rDNA Loci Research Articles

Switch them off or not: selective rRNA gene repression in grasses.

Nucleolar dominance (ND) is selective epigenetic silencing of 35-48S rDNA loci. In allopolyploids, it is frequently manifested at the cytogenetic level by the inactivation of nucleolar organiser region(s) (NORs) inherited from one or several evolutionary ancestors. Grasses are ecologically and economically one of the most important land plant groups, which have frequently evolved through hybridisation and polyploidisation events. Here we review common and unique features of ND phenomena in this monocot family from cytogenetic, molecular, and genomic perspectives. We highlight recent advances achieved by using an allotetraploid model grass, Brachypodium hybridum, where ND commonly occurs at a population level, and we cover modern genomic approaches that decipher structural features of core arrays of NORs.

The retrotransposon R2 maintains Drosophila ribosomal DNA repeats

Ribosomal DNA (rDNA) loci contain hundreds of tandemly repeated copies of ribosomal RNA genes needed to support cellular viability. This repetitiveness makes it highly susceptible to copy number (CN) loss due to intrachromatid recombination between rDNA copies, threatening multigenerational maintenance of rDNA. How this threat is counteracted to avoid extinction of the lineage has remained unclear. Here, we show that the rDNA-specific retrotransposon R2 is essential for restorative rDNA CN expansion to maintain rDNA loci in the Drosophila male germline. The depletion of R2 led to defective rDNA CN maintenance, causing a decline in fecundity over generations and eventual extinction. We find that double-stranded DNA breaks created by the R2 endonuclease, a feature of R2's rDNA-specific retrotransposition, initiate the process of rDNA CN recovery, which relies on homology-dependent repair of the DNA break at rDNA copies. This study reveals that an active retrotransposon provides an essential function for its host, contrary to transposable elements' reputation as entirely selfish. These findings suggest that benefiting host fitness can be an effective selective advantage for transposable elements to offset their threat to the host, which may contribute to retrotransposons' widespread success throughout taxa.

The Role of Repetitive Sequences in Repatterning of Major Ribosomal DNA Clusters in Lepidoptera.

Genes for major ribosomal RNAs (rDNA) are present in multiple copies mainly organized in tandem arrays. The number and position of rDNA loci can change dynamically and their repatterning is presumably driven by other repetitive sequences. We explored a peculiar rDNA organization in several representatives of Lepidoptera with either extremely large or numerous rDNA clusters. We combined molecular cytogenetics with analyses of second- and third-generation sequencing data to show that rDNA spreads as a transcription unit and reveal association between rDNA and various repeats. Furthermore, we performed comparative long read analyses among the species with derived rDNA distribution and moths with a single rDNA locus, which is considered ancestral. Our results suggest that satellite arrays, rather than mobile elements, facilitate homology-mediated spread of rDNA via either integration of extrachromosomal rDNA circles or ectopic recombination. The latter arguably better explains preferential spread of rDNA into terminal regions of lepidopteran chromosomes as efficiency of ectopic recombination depends on the proximity of homologous sequences to telomeres.

Swc4 protects nucleosome-free rDNA, tDNA and telomere loci to inhibit genome instability

In the baker’s yeast Saccharomyces cerevisiae, NuA4 and SWR1-C, two multisubunit complexes, are involved in histone acetylation and chromatin remodeling, respectively. Eaf1 is the assembly platform subunit of NuA4, Swr1 is the assembly platform and catalytic subunit of SWR1-C, while Swc4, Yaf9, Arp4 and Act1 form a functional module, and is present in both NuA4 and SWR1 complexes. ACT1 and ARP4 are essential for cell survival. Deletion of SWC4, but not YAF9, EAF1 or SWR1 results in a severe growth defect, but the underlying mechanism remains largely unknown. Here, we show that swc4Δ, but not yaf9Δ, eaf1Δ, or swr1Δ cells display defects in DNA ploidy and chromosome segregation, suggesting that the defects observed in swc4Δ cells are independent of NuA4 or SWR1-C integrity. Swc4 is enriched in the nucleosome-free regions (NFRs) of the genome, including characteristic regions of RDN5s, tDNAs and telomeres, independently of Yaf9, Eaf1 or Swr1. In particular, rDNA, tDNA and telomere loci are more unstable and prone to recombination in the swc4Δ cells than in wild-type cells. Taken together, we conclude that the chromatin associated Swc4 protects nucleosome-free chromatin of rDNA, tDNA and telomere loci to ensure genome integrity.

