Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes.

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.

4.5SH RNA counteracts deleterious exonization of SINE B1 in mice.

4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.

Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.

RNA Polymerase I Activators Count and Adjust Ribosomal RNA Gene Copy Number.

Ribosome is the most abundant RNA-protein complex in a cell, and many copies of the ribosomal RNA gene (rDNA) have to be maintained. However, arrays of tandemly repeated rDNA genes can lose the copies by intra-repeat recombination. Loss of the rDNA copies of Saccharomyces cerevisiae is counteracted by gene amplification whereby the number of rDNA repeats stabilizes around 150 copies, suggesting the presence of a monitoring mechanism that counts and adjusts the number. Here, we report that, in response to rDNA copy loss, the upstream activating factor (UAF) for RNA polymerase I that transcribes the rDNA is released and directly binds to a RNA polymerase II-transcribed gene, SIR2, whose gene products silence rDNA recombination, to repress. We show that the amount of UAF determines the rDNA copy number that is stably maintained. UAF ensures rDNA production not only by rDNA transcription activation but also by its copy-number maintenance.

DNA polymerase ε-dependent modulation of the pausing property of the CMG helicase at the barrier.

The proper pausing of replication forks at barriers on chromosomes is important for genome integrity. However, the detailed mechanism underlying this process has not been well elucidated. Here, we successfully reconstituted fork-pausing reactions from purified yeast proteins on templates that had binding sites for the LacI, LexA, and/or Fob1 proteins; the forks paused specifically at the protein-bound sites. Moreover, although the replicative helicase Cdc45-Mcm2-7-GINS (CMG) complex alone unwound the protein-bound templates, the unwinding of the LacI-bound site was impeded by the presence of a main leading strand DNA polymerase: polymerase ε (Polε). This suggests that Polε modulates CMG to pause at these sites.

Acidic growth conditions stabilize the ribosomal RNA gene cluster and extend lifespan through noncoding transcription repression.

Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, jam, and liqueur. Blackcurrant extract is known to relieve cells from DNA damage caused by hydrogen peroxide (H2 O2 ), methyl methane sulfonate (MMS), and ultraviolet (UV) radiation. We found that blackcurrant extract (BCE) stabilizes the ribosomal RNA gene cluster (rDNA), one of the most unstable regions in the genome, through repression of noncoding transcription in the intergenic spacer (IGS) which extended the lifespan in budding yeast. Reduced formation of extrachromosomal circles (ERCs) after exposure to fractionated BCE suggested that acidity of the growth medium impacted rDNA stability. Indeed, alteration of the acidity of the growth medium to pH ~4.5 by adding HCl increased rDNA stability and extended the lifespan. We identified RPD3 as the gene responsible for this change, which was mediated by the RPD3L histone deacetylase complex. In mammals, as inflammation sites in a tissue are acidic, DNA maintenance may be similarly regulated to prevent genome instability from causing cancer.

Ctf4 Prevents Genome Rearrangements by Suppressing DNA Double-Strand Break Formation and Its End Resection at Arrested Replication Forks.

Arrested replication forks lead to DNA double-strand breaks (DSBs), which are a major source of genome rearrangements. Yet DSB repair in the context of broken forks remains poorly understood. Here we demonstrate that DSBs that are formed at arrested forks in the budding yeast ribosomal RNA gene (rDNA) locus are normally repaired by pathways dependent on the Mre11-Rad50-Xrs2 complex but independent of HR. HR is also dispensable for DSB repair at stalled forks at tRNA genes. In contrast, in cells lacking the core replisome component Ctf4, DSBs are formed more frequently, and these DSBs undergo end resection and HR-mediated repair that is prone to rDNA hyper-amplification; this highlights Ctf4 as a key regulator of DSB end resection at arrested forks. End resection also occurs during physiological rDNA amplification even in the presence of Ctf4. Suppression of end resection is thus important for protecting DSBs at arrested forks from chromosome rearrangements.

The human ribosomal DNA array is composed of highly homogenized tandem clusters.

