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

The 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 Summary The 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.

1 Intriguingly, fob1 mutants have a stable rDNA copy number, and lifespan is extended 2 by ~60% compared to the wild-type strain (10)(11). An important factor in suppressing 3 rDNA copy-number change is Sir2, an NAD+-dependent protein deacetylase that is 4 conserved across all kingdoms of life. Interestingly, sir2 mutants of S. cerevisiae 5 display increased unequal sister chromatid recombination, and the rDNA copy number 6 frequently changes (7)(12). Moreover, the lifespan of the sir2 mutant is shortened to 7 approximately half that of the wild-type strain (13)(14). Taken together, these 8 observations suggest that rDNA instability (i.e. frequent copy number alteration) is 9 related to senescence (15). 10 11 In mammals, the rDNA structure is similar to that of yeast. However, the intergenic 12 spacer sequence (IGS) in mammalian cells is larger than in yeast ( Figure 1A) and is an 13 unstable region of the genome (16). The connection between aging and rDNA has been 14 suggested in several studies of tissues from dog, mouse and human (17) (18)  The previous qPCR and Southern analysis showed the copy number of the rDNA 6 tended to increase in older mice. We therefore speculated that the increased copy 7 number of rDNA might result in an elevated level of rDNA transcripts (rRNA). To test 8 this hypothesis, RNA was isolated using cells derived from young and old mice and the 9 level of 28S rRNA measured by RT qPCR. The values were normalized against the 10 transcripts of three housekeeping genes, Actb (actin, beta), B2M (beta-2 microglobulin), 11 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The results are shown in 12 Figure 3B. Although there was a tendency for the young mice cells to have more 28S 13 rRNA, the difference was not significant except for the results normalized against B2M. 14 It is possible that the housekeeping genes are also affected by age. In addition, most of 15 the 28S rRNA are thought to be included in the ribosomes that abundantly accumulate 16 in the cell. Therefore, we measured newly synthesized pre-matured 45S rRNA using a 17 probe that recognizes the promoter region and then calculated the ratio of matured to 18 pre-matured rRNA. As shown in Figure 3C, in BALB/c, the newly synthesized rRNA 19 ratio was reduced in the old mice. However, this difference was not as obvious in the 20 C57BL/6 mice. 21

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Transcription inactivation of rRNA gene in C57BL/6 mice was confirmed using the 23 psoralen crosslinking method (24). Psoralen intercalates into non-nucleosomal rDNA 24 copies that are actively transcribed more efficiently than those that are transcriptionally 25 inactive. Therefore, using this method, we can estimate the proportion of active rDNA 26 copies. Cells are treated with psolaren, UV crosslinked and the DNA isolated. After 27 digestion with AflIII the DNA was subjected to agarose gel electrophoresis. The results and similar results were obtained ( Figure 5C). These results confirmed that rDNA in the 23 old mice is more methylated than in the young mice. Taken together, our findings 24 suggest DNA methylation causes the reduced level of transcription of rDNA. 25 Finally, we determined the rDNA sequence in the young and old mice. Bone marrow 1 cells, including hematopoietic stem cells that produce leukocytes, erythrocytes and 2 platelets, are known to divide frequently. Thus, we speculated that mutations in the 3 older mice cells accumulate and affect the function of the ribosome causing aging 4 phenomena, such as slow growth and reduced viability. DNA from young and old mice 5 was isolated and the 18S, 5.8S and 28S genes PCR amplified for analysis by deep 6 sequencing. All of the reads were aligned and compared with the mouse reference 7 sequences (28) to identify mutation sites. The results are shown in Figure 6A-C. The 8 "mutation rate" is the ratio of mutations identified in the sequences to the total reads. 9 Thus, a "mutation rate of 1 (100%)" means the sequence is different from the reference 10 sequence. If the mutation rate is 0.5 (50%), half of the rDNA copies display a variation 11 at that site. As a control, we also analyzed a housekeeping gene ATP5b (ATP synthase 12 gene) ( Figure 6D). 13 14 As shown in Figure 6, the overlapping black and yellow marks indicate the mutation 15 rates in the young and old mice cells were similar. The average mutation rates in both 16 young and old mice cells were similar ( Figure S3 and S4E). Thus, any age-dependent 17 alteration of rDNA sequence was not immediately apparent. Nonetheless, the average 18 mutation rate of 28S rDNA (BA:0.00341, BL:0.00321) was higher than that of 18S 19 rDNA (0.00236, 0.00222) and much higher than ATP5b and 5.8S (0.00054~0.00066). 20 Indeed, sequence variation among copies of 28S rDNA has been reported previously 21 (29). All of the high rate variations in 28S and 18S rDNA were found in DNA from 22 both young and old mice ( Figure 6). 23

