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

Out of the several hundred copies of 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 genes within each NOR in mammals is yet to be determined.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

The shared genomic architecture of human nucleolar organizer regions.

The short arms of the five acrocentric human chromosomes harbor sequences that direct the assembly and function of the nucleolus, one of the key functional domains of the nucleus, yet they are absent from the current human genome assembly. Here we describe the genomic architecture of these human nucleolar organizers. Sequences distal and proximal to ribosomal gene arrays are conserved among the acrocentric chromosomes, suggesting they are sites of frequent recombination.

NOL11, implicated in the pathogenesis of North American Indian childhood cirrhosis, is required for pre-rRNA transcription and processing.

The fundamental process of ribosome biogenesis requires hundreds of factors and takes place in the nucleolus. This process has been most thoroughly characterized in baker's yeast and is generally well conserved from yeast to humans. However, some of the required proteins in yeast are not found in humans, raising the possibility that they have been replaced by functional analogs.

Replication timing of pseudo-NORs.

In mammalian cells, transcriptionally active ribosomal genes are replicated in the early S phase, and the silent ribosomal genes in the late S phase, though mechanisms of this timing remain unknown. UBF (Upstream Binding Factor), a DNA binding protein and component of the pol I transcription machinery, is considered to be responsible for the loose chromatin structure of the active rDNA. Here we question whether such structure alone can ensure early replication of DNA.

A novel small-subunit processome assembly intermediate that contains the U3 snoRNP, nucleolin, RRP5, and DBP4.

Eukaryotic 18S rRNA processing is mediated by the small subunit (SSU) processome, a machine comprised of the U3 small nucleolar RNP (U3 snoRNP), tUTP, bUTP, MPP10, and BMS1/RCL1 subcomplexes. We report that the human SSU processome is a dynamic structure with the recruitment and release of subcomplexes occurring during the early stages of ribosome biogenesis. A novel 50S U3 snoRNP accumulated when either pre-rRNA transcription was blocked or the tUTP proteins were depleted.