scholarly journals Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells

2020 ◽  
Author(s):  
Eriko Watada ◽  
Sihan Li ◽  
Yutaro Hori ◽  
Katsunori Fujiki ◽  
Katsuhiko Shirahige ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Copy Number ◽  
Mammalian Cells ◽  
Bone Marrow Cells ◽  
Budding Yeast ◽  
Rdna Repeat ◽  
Age Dependent ◽  
Old Mice ◽  
Rdna Copy ◽  
Rdna Copy Number

AbstractThe ribosomal RNA gene, which consists of tandem repetitive arrays (rDNA repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in ageing mammalian cells. Using quantitative analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations among the repeats with two mutations being unique to old mice. These sequences were conserved in budding yeast and equivalent mutations shortened the yeast chronological lifespan. Our findings suggest that rDNA is also fragile in mammalian cells and alterations within this region have a profound effect on cellular function.Author SummaryThe ribosomal RNA gene (rDNA) is one of the most unstable regions in the genome due to its tandem repetitive structure. rDNA copy number in the budding yeast increases and becomes unstable as the cell ages. It is speculated that the rDNA produces an “aging signal” inducing senescence and death. However, it is unclear how the rDNA repeat changes during the aging process in mammalian cells. In this study, we attempted to identify the age-dependent alteration of rDNA in mice. Using quantitative single cell analysis, we show that rDNA copy number increases in old mice bone marrow cells. By contrast, the level of ribosomal RNA production was reduced because of increased levels of DNA methylation that represses transcription. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice and two of them were found in the conserved region in budding yeast. We then established a yeast strain with the old mouse-specific mutations and found this shortened the lifespan of the cells. These findings suggest that rDNA is also fragile in mammalian cells and alteration to this region of the genome affects cellular senescence.

scholarly journals Age-Dependent Ribosomal DNA Variations in Mice

2020 ◽  
Vol 40 (22) ◽  
Author(s):  
Eriko Watada ◽  
Sihan Li ◽  
Yutaro Hori ◽  
Katsunori Fujiki ◽  
Katsuhiko Shirahige ◽  
...  
Keyword(s):  
Ribosomal Dna ◽  
Copy Number ◽  
Mammalian Cells ◽  
Bone Marrow Cells ◽  
Budding Yeast ◽  
Mouse Strains ◽  
Rrna Gene ◽  
Rdna Repeat ◽  
Age Dependent ◽  
Old Mice

ABSTRACT The rRNA gene, which consists of tandem repetitive arrays (ribosomal DNA [rDNA] repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast Saccharomyces cerevisiae is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in aging mammalian cells. Using quantitative single-cell analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to levels in 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice, and two of them were found in the conserved region in budding yeast. We established yeast strains with the old-mouse-specific mutations and found that they shortened the life span of the cells. Our findings suggest that rDNA is also fragile in mammalian cells and that alterations within this region have a profound effect on cellular function.

scholarly journals Transcription of ribosomal genes can cause nondisjunction

2006 ◽  
Vol 173 (6) ◽  
pp. 893-903 ◽  
Author(s):  
Felix Machín ◽  
Jordi Torres-Rosell ◽  
Giacomo De Piccoli ◽  
Jesús A. Carballo ◽  
Rita S. Cha ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Copy Number ◽  
Ribosomal Genes ◽  
Rrna Genes ◽  
Normal Length ◽  
Very High ◽  
Rdna Copy ◽  
Rdna Copy Number ◽  
Genetic Rearrangements ◽  
Chromosome Nondisjunction

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction.

scholarly journals Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research

2020 ◽  
Vol 8 (3) ◽  
pp. 316 ◽  
Author(s):  
Yurui Wang ◽  
Yaohan Jiang ◽  
Yongqiang Liu ◽  
Yuan Li ◽  
Laura A. Katz ◽  
...  
Keyword(s):  
Copy Number Variation ◽  
Ribosomal Dna ◽  
Copy Number ◽  
Sequence Variation ◽  
Small Subunit ◽  
Microbial Eukaryotes ◽  
Number Variation ◽  
Small Subunit Ribosomal Dna ◽  
Rdna Copy ◽  
Rdna Copy Number