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For the purpose of identifying old mice-specific mutations, we searched for variations 25 with a mutation rate of >0.0028 (0.28%), which was equivalent to the maximum value identified three old mice-specific mutations in the old BALB/cA strain (Table 1). By 1 contrast, no old mice-specific mutations were identified in the C57BL/6 strain. Indeed, 2 no old mice-specific variations were found after increasing the number of mice that 3 were sequenced ( Figure S4). 4 5 Accuracy of the sequencing data was verified by analyzing variation of the BamHI 6 recognition sequence that was detected in Figure 2 and Figure 5. The anticipated 7 variation in the sequencing data corresponding to the BamHI site (GGATCC) in both 8 mouse strains was observed together with the changes seen in the old BALB/cA mice 9 (0.25 to 0.685) (Table S1). Thus, the sequencing data correlate with the Southern 10 analysis in which the intensities of the upper bands increased in the old BALB/cA mice 11 ( Figure 2B). 12

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The old mouse-specific mutations of rDNA affect yeast ribosomal function 14

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To analyze the relationship between rDNA variation and function, we summed up the 16 mutation rates in 20 bp windows and plotted the values (Figure 7). In the graph, several 17 variations, or "hotspots", were identified over the 28S rDNA. Interestingly, most of the 18 hotspots (highlighted in yellow) were located in the non-conserved regions between 19 mouse and budding yeast rDNA (red line, top). These observations suggest that most of 20 the variations are present in the non-functional region of the 28S rRNA gene. 21 We also mapped the positions of the three old mouse-specific mutations identified in 23 the BALB/cA mice to yeast 25S rDNA. Interestingly, two sites (3291 and 4614) were 24 plotted in the conserved region between mouse and yeast, suggesting they might be 25 located in the functional domains in the rRNA. One approach to study the consequence 26 of these mutations is to examine their impact in yeast. Thus, we generated budding 27 yeast strains carrying the corresponding mutations in the 25S rDNA. For specific expression of the mutated rDNA, we used a yeast strain without rDNA in the 1 chromosome (rdn strain) (30). The strain initially carried a helper rDNA plasmid, 2 which was then shuffled with plasmids containing mutations in the 25S region. The 3 plasmid-borne mutated rDNA thus became the sole source of rRNA. Strains with either 4 plasmid-derived wild-type rDNA, A2131G (mouse A3291G), or A3295G (mouse 5 A4614G) mutated rDNA showed comparable cell growth in both solid and liquid 6 medium. To test the relationship between these mutations and senescence, we measured 7 the chronological lifespan by calculating survival rates every two days after the cells 8 entered the stationary phase. As shown in Figure 8, one of the mutations (A3295G) 9 lowered the proportion of surviving cells at all time points from day 5 onwards, 10 indicating a shortened chronological lifespan. By contrast, another mutant (A2131G) 11 showed similar survival rates to that of the wild-type yeast until day 15, but then the 12 rate dropped on day 17. These observations suggest that although both mutations 13 identified in the old mouse rDNA support cell growth in yeast, they may be harmful 14 during chronological aging, particularly A3295G (mouse A4614G). The rDNA has the following unique features that make it possible to monitor age-19 dependent alterations in the genome. Firstly, because rDNA is a highly repetitive and 20 recombinogenic region it is easy to assess instability by monitoring alterations in copy 21 number. Secondly, as approximately half of the rDNA copies are not transcribed 22 (24)(31), these repetitive non-transcribed regions are targets for both methylation (32) 23 and mutation (33). Indeed, our analyses detected alterations in copy number and 24 methylation level in old mice, as well as putative old mouse-specific mutations. 25 observed by many researchers, but in some reports the number goes up and in others it 1 goes down. Moreover, copy number alteration has also been observed in tissues (18). 2 Some of these discrepancies may arise from problems related to hybridization during 3 Southern blot analysis. The repetitive nature of the DNA combined with the high level 4 of bound proteins from the nucleolus may affect the detection efficiency. Indeed, our 5 results showed that although the rDNA copy number in old mice increased as detected 6 by single cell analysis by qPCR, this increase was not obvious by Southern blot analysis 7 in either of the two mouse strains (Figure 1 and Figure 2). For budding yeast, rDNA 8 copies in the old cells dramatically increases (~10 times) as extra chromosomal rDNA 9 circles (ERC) and their presence is a big burden on the cells because ERCs consume 10 factors that are required for chromosome maintenance (34)(35). Therefore, the copious 11 amount of ERC is thought to be a passive accelerator of cellular aging. In the case of 12 mammals, this age-dependent increase of rDNA copies is not as dramatic (< 2 times, 13 In terms of genome instability, it may be possible to connect age-dependent changes in 17 rDNA to the aging process. To address this issue, we previously established a strain of 18 see (37)(38)). A similar fork arrest induces rDNA instability to promote senescence by 1 distributing the aging signal. Further studies are required to investigate this hypothesis. 2 3 In the single cell analysis, we found that the copy number of rDNA increased and the 4 variation decreased in older cells. As far as we are aware, there is no previous report 5 showing alteration of rDNA copy number at the single cell level. One possible reason to 6 explain the reduced variation phenotype in the old cells is that the number of stem cells 7 for bone marrow goes down with age. Indeed, it is known that the number of 8 the hematopoietic stem cells in the bone marrow gradually decreases during the process 9 of aging (39). As bone marrow cells are produced from the stem cells, the variation of 10 rDNA copies is reduced. 11

