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.
Subject(s)
Eukaryota , RNA, Ribosomal , RNA, Ribosomal/genetics , Eukaryota/genetics , Genes, rRNA/genetics , DNA Copy Number Variations , Gene Dosage , DNA, Ribosomal/geneticsABSTRACT
Ribosome biogenesis is a complex and highly energy-demanding process that requires the concerted action of all three nuclear RNA polymerases (Pol I-III) in eukaryotes. The three largest ribosomal RNAs (rRNAs) originate from a precursor transcript (pre-rRNA) that is encoded by multicopy genes located in the nucleolus. Transcription of these rRNA genes (rDNA) by Pol I is the key regulation step in ribosome production and is tightly controlled by an intricate network of signaling pathways and epigenetic mechanisms. In this article, we give an overview of the composition of the basal Pol I machinery and rDNA chromatin. We discuss rRNA gene regulation in response to environmental signals and developmental cues and focus on perturbations occurring in diseases linked to either excessive or limited rRNA levels. Finally, we discuss the emerging view that rDNA integrity and activity may be involved in the aging process.
Subject(s)
RNA Polymerase I/genetics , RNA Polymerase I/metabolism , Aging/genetics , Aging/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Chromatin/genetics , Chromatin/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Epigenesis, Genetic , Humans , Models, Biological , Multigene Family , Neoplasms/genetics , Neoplasms/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Signal Transduction , Transcription, GeneticABSTRACT
Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of C/EBP alpha (CEBPA), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced RNA Pol I occupancy. Our work identifies numerous potential rRNA regulators and provides a template for dissection of TF roles in rRNA transcription.
Subject(s)
RNA Polymerase I , Transcription Factors , Humans , Mice , Animals , RNA Polymerase I/genetics , RNA Polymerase I/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , RNA, Ribosomal/genetics , Transcription, Genetic , DNA, Ribosomal/genetics , RNA , ChromatinABSTRACT
Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes â¼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.
Subject(s)
RNA Polymerase II , RNA Precursors , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , DNA, Intergenic , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Homeostasis/genetics , Macromolecular Substances/metabolism , RNA Polymerase I/genetics , RNA Polymerase I/metabolism , RNA Polymerase II/metabolism , RNA Precursors/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Transcription, GeneticABSTRACT
The short arms of the human acrocentric chromosomes 13, 14, 15, 21 and 22 (SAACs) share large homologous regions, including ribosomal DNA repeats and extended segmental duplications1,2. Although the resolution of these regions in the first complete assembly of a human genome-the Telomere-to-Telomere Consortium's CHM13 assembly (T2T-CHM13)-provided a model of their homology3, it remained unclear whether these patterns were ancestral or maintained by ongoing recombination exchange. Here we show that acrocentric chromosomes contain pseudo-homologous regions (PHRs) indicative of recombination between non-homologous sequences. Utilizing an all-to-all comparison of the human pangenome from the Human Pangenome Reference Consortium4 (HPRC), we find that contigs from all of the SAACs form a community. A variation graph5 constructed from centromere-spanning acrocentric contigs indicates the presence of regions in which most contigs appear nearly identical between heterologous acrocentric chromosomes in T2T-CHM13. Except on chromosome 15, we observe faster decay of linkage disequilibrium in the pseudo-homologous regions than in the corresponding short and long arms, indicating higher rates of recombination6,7. The pseudo-homologous regions include sequences that have previously been shown to lie at the breakpoint of Robertsonian translocations8, and their arrangement is compatible with crossover in inverted duplications on chromosomes 13, 14 and 21. The ubiquity of signals of recombination between heterologous acrocentric chromosomes seen in the HPRC draft pangenome suggests that these shared sequences form the basis for recurrent Robertsonian translocations, providing sequence and population-based confirmation of hypotheses first developed from cytogenetic studies 50 years ago9.
Subject(s)
Centromere , Chromosomes, Human , Recombination, Genetic , Humans , Centromere/genetics , Chromosomes, Human/genetics , DNA, Ribosomal/genetics , Recombination, Genetic/genetics , Translocation, Genetic/genetics , Cytogenetics , Telomere/geneticsABSTRACT
The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated "promoter and pre-rRNA antisense" RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.
Subject(s)
R-Loop Structures , Replication Protein A , Cell Nucleolus/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Humans , Multifunctional Enzymes , RNA Helicases/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Replication Protein A/genetics , Replication Protein A/metabolism , Transcription, GeneticABSTRACT
Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.