Construction and validation of customized genomes for human and mouse ribosomal DNA mapping

rRNAs are transcribed from ribosomal DNA (rDNA) repeats, the most intensively transcribed loci in the genome. Due to their repetitive nature, there is a lack of genome assemblies suitable for rDNA mapping, creating a vacuum in our understanding of how the most abundant RNA in the cell is regulated. Our recent work revealed binding of numerous mammalian transcription and chromatin factors to rDNA. Several of these factors were known to play critical roles in development, tissue function, and malignancy, but their potential roles in rDNA regulation remained unexplored. This demonstrated the blind spot into which rDNA has fallen in genetic and epigenetic studies and highlighted an unmet need for public rDNA-optimized genome assemblies. Here, we customized five human and mouse assemblies-hg19 (GRCh37), hg38 (GRCh38), hs1 (T2T-CHM13), mm10 (GRCm38), and mm39 (GRCm39)-to render them suitable for rDNA mapping. The standard builds of these genomes contain numerous fragmented or repetitive rDNA loci. We identified and masked all rDNA-like regions, added a single rDNA reference sequence of the appropriate species as a ∼45 kb chromosome designated "chromosome R," and created annotation files to aid visualization of rDNA features in browser tracks. We validated these customized genomes for mapping of known rDNA-binding proteins and present a simple workflow for mapping chromatin immunoprecipitation-sequencing datasets. Customized genome assemblies, annotation files, positive and negative control tracks, and Snapgene files of standard rDNA reference sequences have been deposited to GitHub. These resources make rDNA mapping and visualization more readily accessible to a broad audience.

Tranzscheliella helutae sp. nov. on Agropyron cristatum from Ukraine

The morphology and phylogeny of Tranzscheliella on Agropyron cristatum (Triticeae) collected in Ukraine were studied by light microscopy, scanning electron microscopy and a molecular phylogenetic data of two rDNA loci (ITS and 28S). As a result, T. helutae sp. nov. is described. The fungus differs from other species from the T. hypodytes complex by a combination of morphological and molecular data.

Fob1-dependent condensin recruitment and loop extrusion on yeast chromosome III.

Despite recent advances in single-molecule and structural analysis of condensin activity in vitro, mechanisms of functional condensin loading and loop extrusion that lead to specific chromosomal organization remain unclear. In Saccharomyces cerevisiae, the most prominent condensin loading site is the rDNA locus on chromosome XII, but its repetitiveness deters rigorous analysis of individual genes. An equally prominent non-rDNA condensin site is located on chromosome III (chrIII). It lies in the promoter of a putative non-coding RNA gene called RDT1, which is in a segment of the recombination enhancer (RE) that dictates MATa-specific chrIII organization. Here, we unexpectedly find that condensin is recruited to the RDT1 promoter in MATa cells through hierarchical interactions with Fob1, Tof2, and cohibin (Lrs4/Csm1), a set of nucleolar factors that also recruit condensin to the rDNA. Fob1 directly binds to this locus in vitro, while its binding in vivo depends on an adjacent Mcm1/α2 binding site that provides MATa cell specificity. We also uncover evidence for condensin-driven loop extrusion anchored by Fob1 and cohibin at RDT1 that unidirectionally extends toward MATa on the right arm of chrIII, supporting donor preference during mating-type switching. S. cerevisiae chrIII therefore provides a new platform for the study of programmed condensin-mediated chromosome conformation.

New insights into the genome and transmission of the microsporidian pathogen Nosema muscidifuracis.

