genome assembly. Acrocentric p-arms vary from 10.1 to 16.7 Mb, accounting for ∼2.2% of the genome. Bordering rDNA arrays, distal junctions, and proximal junctions are shared among the p-arms, with distal junctions showing evidence of functionality. The remaining p-arm sequences comprise multiple satellite DNA classes and segmental duplications that facilitate recombination between heterologous chromosomes, which is likely also involved in Robertsonian translocations.

The dual nature of the nucleolus.

The nucleolus is best known for housing the highly ordered assembly line that produces ribosomal subunits. The >100 ribosome assembly factors in the nucleolus are thought to cycle between two states: an operative state (when integrated into subunit assembly intermediates) and a latent state (upon release from intermediates). Although it has become commonplace to refer to the nucleolus as "being a multilayered condensate," and this may be accurate for latent factors, there is little reason to think that such assertions pertain to the operative state of assembly factors.

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.

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.

Human NORs, comprising rDNA arrays and functionally conserved distal elements, are located within dynamic chromosomal regions.

Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.

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.

Resolution of human ribosomal DNA occurs in anaphase, dependent on tankyrase 1, condensin II, and topoisomerase IIα.

Formation of individualized sister chromatids is essential for their accurate segregation. In budding yeast, while most of the genome segregates at the metaphase to anaphase transition, resolution of the ribosomal DNA (rDNA) repeats is delayed. The timing and mechanism in human cells is unknown. Here we show that resolution of human rDNA occurs in anaphase after the bulk of the genome, dependent on tankyrase 1, condensin II, and topoisomerase IIα. Defective resolution leads to rDNA bridges, rDNA damage, and aneuploidy of an rDNA-containing acrocentric chromosome. Thus, temporal regulation of rDNA segregation is conserved between yeast and man and is essential for genome integrity.

Integrating the genomic architecture of human nucleolar organizer regions with the biophysical properties of nucleoli.

Nucleoli are the sites of ribosome biogenesis and the largest membraneless subnuclear structures. They are intimately linked with growth and proliferation control and function as sensors of cellular stress. Nucleoli form around arrays of ribosomal gene (rDNA) repeats also called nucleolar organizer regions (NORs). In humans, NORs are located on the short arms of all five human acrocentric chromosomes. Multiple NORs contribute to the formation of large heterochromatin-surrounded nucleoli observed in most human cells. Here we will review recent findings about their genomic architecture. The dynamic nature of nucleoli began to be appreciated with the advent of photodynamic experiments using fluorescent protein fusions. We review more recent data on nucleoli in Xenopus germinal vesicles (GVs) which has revealed a liquid droplet-like behavior that facilitates nucleolar fusion. Further analysis in both XenopusGVs and Drosophila embryos indicates that the internal organization of nucleoli is generated by a combination of liquid-liquid phase separation and active processes involving rDNA. We will attempt to integrate these recent findings with the genomic architecture of human NORs to advance our understanding of how nucleoli form and respond to stress in human cells.

Nucleolar reorganization in response to rDNA damage.

Nucleoli, sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) comprising rDNA arrays, located on human acrocentric chromosome p-arms. NORs provide an opportunity to investigate the DNA double strand break (DSB) response at highly transcribed, repetitive, essential loci. Targeted introduction of DSBs into rDNA results in ATM-dependent inhibition of RNA-polymerase I transcription, coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors, normally excluded from nucleoli. Importantly, rDNA DSBs recruit the accurate homologous recombination (HR) repair machinery throughout the cell cycle, suggesting that HR can be templated in cis. We discuss recent findings regarding the biophysical properties of nucleoli and suggest a mechanism for stress-induced nucleolar reorganization.

The Relationship Between Human Nucleolar Organizer Regions and Nucleoli, Probed by 3D-ImmunoFISH.

3D-immunoFISH is a valuable technique to compare the localization of DNA sequences and proteins in cells where three-dimensional structure has been preserved. As nucleoli contain a multitude of protein factors dedicated to ribosome biogenesis and form around specific chromosomal loci, 3D-immunoFISH is a particularly relevant technique for their study. In human cells, nucleoli form around transcriptionally active ribosomal gene (rDNA) arrays termed nucleolar organizer regions (NORs) positioned on the p-arms of each of the acrocentric chromosomes. Here, we provide a protocol for fixing and permeabilizing human cells grown on microscope slides such that nucleolar proteins can be visualized using antibodies and NORs visualized by DNA FISH. Antibodies against UBF recognize transcriptionally active rDNA/NORs and NOP52 antibodies provide a convenient way of visualizing the nucleolar volume. We describe a probe designed to visualize rDNA and introduce a probe comprised of NOR distal sequences, which can be used to identify or count individual NORs.

Nucleolar organizer regions: genomic 'dark matter' requiring illumination.

Nucleoli form around tandem arrays of a ribosomal gene repeat, termed nucleolar organizer regions (NORs). During metaphase, active NORs adopt a characteristic undercondensed morphology. Recent evidence indicates that the HMG-box-containing DNA-binding protein UBF (upstream binding factor) is directly responsible for this morphology and provides a mitotic bookmark to ensure rapid nucleolar formation beginning in telophase in human cells. This is likely to be a widely employed strategy, as UBF is present throughout metazoans. In higher eukaryotes, NORs are typically located within regions of chromosomes that form perinucleolar heterochromatin during interphase. Typically, the genomic architecture of NORs and the chromosomal regions within which they lie is very poorly described, yet recent evidence points to a role for context in their function. In Arabidopsis, NOR silencing appears to be controlled by sequences outside the rDNA (ribosomal DNA) array. Translocations reveal a role for context in the expression of the NOR on the X chromosome in Drosophila Recent work has begun on characterizing the genomic architecture of human NORs. A role for distal sequences located in perinucleolar heterochromatin has been inferred, as they exhibit a complex transcriptionally active chromatin structure. Links between rDNA genomic stability and aging in Saccharomyces cerevisiae are now well established, and indications are emerging that this is important in aging and replicative senescence in higher eukaryotes. This, combined with the fact that rDNA arrays are recombinational hot spots in cancer cells, has focused attention on DNA damage responses in NORs. The introduction of DNA double-strand breaks into rDNA arrays leads to a dramatic reorganization of nucleolar structure. Damaged rDNA repeats move from the nucleolar interior to form caps at the nucleolar periphery, presumably to facilitate repair, suggesting that the chromosomal context of human NORs contributes to their genomic stability. The inclusion of NORs and their surrounding chromosomal environments in future genome drafts now becomes a priority.

A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage.

DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.

Construction of synthetic nucleoli and what it tells us about propagation of sub-nuclear domains through cell division.

The cell nucleus is functionally compartmentalized into numerous membraneless and dynamic, yet defined, bodies. The cell cycle inheritance of these nuclear bodies (NBs) is poorly understood at the molecular level. In higher eukaryotes, their propagation is challenged by cell division through an "open" mitosis, where the nuclear envelope disassembles along with most NBs. A deeper understanding of the mechanisms involved can be achieved using the engineering principles of synthetic biology to construct artificial NBs. Successful biogenesis of such synthetic NBs demonstrates knowledge of the basic mechanisms involved. Application of this approach to the nucleolus, a paradigm of nuclear organization, has highlighted a key role for mitotic bookmarking in the cell cycle propagation of NBs.

Construction of synthetic nucleoli in human cells reveals how a major functional nuclear domain is formed and propagated through cell division.

Human cell nuclei are functionally organized into structurally stable yet dynamic bodies whose cell cycle inheritance is poorly understood. Here, we investigate the biogenesis and propagation of nucleoli, sites of ribosome biogenesis and key regulators of cellular growth. Nucleolar and cell cycles are intimately connected. Nucleoli disappear during mitosis, reforming around prominent uncharacterized chromosomal features, nucleolar organizer regions (NORs). By examining the effects of UBF depletion on both endogenous NORs and synthetic pseudo-NORs, we reveal its essential role in maintaining competency and establishing a bookmark on mitotic NORs. Furthermore, we demonstrate that neo-NORs, UBF-binding site arrays coupled with rDNA transcription units, direct the de novo biogenesis of functional compartmentalized neonucleoli irrespective of their site of chromosomal integration. For the first time, we establish the sequence requirements for nucleolar biogenesis and provide proof that this is a staged process where UBF-dependent mitotic bookmarking precedes function-dependent nucleolar assembly.

Structure-function analysis of Hmo1 unveils an ancestral organization of HMG-Box factors involved in ribosomal DNA transcription from yeast to human.

Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of ∼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.