The structure of the human ribosomal DNA (rDNA) cluster has traditionally been hard to analyze owing to its highly repetitive nature. However, the recent development of long-read sequencing technology, such as Oxford Nanopore sequencing, has enabled us to study the large-scale structure of the genome. Using this technology, we found that human cells have a quite regular rDNA structure. Although each human rDNA copy has some variations in its noncoding region, contiguous copies of rDNA are similar, suggesting that homogenization through gene conversion frequently occurs between copies. Analysis of rDNA methylation by Nanopore sequencing further showed that all the noncoding regions are heavily methylated, whereas about half of the coding regions are clearly unmethylated. The ratio of unmethylated copies, which are speculated to be transcriptionally active, was lower in individuals with a higher rDNA copy number, suggesting that there is a mechanism that keeps the active copy number stable. In addition, the rDNA in progeroid syndrome patient cells with reduced DNA repair activity had more unstable copies compared with control normal cells, although the rate was much lower than previously reported using a fiber-FISH method. Collectively, our results clarify the view of rDNA stability and transcription regulation in human cells, indicating the presence of mechanisms for both homogenizations to ensure sequence quality and maintenance of active copies for cellular functions.

Efficacy of an oscillating positive expiratory pressure device in patients with Mycobacterium avium complex pulmonary disease.

Patients with Mycobacterium avium complex pulmonary disease (MAC-PD) often suffer from chronic symptoms such as sputum production, which reduces quality of life. Oscillatory positive expiratory pressure (OPEP) devices are used in physiotherapy to promote the clearance of respiratory secretions. We report two cases of improved lung function and improved scores on the Leicester Cough Questionnaire (LCQ) and the Breathlessness, Cough and Sputum Scale (BCSS) after the use of OPEP in patients with MAC-PD where treatment with guideline-based therapy, including amikacin liposome inhalation suspension, had proved ineffective for symptoms. Use of OPEP might maximize the efficacy of therapy and thereby improves outcomes in patients with MAC-PD. It is important to use both guideline-based therapy and OPEP, especially in patients whose health-related quality of life is affected by sputum symptoms. Further prospective studies are warranted to assess the benefit of adding OPEP to guidelines concerning therapy for patients with MAC-PD and sputum symptoms.

Changes in skin discoloration according to clofazimine dosage in nontuberculous mycobacterial pulmonary disease.

Although clofazimine is currently one of the standard regimens for Mycobacterium abscessus, it frequently causes skin discoloration, posing esthetic concerns for patients. We studied thirteen Asian patients with pulmonary nontuberculous mycobacterial disease treated with clofazimine at the NHO Kinki Chuo Chest Medical Center. In three patients (two women and one man) whose dosing regimens were altered owing to skin discoloration, we continuously measured luminance (L*), red-green (a*), and yellow-blue (b*) values (using a colorimeter) in both sun-exposed and sun-unexposed skin areas at each visit. Compared to baseline L* and a* values, the ΔL* values were negative (decreased brightness) and Δa* values were positive (increased redness) while patients received daily clofazimine. After switching to intermittent or reduced dosing, these changes gradually diminished. If such a dose reduction does not affect the therapeutic outcome, an even lower clofazimine dose may be attempted to minimize skin adverse effects.

Cases of severe Mycobacterium avium lung disease occurred in a married couple caused by identical strain.

Bacteria of the Mycobacterium avium complex, which are environmental organisms found in soil and water, have been found to cause human lung diseases. Although infection is reported to occur in cohabiting patients, the incidence of infection from the single clone remains rarely documented. Herein, we report a case of M. avium lung disease caused by specimens with the same clone strains in a married couple. The wife, a 67-year-old female, had severe M. avium lung disease despite receiving multidrug chemotherapy for eleven years. The husband, a 68-year-old male, died of acute lung injury complicated by M. avium pleurisy. The result of the variable-number tandem-repeat analysis of isolates from serial sputum specimens of both patients indicated that the severe M. avium lung disease in a married couple was caused by the isolates with identical pattern. This case were considered to have acquired clarithromycin resistance during each clinical course, revealing the possibility of infection with a strain that may induce severe pulmonary condition.