Nosema is a diverse genus of unicellular microsporidian parasites of insects and other arthropods. Nosema muscidifuracis infects parasitoid wasp species of Muscidifurax zaraptor and M. raptor (Hymenoptera: Pteromalidae), causing ~50% reduction in longevity and ~90% reduction in fecundity. Here, we report the first assembly of the N. muscidifuracis genome (14,397,169 bp in 28 contigs) of high continuity (contig N50 544.3 Kb) and completeness (BUSCO score 97.0%). A total of 2,782 protein-coding genes were annotated, with 66.2% of the genes having two copies and 24.0% of genes having three copies. These duplicated genes are highly similar, with a sequence identity of 99.3%. The complex pattern suggests extensive gene duplications and rearrangements across the genome. We annotated 57 rDNA loci, which are highly GC-rich (37%) in a GC-poor genome (25% genome average). Nosema-specific qPCR primer sets were designed based on 18S rDNA annotation as a diagnostic tool to determine its titer in host samples. We discovered high Nosema titers in Nosema-cured M. raptor and M. zaraptor using heat treatment in 2017 and 2019, suggesting that the remedy did not completely eliminate the Nosema infection. Cytogenetic analyses revealed heavy infections of N. muscidifuracis within the ovaries of M. raptor and M. zaraptor, consistent with the titer determined by qPCR and suggesting a heritable component of infection and per ovum vertical transmission. The parasitoids-Nosema system is laboratory tractable and, therefore, can serve as a model to inform future genome manipulations of Nosema-host system for investigations of Nosemosis.

TELOMERE BIOLOGY AND RIBOSOME BIOGENESIS: STRUCTURAL AND FUNCTIONAL INTERCONNECTIONS.

Telomeres are the nucleoprotein structures which play a pivotal role in the protection and maintenance of eukaryotic chromosomes. Telomeres and the enzyme telomerase which replenishes telomeric DNA lost during replication are important factors necessary to ensure continued cell proliferation. Cell proliferation is also dependent on proper and efficient protein synthesis, which is carried out by ribosomes. Mutations in genes involved in either ribosome biogenesis or telomere biology result in cellular abnormalities and can cause human genetic diseases, defined as ribosomopathies and telomeropathies, respectively. Interestingly, recent discoveries indicate that many of the ribosome assembly and rRNA maturation factors have additional non-canonical functions in telomere biology. Similarly, several key proteins and enzymes involved in telomere biology, including telomerase, have unexpected roles in rRNA transcription and maturation. These observations point to an intriguing crosstalk mechanism potentially explaining the multiple pleiotropic symptoms of mutations in many causal genes identified in various telomeropathy and ribosomopathy diseases. In this review, we provide a brief summary of eukaryotic telomere and rDNA loci structures, highlight several universal features of rRNA and telomerase biogenesis, evaluate intriguing interconnections between telomere biology and ribosome assembly, and conclude with an assessment of overlapping features of human diseases of telomeropathies and ribosomopathies.

Expanding the Chromosomal Evolution Understanding of Lygaeioid True Bugs (Lygaeoidea, Pentatomomorpha, Heteroptera) by Classical and Molecular Cytogenetic Analysis.

The Lygaeoidea comprise about 4660 species in 790 genera and 16 families. Using standard chromosome staining and FISH with 18S rDNA and telomeric (TTAGG)n probes, we studied male karyotypes and meiosis in 10 species of Lygaeoidea belonging to eight genera of the families Blissidae, Cymidae, Heterogastridae, Lygaeidae, and Rhyparochromidae. Chromosome numbers were shown to range from 12 to 28, with 2n = 14 being predominant. All species have an XY system and all but one have a pair of m-chromosomes. The exception is Spilostethus saxatilis (Lygaeidae: Lygaeinae); in another species of Lygaeinae, Thunbergia floridulus, m-chromosomes were present, which represents the first finding for this subfamily. All species have an inverted sequence of sex chromosome divisions ("post-reduction"). The 18S rDNA loci were observed on one or both sex chromosomes in Kleidocerys resedae and Th. floridulus, respectively (Lygaeidae), while on an autosomal bivalent in all other species. The rDNA loci tended to be close to the end of the chromosome. Using (TTAGG)n-FISH, we were able to show for the first time that the Lygaeoidea lack the canonical "insect" telomere motif (TTAGG)n. We speculate that this ancestral motif is absent from the entire infraorder Pentatomomorpha being replaced by some other telomere repeat motif sequences.

Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast

Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.

Two differentially stable rDNA loci coexist on the same chromosome and form a single nucleolus

The nucleolus is the most prominent membraneless compartment within the nucleus-dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations-where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location-the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This "megablock" rDNA engineering was performed in a fused-karyotype strain of Saccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to the CEN, in that the locus that is CEN-distal undergoes size reduction at a higher frequency compared to the CEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogous cis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.

A nucleolar long "non-coding" RNA encodes a novel protein that functions in response to stress.

Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar "non-coding" RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.

Nuclear DNA content and comparative FISH mapping of the 5s and 45s rDNA in wild and cultivated populations of Physalis peruviana L.

Physalis peruviana L. often known as goldenberry, has increased its commercial growth in the international market in recent years due to its nutritional value and antioxidant potential. This situation has enabled countries such as Peru to increase their production in order to meet the global demand. However, investigations about the genetic diversity of cultivated and wild populations of goldenberry are still in their early stages. FISH mapping of 5s and 45s rDNA loci and flow cytometry estimation of nuclear DNA content were used to assess genetic differences between wild and cultivated goldenberry populations from Ayacucho and Cajamarca. The majority of metaphases had six 5s rDNA sites for all populations and two and four 45s rDNA sites for the cultivated and wild populations, respectively. We were able to characterize nine different types of chromosomes based on their morphology, fluorescence, rDNA location, and conservation across populations by analyzing the chromosomes that contained rDNA. Furthermore, cultivated populations had more nuclear DNA (13.262±0.087 pg) than wild populations (12.955±0.086 pg). The results show genetic differences between wild and cultivated populations of goldenberry at molecular cytogenetic level as well as in genome size. These findings establish a precedent for future cytogenetic and genomic studies in goldenberry populations, enabling future breeding programs.

Non-Rabl chromosome organization in endoreduplicated nuclei of barley embryo and endosperm tissues.

Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner.

Living on the edge: morphological, karyological and genetic diversity studies of the Hungarian Plantago maxima populations and established ex situ collection

BackgroundThe analysis of genetic diversity of protected plant species can greatly support conservation efforts. Plantago maxima Juss. ex Jacq. is a perennial species distributed along the Eurasian steppe. The westernmost range edge of the species’ distribution is located in the Pannonian basin, in Hungary where it is represented by a few, fragmented and highly endangered populations. We studied population diversity of all Hungarian range edge, natural populations, and one established ex situ population. One population from the centre of distribution (Kazakhstan) was implemented in the cpDNA haplotype study to compare the peripheral vs. central populations. We performed morphometric trait-based analysis, chromosome studies (morphometric analyses and FISH) and genetic diversity evaluations using inter simple sequence repeats (ISSR) and cpDNA trnL-trnF to evaluate differences between the in situ and ex situ populations as well as central vs. peripheral populations.ResultsOur results showed no obvious morphological differences among the in situ and ex situ populations in the period between 2018 and 2020. One ex situ subpopulation develops flowers three years in a row from 2019, which is a favourable indicator of the introduction success. Hungarian populations are exclusively diploids (2n = 2x = 12). The karyogram consists of 5 metacentric and 1 acrocentric chromosome pair. Plantago maxima has one 35S and two 5S rDNA loci, located on the acrocentric chromosome pair. Eight variable ISSR primers yielded 100 fragments, of which 74.6% were polymorphic (mean He = 0.220). A high level of genetic variation within population was observed (92%) while the genetic differentiation among the populations was only 8%. STRUCTURE analysis revealed that the largest Kunpeszér population separated from the rest of the Hungarian populations, indicating a high rate of admixture among the other ones. Based on the trnL-trnF sequence analysis the Hungarian populations represent a single haplotype, which can indicate a reduced diversity due to isolation and recent population decline. By contrast, Kazakh population represents a distinct haplotype compared to the Hungarian samples.ConclusionsThe present study draws the attention to the high conservation value of the Plantago maxima populations from the westernmost range edge of the species’ distribution.

Cytogenetic Analysis of Seven Species of Gekkonid and Phyllodactylid Geckos.