Subject(s)
RNA Polymerase III/genetics , RNA Polymerase II/genetics , RNA Polymerase I/genetics , RNA, Fungal/chemistry , Saccharomyces cerevisiae/genetics , Transcription Elongation, Genetic , Base Composition , Base Sequence , Binding Sites , Chromatin/chemistry , Chromatin/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Gene Expression Regulation, Fungal , Protein Binding , RNA Folding , RNA Polymerase I/metabolism , RNA Polymerase II/metabolism , RNA Polymerase III/metabolism , RNA Splice Sites , RNA Splicing , RNA, Fungal/genetics , RNA, Fungal/metabolism , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , ThermodynamicsABSTRACT
DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae, we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological-stress-linked damage in these regions. At cohesin-enriched centromeres, cohesin activity causes the accumulation of DNA damage, RF rotation, and pre-catenation, confirming that cohesin-dependent DNA topological stress impacts on normal replication progression. In contrast, at the rDNA, cohesin and condensin activity inhibit the repair of damage caused by DNA topological stress. We propose that, as well as generally acting to ensure faithful genetic inheritance, SMCs can disrupt genome stability by trapping DNA topological stress.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal , DNA Damage , DNA Replication , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , DNA-Binding Proteins/genetics , Multiprotein Complexes/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
DNA methylation is an important epigenetic mark implicated in selective rRNA gene expression, but the DNA methylation readers and effectors remain largely unknown. Here, we report a protein complex that reads DNA methylation to regulate variant-specific 45S ribosomal RNA (rRNA) gene expression in Arabidopsis (Arabidopsis thaliana). The complex, consisting of METHYL-CpG-BINDING DOMAIN PROTEIN5 (MBD5), MBD6, ALPHA-CRYSTALLIN DOMAIN PROTEIN15.5 (ACD15.5), and ACD21.4, directly binds to 45S rDNA. While MBD5 and MBD6 function redundantly, ACD15.5 and ACD21.4 are indispensable for variant-specific rRNA gene expression. These 4 proteins undergo phase separation in vitro and in vivo and are interdependent for their phase separation. The α-crystallin domain of ACD15.5 and ACD21.4, which is essential for their function, enables phase separation of the complex, likely by mediating multivalent protein interactions. The effector MICRORCHIDIA6 directly interacts with ACD15.5 and ACD21.4, but not with MBD5 and MBD6, and is recruited to 45S rDNA by the MBD-ACD complex to regulate variant-specific 45S rRNA expression. Our study reveals a pathway in Arabidopsis through which certain 45S rRNA gene variants are silenced, while others are activated.
Subject(s)
Arabidopsis Proteins , Arabidopsis , alpha-Crystallins , Arabidopsis/genetics , Arabidopsis/metabolism , Genes, rRNA , DNA Methylation/genetics , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , alpha-Crystallins/genetics , alpha-Crystallins/metabolismABSTRACT
Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNA's repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells' viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as 'rDNA magnification', has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary.
Subject(s)
DNA, Ribosomal , RNA Polymerase II , Transcription, Genetic , Animals , DNA, Ribosomal/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , DNA Breaks, Double-Stranded , Drosophila melanogaster/genetics , Sister Chromatid Exchange/genetics , Germ Cells/metabolism , DNA Copy Number VariationsABSTRACT
Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.
Subject(s)
DNA, Ribosomal , Ribosomes , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , DNA, Ribosomal/genetics , Ribosomes/metabolism , Ribosomes/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , RNA Polymerase I/genetics , RNA Polymerase I/metabolism , Gene Expression Regulation, Fungal , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Signal Transduction/genetics , Cell Cycle/genetics , Gene DeletionABSTRACT
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, â¼3 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.
Subject(s)
Chromosomes/chemistry , Chromosomes/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Nucleolus Organizer Region/chemistry , Nucleolus Organizer Region/genetics , Animals , Base Sequence , Cell Line , Conserved Sequence/genetics , Genetic Structures/genetics , Genetic Variation , Humans , Hybrid Cells , Mice , Pan troglodytes/geneticsABSTRACT
The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. DNA double-strand breaks (DSBs) within rDNA induce both rDNA transcriptional repression and nucleolar segregation, but the link between the two events remains unclear. Here we found that DSBs induced on rDNA trigger transcriptional repression in a cohesin- and HUSH (human silencing hub) complex-dependent manner throughout the cell cycle. In S/G2 cells, transcriptional repression is further followed by extended resection within the interior of the nucleolus, DSB mobilization at the nucleolar periphery within nucleolar caps, and repair by homologous recombination. We showed that nuclear envelope invaginations frequently connect the nucleolus and that rDNA DSB mobilization, but not transcriptional repression, involves the nuclear envelope-associated LINC complex and the actin pathway. Altogether, our data indicate that rDNA break localization at the nucleolar periphery is not a direct consequence of transcriptional repression but rather is an active process that shares features with the mobilization of persistent DSB in active genes and heterochromatin.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Breaks, Double-Stranded , DNA Repair/genetics , DNA, Ribosomal/genetics , Gene Expression Regulation/genetics , RNA, Long Noncoding/metabolism , Cell Nucleolus/metabolism , Histones/metabolism , Homologous Recombination/genetics , Nuclear Envelope/metabolism , CohesinsABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/metabolism , Anaphase/physiology , DNA Topoisomerases, Type II/metabolism , DNA, Ribosomal/metabolism , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Tankyrases/metabolism , Aneuploidy , Chromosome Segregation , DNA Damage/genetics , DNA, Ribosomal/genetics , Humans , Saccharomyces cerevisiae/geneticsABSTRACT
The rRNA genes [ribosomal DNA (rDNA)] are organized in a prominent nuclear compartment, the nucleolus. It is now well established that the nucleolus functions beyond ribosome biosynthesis, regulating several physiological cellular responses. The nucleoli constitute dynamic genomic/nuclear hubs and demonstrate unique inherent characteristics, rendering them ideal to sense, signal, and respond to various intrinsic and environmental insults. Here, we discuss emerging findings supporting direct links between rDNA/nucleolar instability and cellular senescence/organismal aging from yeast to mammals. Moreover, we highlight evidence that nucleolar functionality and rDNA architecture impact on meiotic/transgenerational rejuvenation, thus revealing causality underlying connections between rDNA/nucleolar instability and aging.
