Ribosomal RNA gene repeats (rDNA) encode ribosomal RNA, a major component of ribosomes. Ribosome biogenesis is central to cellular metabolic regulation, and several diseases are associated with rDNA ...dysfunction, notably cancer, However, its highly repetitive nature has severely limited characterization of the elements responsible for rDNA function. Here we make use of phylogenetic footprinting to provide a comprehensive list of novel, potentially functional elements in the human rDNA. Complete rDNA sequences for six non-human primate species were constructed using de novo whole genome assemblies. These new sequences were used to determine the conservation profile of the human rDNA, revealing 49 conserved regions in the rDNA intergenic spacer (IGS). To provide insights into the potential roles of these conserved regions, the conservation profile was integrated with functional genomics datasets. We find two major zones that contain conserved elements characterised by enrichment of transcription-associated chromatin factors, and transcription. Conservation of some IGS transcripts in the apes underpins the potential functional significance of these transcripts and the elements controlling their expression. Our results characterize the conservation landscape of the human IGS and suggest that noncoding transcription and chromatin elements are conserved and important features of this unique genomic region.
Genomic instability is a conserved factor in lifespan reduction, although the molecular mechanism is not known 1. Studies in the yeast Saccharomyces cerevisiae over the past 20 years have found a ...connection between the ribosomal RNA gene cluster (rDNA) and lifespan 2. The highly repetitive rDNA exhibits genomic instability, and the antiaging histone deacetylase gene SIR2 regulates this instability. We previously proposed that SIR2 governs lifespan by repressing rDNA noncoding transcription and rDNA instability, but the extent to which lifespan is affected by SIR2 acting at the rDNA versus other genomic regions, and the relationship between rDNA noncoding transcription/rDNA stability and lifespan have remained controversial. To control rDNA noncoding transcription and rDNA instability, we use a strain in which the rDNA noncoding promoter is replaced with an inducible promoter. Here, we show that repression of noncoding transcription extends lifespan and makes SIR2 dispensable for lifespan extension. These results indicate that Sir2 maintains lifespan through repression of E-pro noncoding transcription in the rDNA cluster, rather than pleiotropically at other loci. The observation of rDNA instability in other organisms, including humans, suggests that this may be a conserved aging pathway.
•The ribosomal RNA gene repeat array is the most unstable region in the genome•We succeeded in regulating rDNA stability and manipulating yeast lifespan•SIR2, an antiaging gene, maintains lifespan solely through rDNA stability
Organisms maintain ribosomal RNA gene repeats (rDNA) at stable copy numbers by recombination; the loss of repeats results in gene amplification. Here we report a mechanism of amplification ...regulation. We show that amplification is dependent on transcription from a noncoding bidirectional promoter (E-pro) within the rDNA spacer. E-pro transcription stimulates the dissociation of cohesin, a DNA binding protein complex that suppresses sister-chromatid-based changes in rDNA copy number. This transcription is regulated by the silencing gene, SIR2, and by copy number. Transcription-induced cohesin dissociation may be a general mechanism of recombination regulation.
Repeat families within genomes are often maintained with similar sequences. Traditionally, this has been explained by concerted evolution, where repeats in an array evolve "in concert" with the same ...sequence via continual turnover of repeats by recombination. Another form of evolution, birth-and-death evolution, can also explain this pattern, although in this case selection is the critical force maintaining the repeats. The level of intragenomic variation is the key difference between these two forms of evolution. The prohibitive size and repetitive nature of large repeat arrays have made determination of the absolute level of intragenomic repeat variability difficult, thus there is little evidence to support concerted evolution over birth-and-death evolution for many large repeat arrays. Here we use whole-genome shotgun sequence data from the genome projects of five fungal species to reveal absolute levels of sequence variation within the ribosomal RNA gene repeats (rDNA). The level of sequence variation is remarkably low. Furthermore, the polymorphisms that are detected are not functionally constrained and seem to exist beneath the level of selection. These results suggest the rDNA is evolving via concerted evolution. Comparisons with a repeat array undergoing birth-and-death evolution provide a clear contrast in the level of repeat array variation between these two forms of evolution, confirming that the rDNA indeed does evolve via concerted evolution. These low levels of intra-genomic variation are consistent with a model of concerted evolution in which homogenization is very rapid and efficiently maintains highly similar repeat arrays.
Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on ...how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.
Sound is a physical stimulus that has the potential to affect various growth parameters of microorganisms. However, the effects of audible sound on microbes reported in the literature are ...inconsistent. Most published studies involve transmitting sound from external speakers through air toward liquid cultures of the microorganisms. However, the density differential between air and liquid culture could greatly alter the sound characteristics to which the microorganisms are exposed. In this study we apply white noise sound in a highly controlled experimental system that we previously established for transmitting sound underwater directly into liquid cultures to examine the effects of two key sound parameters, frequency and intensity, on the fermentation performance of a commercial Saccharomyces cerevisiae ale yeast growing in a maltose minimal medium. We performed these experiments in an anechoic chamber to minimise extraneous sound, and find little consistent effect of either sound frequency or intensity on the growth rate, maltose consumption, or ethanol production of this yeast strain. These results, while in contrast to those reported in most published studies, are consistent with our previous study showing that direct underwater exposure to white noise sound has little impact on S. cerevisiae volatile production and sugar utilization in beer medium. Thus, our results suggest the possibility that reported microorganism responses to sound may be an artefact associated with applying sound to cultures externally via transmission through air.
Abstract
The yeast Saccharomyces cerevisiae is a powerful model system to understand the molecular basis of aging. It has been known for over 50 years that yeast cells have a finite replicative ...capacity and develop an aging phenotype, and much recent research has focused on the molecular changes that underlie this replicative senescence. A common thread in many yeast replicative aging studies is the involvement of the ribosomal DNA gene repeats (rDNA), beginning with the discovery that the rDNA silencing gene, SIR2, regulates life span. In 2008, a novel aging hypothesis, termed the rDNA theory of aging, was presented where the high level of genomic instability at the rDNA repeats was proposed to dominate global genome stability and determine the life span. Here, we review the rDNA theory of aging and discuss a number of recent studies that provide important new data on the roles of the rDNA in yeast replicative aging. Based on these recent results, we propose an integrative model of the rDNA theory of aging that encompasses genomic instability, chromatin relocalization following DNA repair, and replication stress in a self-reinforcing cyclical pathway that is primarily manifested at the rDNA repeats and results in the aging phenotype.
This review discusses recent evidence that supports a fundamental role for instability of the ribosomal DNA repeats in yeast replicative aging and offers a revised model for this rDNA theory of aging.
This review discusses recent evidence that supports a fundamental role for instability of the ribosomal DNA repeats in yeast replicative aging and offers a revised model for this rDNA theory of aging.
The short arms of the five acrocentric human chromosomes harbor sequences that direct the assembly and function of the nucleolus, one of the key functional domains of the nucleus, yet they are absent ...from the current human genome assembly. Here we describe the genomic architecture of these human nucleolar organizers. Sequences distal and proximal to ribosomal gene arrays are conserved among the acrocentric chromosomes, suggesting they are sites of frequent recombination. Although previously believed to be heterochromatic, characterization of these two flanking regions reveals that they share a complex genomic architecture similar to other euchromatic regions of the genome, but they have distinct genomic characteristics. Proximal sequences are almost entirely segmentally duplicated, similar to the regions bordering centromeres. In contrast, the distal sequence is predominantly unique to the acrocentric short arms and is dominated by a very large inverted repeat. We show that the distal element is localized to the periphery of the nucleolus, where it appears to anchor the ribosomal gene repeats. This, combined with its complex chromatin structure and transcriptional activity, suggests that this region is involved in nucleolar organization. Our results provide a platform for investigating the role of NORs in nucleolar formation and function, and open the door for determining the role of these regions in the well-known empirical association of nucleoli with pathology.
Concerted evolution describes the unusual evolutionary pattern exhibited by certain repetitive sequences, whereby all the repeats are maintained in the genome with very similar sequences but differ ...between related species. The pattern of concerted evolution is thought to result from continual turnover of repeats by recombination, a process known as homogenization. Approaches to studying concerted evolution have largely been observational because of difficulties investigating repeat evolution in an experimental setting with large arrays of identical repeats. Here, we establish an experimental evolution approach to look at the rate and dynamics of concerted evolution in the ribosomal DNA (rDNA) repeats. A small targeted mutation was made in the spacer of a single rDNA unit in Saccharomyces cerevisiae so we could monitor the fate of this unit without the need for a selectable marker. The rate of loss of this single unit was determined, and the frequency of duplication was also estimated. The results show that duplication and deletion events occur at similar rates and are very common: An rDNA unit may be gained or lost as frequently as once every cell division. Investigation of the spatial dynamics of rDNA turnover showed that when the tagged repeat unit was duplicated, the copy predominantly, but not exclusively, ended up near to the tagged repeat. This suggests that variants in the rDNA spread in a semiclustered fashion. Surprisingly, large deletions that remove a significant fraction of total rDNA repeats were frequently found. We propose these large deletions are a driving force of concerted evolution, acting to increase homogenization efficiency over-and-above that afforded by turnover of individual rDNA units. Thus, the results presented here enhance our understanding of concerted evolution by offering insights into both the spatial and temporal dynamics of the homogenization process and suggest an important new aspect in our understanding of concerted evolution.
Polyploidy, a state in which the chromosome complement has undergone an increase, is a major force in evolution. Understanding the consequences of polyploidy has received much attention, and ...allopolyploids, which result from the union of two different parental genomes, are of particular interest because they must overcome a suite of biological responses to this merger, known as "genome shock." A key question is what happens to gene expression of the two gene copies following allopolyploidization, but until recently the tools to answer this question on a genome-wide basis were lacking. Here we utilize high throughput transcriptome sequencing to produce the first genome-wide picture of gene expression response to allopolyploidy in fungi. A novel pipeline for assigning sequence reads to the gene copies was used to quantify their expression in a fungal allopolyploid. We find that the transcriptional response to allopolyploidy is predominantly conservative: both copies of most genes are retained; over half the genes inherit parental gene expression patterns; and parental differential expression is often lost in the allopolyploid. Strikingly, the patterns of gene expression change are highly concordant with the genome-wide expression results of a cotton allopolyploid. The very different nature of these two allopolyploids implies a conserved, eukaryote-wide transcriptional response to genome merger. We provide evidence that the transcriptional responses we observe are mostly driven by intrinsic differences between the regulatory systems in the parent species, and from this propose a mechanistic model in which the cross-kingdom conservation in transcriptional response reflects conservation of the mutational processes underlying eukaryotic gene regulatory evolution. This work provides a platform to develop a universal understanding of gene expression response to allopolyploidy and suggests that allopolyploids are an exceptional system to investigate gene regulatory changes that have evolved in the parental species prior to allopolyploidization.
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