Geckos (Gekkota), the species-rich clade of reptiles with more than 2200 currently recognized species, demonstrate a remarkable variability in diploid chromosome numbers (2n = 16-48) and mode of sex determination. However, only a small fraction of gekkotan species have been studied with cytogenetic methods. Here, we applied both conventional (karyotype reconstruction and C-banding) and molecular (fluorescence in situ hybridization with probes for rDNA loci and telomeric repeats) cytogenetic analyses in seven species of geckos, namely Blaesodactylus boivini, Chondrodactylus laevigatus, Gekko badenii, Gekko cf. lionotum, Hemidactylus sahgali, Homopholis wahlbergii (Gekkonidae) and Ptyodactylus togoensis (Phyllodactylidae), in order to provide further insights into the evolution of karyotypes in geckos. Our analysis revealed the presence of interstitial telomeric repeats in four species, but we were not able to conclude if they are remnants of previous chromosome rearrangements or were formed by an accumulation of telomeric-like satellite motifs. Even though sex chromosomes were previously identified in several species from the genera Hemidactylus and Gekko by cytogenetic and/or genomic methods, they were not detected by us in any examined species. Our examined species either have poorly differentiated sex chromosomes or, possibly, environmental sex determination. Future studies should explore the effect of temperature and conduct genome-wide analyses in order to identify the mode of sex determination in these species.

Investigating the Origin and Evolution of Polyploid Trifolium medium L. Karyotype by Comparative Cytogenomic Methods.

Trifolium medium L. is a wild polyploid relative of the agriculturally important red clover that possesses traits promising for breeding purposes. To date, T. medium also remains the only clover species with which agriculturally important red clover has successfully been hybridized. Even though allopolyploid origin has previously been suggested, little has in fact been known about the T. medium karyotype and its origin. We researched T. medium and related karyotypes using comparative cytogenomic methods, such as fluorescent in situ hybridization (FISH) and RepeatExplorer cluster analysis. The results indicate an exceptional karyotype diversity regarding numbers and mutual positions of 5S and 26S rDNA loci and centromeric repeats in populations of T. medium ecotypes and varieties. The observed variability among T. medium ecotypes and varieties suggests current karyotype instability that can be attributed to ever-ongoing battle between satellite DNA together with genomic changes and rearrangements enhanced by post-hybridization events. Comparative cytogenomic analyses of a T. medium hexaploid variety and diploid relatives revealed stable karyotypes with a possible case of chromosomal rearrangement. Moreover, the results provided evidence of T. medium having autopolyploid origin.

The first chromosome study of the genera Calchas Birula, 1899 and Neocalchas Yağmur, Soleglad, Fet & Kovařík, 2013 (Scorpiones: Iuridae)

The chromosomes of two species belonging to the scorpion family Iuridae are studied for the first time. Both analysed species displayed achiasmatic meiosis and no morphologically differentiated sex chromosomes; these features are typical for all scorpions. Both species possessed monocentric chromosomes, a characteristic specific to the entire parvorder Iurida. The karyotype of Calchas nordmanni Birula, 1899 is composed of 82 chromosomes, and the diploid number of chromosomes in Neocalchas gruberi is 40. Despite this conspicuous difference in 2n chromosome morphology is mainly acrocentric. Both species possess two pairs of the 18S rDNA clusters identified by FISH. A higher number of rDNA loci may represent an ancestral state for the parvorder Iurida.

The Use of Ribosomal DNA for Comparative Cytogenetics.

Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) sequences provides excellent chromosome markers for comparative cytogenetic analyses, especially in non-model plant species. The tandem repeat nature of a sequence and the presence of a highly conserved genic region make rDNA sequences relatively easy to isolate and clone. In this chapter, we describe the use of rDNA as markers for comparative cytogenetics studies. Traditionally, cloned probes labeled with Nick-translation have been used to detect rDNA loci. Recently, pre-labeled oligonucleotides are also employed quite frequently to detect both 35S and 5S rDNA loci. Ribosomal DNA sequences, together with other DNA probes in FISH/GISH or with fluorochromes such as CMA3 banding or silver staining, are very useful tools in comparative analyses of plant karyotypes.