Evaluation of repair activity by quantification of ribonucleotides in the genome.

Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ-seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ-seq analysis of cells harboring wild-type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3'-shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild-type and repair-deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ-seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ-seq can provide an estimate of indel risk.

The feasibility of rifampicin Re-administration in patients with tuberculosis and Clostridioides difficile infection.

BACKGROUND: The effects of a rifampicin (RIF) on the evolution of Clostridioides difficile infection (CDI) have not previously been investigated and there is currently no consensus on whether RIF re-administration is feasible. METHODS: This retrospective observational study included consecutive tuberculosis (TB) patients diagnosed with comorbid RIF-associated CDI (RA-CDI) using strict diagnostic criteria. We investigated the association between RA-CDI and clinical outcomes, and also examined the feasibility of re-administering RIF. RESULTS: Out of the 11,230 patients were admitted to TB ward at our hospital, 156 TB patients (1.4%) were diagnosed with CDI and the overall incidence of CDI was calculated as 2.1 cases per 10,000 patient-days. Of 156 patients with CDI, 86 were diagnosed with RA-CDI, of whom 28 (32.6%) were re-administered with RIF. In the re-administration group, time to initial sputum smear conversion was significantly shorter than for patients who were not re-administered with RIF (42 days [interquartile range, IQR: 35-65] vs. 55 days [IQR: 44-70], p = 0.041). Further, RIF re-administration significantly reduced length of hospital stay (69 days [IQR: 66-82] vs. 81 days [IQR: 72-89], p = 0.014). Ten patients (35.7%) had recurrent CDI after RIF re-administration. On the other hand, 15 patients (53.6%) were able to continue their TB treatment, including the RIF regimen. CONCLUSIONS: The present study strengthens the argument for including RIF in the list of antibiotics that can induce CDI, particularly in elderly men suffering from underlying conditions. Although careful attention must be paid to the possibility of CDI recurrence, a strategy of re-administration of RIF is feasible.

Ribosomal RNA gene repeats associate with the nuclear pore complex for maintenance after DNA damage.

The ribosomal RNA genes (rDNA) comprise a highly repetitive gene cluster. The copy number of genes at this locus can readily change and is therefore one of the most unstable regions of the genome. DNA damage in rDNA occurs after binding of the replication fork blocking protein Fob1 in S phase, which triggers unequal sister chromatid recombination. However, the precise mechanisms by which such DNA double-strand breaks (DSBs) are repaired is not well understood. Here, we demonstrate that the conserved protein kinase Tel1 maintains rDNA stability after replication fork arrest. We show that rDNA associates with nuclear pores, which is dependent on DNA damage checkpoint kinases Mec1/Tel1 and replisome component Tof1. These findings suggest that rDNA-nuclear pore association is due to a replication fork block and subsequent DSB. Indeed, quantitative microscopy revealed that rDNA is relocated to the nuclear periphery upon induction of a DSB. Finally, rDNA stability was reduced in strains where this association with the nuclear envelope was prevented, which suggests its importance for avoiding improper recombination repair that could induce repeat instability.

Two New Cases of Pulmonary Infection by Mycobacterium shigaense, Japan.

We report 2 case-patients in Japan with Mycobacterium shigaense pulmonary infections. One patient was given aggressive treatment and the other conservative treatment, according to distinctive radiologic evidence. A close phylogenetic relationship based on whole-genome sequencing was found between strain from the conservatively treated patient and a reference strain of cutaneous origin.

Rejuvenation of ribosomal RNA gene repeats at the nuclear pore.