While nuclear small subunit ribosomal DNA (nSSU rDNA) is the most commonly-used gene marker in studying phylogeny, ecology, abundance, and biodiversity of microbial eukaryotes, mitochondrial small subunit ribosomal DNA (mtSSU rDNA) provides an alternative. Recently, both copy number variation and sequence variation of nSSU rDNA have been demonstrated for diverse organisms, which can contribute to misinterpretation of microbiome data. Given this, we explore patterns for mtSSU rDNA among 13 selected ciliates (representing five classes), a major component of microbial eukaryotes, estimating copy number and sequence variation and comparing to that of nSSU rDNA. Our study reveals: (1) mtSSU rDNA copy number variation is substantially lower than that for nSSU rDNA; (2) mtSSU rDNA copy number ranges from 1.0 × 104 to 8.1 × 105; (3) a most common sequence of mtSSU rDNA is also found in each cell; (4) the sequence variation of mtSSU rDNA are mainly indels in poly A/T regions, and only half of species have sequence variation, which is fewer than that for nSSU rDNA; and (5) the polymorphisms between haplotypes of mtSSU rDNA would not influence the phylogenetic topology. Together, these data provide more insights into mtSSU rDNA as a powerful marker especially for microbial ecology studies.

scholarly journals The Shu complex, which contains Rad51 paralogues, promotes DNA repair through inhibition of the Srs2 anti-recombinase

2011 ◽  
Vol 22 (9) ◽  
pp. 1599-1607 ◽  
Author(s):  
Kara A. Bernstein ◽  
Robert J.D. Reid ◽  
Ivana Sunjevaric ◽  
Kimberly Demuth ◽  
Rebecca C. Burgess ◽  
...  
Keyword(s):  
Copy Number ◽  
Dna Helicase ◽  
Double Strand Breaks ◽  
General Function ◽  
Rdna Transcription ◽  
Strand Breaks ◽  
Important Regulator ◽  
Error Prone Repair ◽  
Rdna Copy ◽  
Rdna Copy Number

The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.

scholarly journals Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error

2017 ◽  
Vol 284 (1859) ◽  
pp. 20170425 ◽  
Author(s):  
Chundi Wang ◽  
Tengteng Zhang ◽  
Yurui Wang ◽  
Laura A. Katz ◽  
Feng Gao ◽  
...  
Keyword(s):  
Single Cell ◽  
Copy Number ◽  
Sequence Variation ◽  
Ssu Rdna ◽  
Copy Numbers ◽  
Number Variation ◽  
The Impact ◽  
Rdna Copy ◽  
Rdna Polymorphism ◽  
Rdna Copy Number

Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: Halteria grandinella , Blepharisma americanum and Strombidium stylifer . We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of Halteria grandinella is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size.

scholarly journals Gene dosage compensation of rRNA transcript levels in Arabidopsis thaliana lines with reduced ribosomal gene copy number

2021 ◽  
Author(s):  
Francesca B Lopez ◽  
Antoine Fort ◽  
Luca Tadini ◽  
Aline V Probst ◽  
Marcus McHale ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Genome Editing ◽  
Dosage Compensation ◽  
Copy Number ◽  
Gene Dosage ◽  
Rrna Genes ◽  
Gene Copy ◽  
Transcript Levels ◽  
Rdna Copy ◽  
Rdna Copy Number

Abstract The 45S rRNA genes (rDNA) are amongst the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as ‘back-up’ copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte we reduced 45S rDNA copy number to a plateau of ∼10%. Two independent lines had rDNA copy numbers reduced by up to 90% at the T7 generation, named Low Copy Number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates and steady-state levels remained the same as wild type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. While overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA copy number on genome stability, development, and cellular processes.

scholarly journals 53BP1 mediated recruitment of RASSF1A at ribosomal DNA breaks facilitates local ATM signal amplification

2021 ◽  
Author(s):  
Stavroula Tsaridou ◽  
Georgia Velimezi ◽  
Frances Willenbrock ◽  
Maria Chatzifrangkeskou ◽  
Andreas Panagopoulos ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Viability ◽  
Copy Number ◽  
Adaptor Proteins ◽  
Fragile Sites ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Damage Response ◽  
Rdna Copy ◽  
Rdna Copy Number

DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a disproportionally greater impact on the overall genome stability. The ribosomal RNA gene repeats (rDNA) are emerging fragile sites due to repetitive nature, clustering and high transcriptional activity. Recent progress in understanding how the rDNA damage response is organized has highlighted the key role of adaptor proteins in the response. Here we identify that the scaffold and tumor suppressor, RASSF1A is recruited at sites of damage and enriched at rDNA breaks. Employing targeted nucleolar DNA damage, we find that RASSF1A recruitment requires ATM activity and depends on 53BP1. At sites of damage RASSF1A facilitates local ATM signal establishment and rDNA break repair. RASSF1A silencing, a common epigenetic event during malignant transformation, results in persistent breaks, rDNA copy number alterations and decreased cell viability. Meta-analysis of a lung adenocarcinoma cohort showed that RASSF1A epigenetic silencing leads in rDNA copy number discrepancies. Overall, we present evidence that RASSF1A acts as a DNA repair factor and offer mechanistic insight in how the nucleolar DNA damage response is organized.

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