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The relationship between rDNA methylation and senescence has been discussed in 13 previous reports (25)(26). The present results are consistent with these previous studies 14 in showing that rDNA is more highly methylated in older mice ( Figure 5). DNA 15 methylation is known to repress transcriptional activity (25). Indeed, the ratio of 45S to 16 28S transcripts reduced in the old BALB/cA mice. The underlying reason for the age-17 dependent increase in methylation has not been elucidated. However, repetitive non-18 coding elements, such as retrotransposons, are known targets for DNA methylation 19 enzymes (32). In addition, rDNA is subject to DNA damage and has a high GC content, 20 which are known to be related to age-dependent methylation (40)(41). Hence, a similar 21 mechanism may recognize the repetitive rDNA as a target for methylation. Moreover, in 22 terms of the relationship between reduced rDNA transcription and increased copy 23 number in old cells, one possible explanation is that cells can compensate for lowering 24 the production of rRNA by elevated copies of rDNA to enable them to survive. As a 25 result, the rDNA copy number in the old mice is more than in the young mice. 26 We anticipated more mutations in the older mice because there are many untranscribed 1 non-canonical rDNA copies (22) and hematopoietic stem cells are subject to DNA 2 replication stress (23). The untranscribed copies can accumulate mutations and 3 replication stress increases DNA damage. However, the mutation rate in old mice was 4 similar to that in young mice ( Figure S3 and S4E). Therefore, cells should have an 5 effective repair system and/or mechanism to avoid mutation accumulation such as gene 6 conversion for homogenization (33). In this study, we identified three such mutations in 7 the old mice. Although these mutations were present only in the old mice, it is not 8 known whether they occurred during the aging process. Moreover, it is not known 9 whether the rDNA copies with the mutation are actually transcribed or not. Thus, these 10 mutations may not be related to senescence in the mice. Nonetheless, we found that two 11 equivalent mutations in the budding yeast permitted cell growth, but one of the 12 mutations (A3295G) apparently shortened the chronological lifespan. These findings 13 indicate that the mutated rDNA, when present as the only source of rRNA, is 14 transcribed and can support the essential functions of the ribosome, but viability during 15 aging is negatively impacted, at least in yeast. Therefore, one could infer that if such 16 harmful mutations accumulate in the rDNA repeats during the course of successive cell 17 divisions, they may cause defects in the ribosomal and cellular functions to induce 18 senescence. 19 20 In this study, we used two mice strains, BALB/cA and C57BL/6, for the analyses and 21 they showed slightly different results. The rDNA copy number in C57BL/6 is twice as 22 large as that in BALB/cA. Age-dependent alterations in the copy number, transcription 23 and methylation levels were more prominent in BALB/cA. The mutation rate in 24 BALB/cA was also higher than that in C57BL/6 and we were only able to identify 25 specific mutations in older mice for the BALB/cA strain. These observations suggest 26 that BALB/cA has a stronger aging phenotype than C57BL/6. Indeed, of the two mouse may have a less efficient DNA repair system and a more unstable rDNA region, 1 resulting in an enhanced level of senescence. week-old C57BL/6JJcl mice were purchased from CLEA Japan, Inc.

Southern blot analysis to detect rDNA 13
For Southern blot analysis 150 ng of mouse DNA was digested with 10 units of BamHI-14 HF (NEB, Figure 2 and 5), NdeI (NEB, Figure 2 and 5) and SacII (NEB, Figure 5) 15 overnight at 37℃. The digested DNA was resolved on a 0.8-1.0% agarose gel (in 16 1xTAE) and blotted onto a filter. The 28S and SWI5 were detected on the same filter 17 using PCR amplified probes with specific primers (Table S3). For the psoralen 18 crosslinking assay, 2 x 107 bone marrow cells were suspended in 8 ml Opti-MEM® I min and crosslinked using UV-A for 4 min (7 cm apart from the UV light). This UV 23 exposure and psoralen addition cycle was repeated four times. Cells were then scraped 24 and collected by centrifugation (1,800 rpm, 5 min), and the DNA isolated. A 500 ng 25 aliquot of DNA was digested with 20 units of AflIII (NEB) overnight at 37℃ and was then exposed to UV (4000 J/cm2 x 100) using a UV Stratalinker to reverse the 1 crosslinking(42), (43), (44). given in Table S2. The PCR conditions were as follows; 40 cycles of 95℃ for 5 sec, 13 60℃ for 30 sec. Strains that had lost the former URA3+ plasmid were then positively selected on SC-20 LEU plates containing 5-FOA. 21

Yeast chronological lifespan analysis 23
Yeast cells were streaked on a 2%-glucose YP plate from a glycerol stock and incubated 24 at 30°C for 3 days. A single colony was grown at 30°C overnight in 2 ml SC medium 25 containing 2% glucose, shaking at 200 rpm. The culture was diluted with fresh 2%-26 glucose SC medium to an optical density of 0.1 (OD600 units) to give a day 0 culture of 27 20 ml. Starting at day 3 and every 2 days, a 100 μl aliquot of the culture was removed 28 and diluted with sterile water to prepare a 1:10,000 dilution. The dilution was spread forming units (CFU) was scored and normalized with that of the day 3 culture to 1 establish the survival rate. All experiments were performed in biological triplicates.   which retards migration during gel electrophoresis. ID # is the identification number of 2 the mice (same as Figure 2B). (C) Ratio of active to inactive rDNA copies. Band 3 intensities of (B) were measured and the ratio of active to inactive rDNA calculated. 4 The error bars are S.D. The p value is shown. rDNA copy numbers of one, two and four cells were measured by qPCR. rDNA copy 24 number in RPE1 determined by digital PCR was used as the standard (Materials & Figure S2. Estimated rDNA copy number from database 1 rDNA copy numbers of BALB/cA (BALB/c 1-3) and C57BL/6 (B6J 1-3) mice were 2 estimated by reanalysis of publicly available whole genome sequencing data. rDNA 3 copy number estimation by whole genome sequencing data were performed as follows. 4 Fastq files obtained from NCBI SRA (PRJNA41995, PRJNA386034) were mapped 5 against mouse whole genome and rDNA sequence using Bowtie2, and the fraction of 6 rDNA reads among all mapped reads were used to calculate the copy numbers.  Non-coding regions are shadowed. Ave. is the average mutation rate. Mutation rate is 23 the difference from the reference sequence as for Figure S3. m3 f1 m3 m4m3 f1 m3m4m3 f1 m3 m4 m1 f1 m1 f1 m1 f1 m1 f1 m1 f1 m1 f1 m1 f1 ID#: Figure S4 rDNA sequence variation in young and old C57BL/6 mice tgggttttaagcaggaggtg Figure 1, 3, ddPCR 28S_Rv gtgaattctgcttcacaatg Figure 1,3 28S_ddPCR_Rv gacggtctaaacccagctca ddPCR probe_28S_Fw gttgccatggtaatcctgct Figure 2, 4, 5 probe_28S_Rv acccagaagcaggtcgtcta Figure 2, 4, 5 probe_Swi5_Fw aggagttgattctctctacc Figure 2, 5 probe_Swi5_Rv gcatcaagacaattgtggtt Figure 2, 5 45S_Fw ctcttagatcgatgtggtgctc