Subject(s)
Aging , Cell Nucleolus , Aging/genetics , Animals , Cell Nucleolus/genetics , Cellular Senescence , DNA, Ribosomal/genetics , Mammals , RNA, Ribosomal/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
The ribosomal DNA locus (rDNA) is central for the functioning of cells because it encodes ribosomal RNAs, key components of ribosomes, and also because of its links to fundamental metabolic processes, with significant impact on genome integrity and aging. The repetitive nature of the rDNA gene units forces the locus to maintain sequence homogeneity through recombination processes that are closely related to genomic stability. The co-presence of basic DNA transactions, such as replication, transcription by major RNA polymerases, and recombination, in a defined and restricted area of the genome is of particular relevance as it affects the stability of the rDNA locus by both direct and indirect mechanisms. This condition is well exemplified by the rDNA of Saccharomyces cerevisiae. In this review we summarize essential knowledge on how the complexity and overlap of different processes contribute to the control of rDNA and genomic stability in this model organism.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Genomic Instability/genetics , DNA Replication/geneticsABSTRACT
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 â¼2.2% 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.
Subject(s)
Chromosomes, Human , Nucleolus Organizer Region , Humans , Chromosomes, Human/genetics , Chromosomes , Cell Nucleolus/genetics , Centromere , DNA, Ribosomal/geneticsABSTRACT
The massive accumulation of extrachromosomal ribosomal DNA circles (ERCs) in yeast mother cells has been long cited as the primary driver of replicative ageing. ERCs arise through ribosomal DNA (rDNA) recombination, and a wealth of genetic data connects rDNA instability events giving rise to ERCs with shortened life span and other ageing pathologies. However, we understand little about the molecular effects of ERC accumulation. Here, we studied ageing in the presence and absence of ERCs, and unexpectedly found no evidence of gene expression differences that might indicate stress responses or metabolic feedback caused by ERCs. Neither did we observe any global change in the widespread disruption of gene expression that accompanies yeast ageing, altogether suggesting that ERCs are largely inert. Much of the differential gene expression that accompanies ageing in yeast was actually associated with markers of the senescence entry point (SEP), showing that senescence, rather than age, underlies these changes. Cells passed the SEP irrespective of ERCs, but we found the SEP to be associated with copy number amplification of a region of chromosome XII between the rDNA and the telomere (ChrXIIr) forming linear fragments up to approximately 1.8 Mb size, which arise in aged cells due to rDNA instability but through a different mechanism to ERCs. Therefore, although rDNA copy number increases dramatically with age due to ERC accumulation, our findings implicate ChrXIIr, rather than ERCs, as the primary driver of senescence during budding yeast ageing.
Subject(s)
Saccharomyces cerevisiae , Telomere , Saccharomyces cerevisiae/genetics , DNA, Ribosomal/genetics , Telomere/genetics , Endosomes , RibosomesABSTRACT
Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.
Subject(s)
Cell Nucleolus/enzymology , Cell Nucleolus/genetics , DNA, Ribosomal/genetics , RNA Polymerase II/metabolism , RNA, Untranslated/biosynthesis , RNA, Untranslated/genetics , Ribosomes/metabolism , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Cell Line, Tumor , Cell Nucleolus/physiology , DNA Helicases/metabolism , DNA, Intergenic/genetics , Humans , Multifunctional Enzymes/metabolism , Protein Biosynthesis , R-Loop Structures , RNA Helicases/metabolism , RNA Polymerase I/antagonists & inhibitors , RNA Polymerase I/metabolism , Ribonuclease H/metabolism , Ribosomes/chemistry , Ribosomes/genetics , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathologyABSTRACT
Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC formation is replicative, separable from repeat loss, and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Last, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.