The ribosomal RNA genes (rDNA) exist as tandem repeats in eukaryotes and are, therefore, highly unstable. Each rDNA unit includes a replication fork barrier site to avoid collisions between DNA replication forks and transcriptional machinery. However, because of this barrier, DNA double-strand breaks are induced at a relatively high frequency. If damage is repaired by the homologous recombination in rDNAs, it may result in frequent copy number changes and the production of extrachromosomal ribosomal DNA circles, both of which are closely linked to the regulation of lifespan. Here, we review recent progress in elucidating a multi-layered repair process of rDNA that occurs in the nucleolus, nucleoplasm and nuclear pores. Binding to nuclear pores appears to be the final strategy for repairing any remaining damage to the rDNA. Here, we propose the possible contribution of nuclear pores to the asymmetric distribution of damaged rDNA between mother and daughter cells as well as its possible impact on aging/rejuvenation.

Effective treatment for clarithromycin-resistant Mycobacterium avium complex lung disease.

Clinical management of macrolide-resistant Mycobacterium avium complex (MR-MAC) lung disease is difficult. To date, there only exist a limited number of reports on the treatment of clarithromycin-resistant MAC (CR-MAC) lung disease. This study aimed to evaluate prognostic factors and identify effective treatments in CR-MAC lung disease. We retrospectively collected clinical data of patients newly diagnosed with CR-MAC lung disease at the Kinki-Chuo Chest Medical Center between August 2010 and June 2018. Altogether, 37 patients with CR-MAC lung disease were enrolled. The median age was 69 years; 30, 22, and 21 patients received clarithromycin, ethambutol, and rifampicin, respectively, on their own or in drug combination. The observed sputum culture conversion rate was 29.7% (11/37 patients). In univariate analysis, ethambutol significantly increased the rate of sputum culture conversion (p = 0.027, odds ratio (OR) 10; 95% confidence interval (CI) 1.11-89.77). Multivariate analysis confirmed that ethambutol increased sputum culture conversion rate (p = 0.026; OR 21.8; 95% CI 1.45-329) while the existence of lung cavities decreased it (p = 0.04; OR 0.088; 95% CI 0.009-0.887). The combined use of ethambutol with other drugs may improve sputum culture conversion rate in CR-MAC lung disease.

How do cells count multi-copy genes?: "Musical Chair" model for preserving the number of rDNA copies.

To supply abundant ribosomes, multiple copies of ribosomal RNA genes (rDNA) are conserved from bacterial to human cells. In eukaryotic genomes, clusters of tandemly repeated rDNA units are present, and their number is stably maintained. Due to high level of transcription of rRNA genes, the repetitive structure is prone to rearrangement. In budding yeast, rDNA homeostasis can compensate for this by the regulation of recombination events that will change the copy number. The histone deacetylase Sir2 plays a key role in rDNA copy maintenance and its expression level determines a state of "maintenance" or "amplification" of rDNA copy number. We recently showed that Upstream Activating Factors (UAF) for RNA polymerase I act as a RNA polymerase II repressor of SIR2 transcription in response to rDNA copy loss. Furthermore, the amount of UAF, which is limited in the cell, determines the stable copy number of rDNA and is a molecular switch for rDNA recovery. In this mini-review, we propose a "Musical Chair" model for rDNA copy counting as mediated by UAF and Sir2. The model describes how a straightforward molecular mechanism can account for the "cellular memory" of the proper rDNA copy number.

More than 10% of yeast genes are related to genome stability and influence cellular senescence via rDNA maintenance.

Genome instability triggers cellular senescence and is a common cause of cancer. The ribosomal RNA genes (rDNA), due to their repetitive structure, form a fragile site with frequent rearrangements. To identify eukaryotic factors that connect reduced genome stability to senescence we screened 4,876 strains of a Saccharomyces cerevisiae deletion library for aberrant rDNA and found 708 genes that contribute to its upkeep. 28 mutants caused abnormalities in non-rDNA chromosomes and among them 12 mutants have abnormalities both in rDNA and in non-rDNA chromosomes. Many mutated genes have not previously been implicated with genome maintenance nor their homologues with tumorigenesis in mammals. The link between rDNA state and senescence was broken after deletion of factors related with DNA polymerase ϵ. These mutations also suppressed the short lifespan phenotype of a sir2 mutant, suggesting a model in which molecular events at the heart of the replication fork induce abnormal rDNA recombination and are responsible for the emergence of an aging signal.

Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity.