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.

Spt4 promotes cellular senescence by activating non-coding RNA transcription in ribosomal RNA gene clusters.

Genome instability can drive aging in many organisms. The ribosomal RNA gene (rDNA) cluster is one of the most unstable regions in the genome and the stability of this region impacts replicative lifespan in budding yeast. To understand the underlying mechanism, we search for yeast mutants with stabler rDNA and longer lifespans than wild-type cells. We show that absence of a transcription elongation factor, Spt4, results in increased rDNA stability, reduced levels of non-coding RNA transcripts from the regulatory E-pro promoter in the rDNA, and extended replicative lifespan in a SIR2-dependent manner. Spt4-dependent lifespan restriction is abolished in the absence of non-coding RNA transcription at the E-pro locus. The amount of Spt4 increases and its function becomes more important as cells age. These findings suggest that Spt4 is a promising aging factor that accelerates cellular senescence through rDNA instability driven by non-coding RNA transcription.

Varying strength of selection contributes to the intragenomic diversity of rRNA genes.

Ribosome biogenesis in eukaryotes is supported by hundreds of ribosomal RNA (rRNA) gene copies that are encoded in the ribosomal DNA (rDNA). The multiple copies of rRNA genes are thought to have low sequence diversity within one species. Here, we present species-wide rDNA sequence analysis in Saccharomyces cerevisiae that challenges this view. We show that rDNA copies in this yeast are heterogeneous, both among and within isolates, and that many variants avoided fixation or elimination over evolutionary time. The sequence diversity landscape across the rDNA shows clear functional stratification, suggesting different copy-number thresholds for selection that contribute to rDNA diversity. Notably, nucleotide variants in the most conserved rDNA regions are sufficiently deleterious to exhibit signatures of purifying selection even when present in only a small fraction of rRNA gene copies. Our results portray a complex evolutionary landscape that shapes rDNA sequence diversity within a single species and reveal unexpectedly strong purifying selection of multi-copy genes.

Morphology and SSU rRNA gene-based phylogeny of three peniculid ciliates (Ciliophora, Oligohymenophorea) from China, including a new Frontonia species.

In the present study, a new freshwater peniculid species, Frontonia apoelegans sp. nov., and two other peniculid species, Frontonia atra (Ehrenberg, 1833) Bütschli, 1889 and Stokesia vernalis Wenrich, 1929, were isolated from Lake Weishan wetland, northern China. Their morphology and infraciliature are described based on live observations and silver staining methods. The SSU rRNA gene sequences are also provided. Frontonia apoelegans sp. nov. is recognized by the following combination of characteristics: two contractile vacuoles located right-dorsally, without collecting canals; peniculi 1 and 2 four-rowed, peniculus 3 three-rowed with leftmost row containing only one kinetosome; 62-76 somatic kineties; three ophryokineties; and four or five postoral kineties. We also provide improved diagnoses for Frontonia atra and Stokesia vernalis based on current and previous reports. Comparisons with sequences of morphologically similar species clearly support the validity of the new species. Phylogenetic analyses based on SSU rRNA gene sequence data reveal that Frontonia species with two contractile vacuoles cluster in a single clade, indicating these species may have a common origin. The family Frontoniidae is non-monophyletic whereas the family Stokesiidae remains monophyletic according to our analyses.

Widespread genetic heterogeneity of human ribosomal RNA genes.

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5' ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5' ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.

16S rRNA gene profiling of rhizospheric microbial community of Eichhornia crassipes.

The rhizosphere of a plant is an important interface for the plant-microbe interaction that plays a significant role in the uptake and removal of heavy metal from contaminated sites. Eichhornia crassipes is a free-floating macrophyte and a well-known metal hyperaccumulator. It is a promising plant, which harbors a diverse microbial community in its rhizosphere. Therefore it is hypothesized that it can be a good habitat for microorganisms that supports plant growth and increases its phytoremediation potential. The rhizospheric DNA was extracted from the procured plant samples. The library was prepared and sequenced using the Illumina platform. 16S rRNA data from the Next Generation Sequencing (NGS) platform was analyzed using the QIIME software package. Alpha diversity was estimated from statistical indices i.e. Shannon index, Chao1 index, and observed species. The rarefaction plots, rank abundance curve, krona graph, and heat map were generated to study the rhizospheric community in detail. Metagenome consisted of 225,408 flash reads, 185,008 non-chimeric sequences with 17,578 Operational Taxonomic Units (OTU's), and 4622 OTU's without singletons. The data of present study are available at NCBI Bioproject (PRJNA631882). The taxonomic analysis of OTU's showed that the sequences belonged to major Phyla revealing the dominance of Proteobacteria, Bacteroidetes, Cyanobacteria, and Verrucomicrobia. The most abundant Genera in the sampled rhizosphere recorded were Thiothrix and Flavobacterium.

A PRC2-independent function for EZH2 in regulating rRNA 2'-O methylation and IRES-dependent translation.

Dysregulated translation is a common feature of cancer. Uncovering its governing factors and underlying mechanism are important for cancer therapy. Here, we report that enhancer of zeste homologue 2 (EZH2), previously known as a transcription repressor and lysine methyltransferase, can directly interact with fibrillarin (FBL) to exert its role in translational regulation. We demonstrate that EZH2 enhances rRNA 2'-O methylation via its direct interaction with FBL. Mechanistically, EZH2 strengthens the FBL-NOP56 interaction and facilitates the assembly of box C/D small nucleolar ribonucleoprotein. Strikingly, EZH2 deficiency impairs the translation process globally and reduces internal ribosome entry site (IRES)-dependent translation initiation in cancer cells. Our findings reveal a previously unrecognized role of EZH2 in cancer-related translational regulation.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast.

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

Metasequencing of V3-V4 Variable Regions of 16S rRNA Gene in Opportunistic Microbiota and Gut Biocenosis in Obese Adolescents.

Opportunistic microorganisms in the gut biocenosis were studied in adolescents with normal body weight and obesity (patients consulted at the Clinical Department of Research Center of Family Health and Human Reproduction Problems). The biological material was studied by standard bacteriological methods, representatives of Enterobacteriaceae family were also characterized using metagenomic sequencing of V3-V4 variable regions of 16S gene rRNA. Gut microbiota of obese adolescents was unbalanced and was characterized by low levels of bifido- and lactoflora representatives, a spectrum of E. coli associations, and high prevalence of opportunistic microorganisms and their associations. Representatives of Enterobacteriaceae family were most often found in the gut microbiota of obese adolescents.

New contributions to the phylogeny of the ciliate class Heterotrichea (Protista, Ciliophora): analyses at family-genus level and new evolutionary hypotheses.

Heterotrichous ciliates play an important role in aquatic ecosystem energy flow processes and many are model organisms for research in cytology, regenerative biology, and toxicology. In the present study, we combine both morphological and molecular data to infer phylogenetic relationships at family-genus level and propose new evolutionary hypotheses for the class Heterotrichea. The main results include: (1) 96 new ribosomal DNA sequences from 36 populations, representing eight families and 13 genera, including three poorly annotated genera, Folliculinopsis, Ampullofolliculina and Linostomella; (2) the earliest-branching families are Spirostomidae in single-gene trees and Peritromidae in the concatenated tree, but the family Peritromidae probably represents the basal lineage based on its possession of many "primitive" morphological characters; (3) some findings in molecular trees are not supported by morphological evidence, such as the family Blepharismidae is one of the most recent branches and the relationship between Fabreidae and Folliculinidae is very close; (4) the systematic positions of Condylostomatidae, Climacostomidae, and Gruberiidae remain uncertain based either on morphological or molecular data; and (5) the monophyly of each genus included in the present study is supported by the molecular phylogenetic trees, except for Blepharisma in the SSU rDNA tree and Folliculina in the ITS1-5.8S-ITS2 tree.

The interaction between nucleophosmin/NPM1 and the large ribosomal subunit precursors contribute to maintaining the nucleolar structure.

Nucleoli are sites where both the large and small ribosomal subunits mature. Biochemical assays have suggested that a multivalent nucleolar protein, NPM1/nucleophosmin contributes to the formation of the outer layer of the nucleolus. Prior works show that NPM1 depletion disorganizes the nucleolar structure. However, the mechanism of how NPM1 regulates the nucleolar structure has been unknown. We demonstrated that NPM1 directly interacts with the large ribosomal subunits and maintains them in the nucleolus. Ectopically localized NPM1 efficiently recruits only the large ribosomal subunit precursors, while ectopically localized large ribosomal subunit by the ribosomal protein RPL4 efficiently recruits NPM1. These results suggest that the nucleolar localization of NPM1 and the large ribosomal subunit precursors are mutually dependent. Furthermore, proteomic and localization analyses suggest that NPM1 plays a crucial role in the accumulation of the late processing machinery of the large ribosomal subunits in the nucleolus. Our results suggest that NPM1 maintains the pre-ribosomes and assembly machinery in the nucleolus, which in turn determines the nucleolar volume.

A Faster Algorithm for Computing the Kernel of Maximum Agreement Subtrees.

The maximum agreement subtree method determines the consensus of a collection of phylogenetic trees by identifying maximum cardinality subsets of leaves for which all input trees agree. The trees induced by these maximum cardinality subsets are maximum agreement subtrees (MASTs). A single MAST may be misleading, since there can exist two MASTs which share almost no leaves; nevertheless, it may be impossible to inspect all MASTs, since the number of MASTs can be exponential in the number of leaves. To overcome this drawback, Swenson et al. suggested to further summarize the information common to all MASTs by their intersection, which is called the kernel agreement subtree (KAST). The construction of the KAST is the focus of this paper. Swenson et al. had an O(kn3 + n4 + nd + 1) time algorithm for computing the KAST of k trees on n leaves, in which at least one tree has maximum degree d. In this paper, an O(kn3 + nd)-time algorithm is presented. We demonstrate the efficiency of our algorithm on simulated trees as well as on ribosomal RNA alignments, where trees with 13,000 taxa took only hours to process, whereas the previous algorithm did not terminate after a week of computation.

FastFung: A novel medium for the culture and isolation of fastidious fungal species from clinical samples.

We developed a novel culture medium, referred to FastFung medium as suitable for the culture of clinical fungi, including fastidious ones, for both research and diagnostic studies. It is based on Schædler agar supplemented with many essential components for the growth of fastidious fungi. It also contains selective antibacterial agents for the inhibition of contaminant bacteria growth. In this preliminary study, the FastFung medium was compared to the gold standard Sabouraud medium for 98 fungal and 20 bacterial strains. The fungal strain positive culture rate was 100% vs. 95% and the bacterial strain inhibition was 100% vs. 20%, for the FastFung and Sabouraud media, respectively. When compared to the Sabouraud medium on 120 clinical samples, the FastFung medium displayed both a higher fungal colonies count, and a lower culture contamination rate. Storage at 4 °C for 4 weeks did not alter the FastFung culture medium performances for the six isolates of Candida, Cryptococcus, and Penicillium tested. These encouraging results suggest future development of using the FastFung medium in clinical mycology and in mycobiome characterization. Further prospective evaluation aiming at assessing whether implementing the FastFung medium in the routine workflow simplifies and strengthen fungal isolation capacities in the clinical laboratory is warranted.

Three redescriptions in Tintinnopsis (Protista: Ciliophora: Tintinnina) from coastal waters of China, with cytology and phylogenetic analyses based on ribosomal RNA genes.

BACKGROUND: The taxonomy of tintinnine ciliates is vastly unresolved because it has traditionally been based on the lorica (a secreted shell) and it has only recently incorporated cytological and molecular information. Tintinnopsis, the most speciose tintinnine genus, is also the most problematic: it is known to be non-monophyletic, but it cannot be revised until more of its species are studied with modern methods. RESULTS: Here, T. hemispiralis Yin, 1956, T. kiaochowensis Yin, 1956, and T. uruguayensis Balech, 1948, from coastal waters of China, were studied. Lorica and cell features were morphometrically investigated in living and protargol-stained specimens, and sequences of three ribosomal RNA (rRNA) loci were phylogenetically analyzed. The three species show a complex ciliary pattern (with ventral, dorsal, and posterior kineties and right, left, and lateral ciliary fields), but differ in lorica morphology, details of the somatic ciliature and rRNA gene sequences. Tintinnopsis hemispiralis is further distinguished by a ciliary tuft (a ribbon of very long cilia originated from the middle portion of the ventral kinety and extending out of the lorica) and multiple macronuclear nodules. Both T. kiaochowensis and T. uruguayensis have two macronuclear nodules, but differ in the number of somatic kineties and the position of the posterior kinety. Two neotypes are fixed for T. hemispiralis and T. kiaochowensis to stabilize the species names objectively, mainly because of the previous unavailability of type materials. By phylogenetic analysis and comparison with closely-related species, we infer that the ciliary tuft and details such as the commencement of the rightmost kinety in the lateral ciliary field are synapomorphies that may help clarify the systematics of Tintinnopsis-like taxa. CONCLUSION: The redescriptions of three poorly known Tintinnopsis species, namely T. hemispiralis, T. kiaochowensis, and T. uruguayensis firstly revealed their ciliary patterns and rRNA sequences. This study expands knowledge and database of tintinnines and helps in identifying potential synapomorphies for future taxonomic rearrangements.

Seasonal dynamics of lotic bacterial communities assessed by 16S rRNA gene amplicon deep sequencing.

Aquatic microbial diversity, composition, and dynamics play vital roles in sustaining water ecosystem functionality. Yet, there is still limited knowledge on bacterial seasonal dynamics in lotic environments. This study explores a temporal pattern of bacterial community structures in lotic freshwater over a 2-year period. The aquatic bacterial communities were assessed using Illumina MiSeq sequencing of 16S rRNA genes. Overall, the communities were dominated by α-, ß-, and γ-Proteobacteria, Bacteroidetes, Flavobacteriia, and Sphingobacteriia. The bacterial compositions varied substantially in response to seasonal changes (cold vs. warm), but they were rather stable within the same season. Furthermore, higher diversity was observed in cold seasons compared to warm periods. The combined seasonal-environmental impact of different physico-chemical parameters was assessed statistically, and temperature, suspended solids, and nitrogen were determined to be the primary abiotic factors shaping the temporal bacterial assemblages. This study enriches particular knowledge on the seasonal succession of the lotic freshwater bacteria.

Longitudinal detection of Cryptosporidium spp. in 1-10-week-old dairy calves on a farm in Xinjiang, China.

Cryptosporidiosis is an important cause of morbidity and mortality in the cattle industry and leads to severe economic losses. Fecal samples were collected from 25 dairy calves once a week for 10 weeks for continuous longitudinal detection of Cryptosporidium spp. Cryptosporidium spp. were detected via nested PCR amplification of the ribosomal small subunit RNA gene, followed by restriction fragment length polymorphism analysis with enzymes SspI and MboII to identify the species. PCR results indicated that all calves were infected with Cryptosporidium spp. at least once, with an average overall prevalence rate of 52.0% (130/250). One-week-old calves had the highest occurrences of Cryptosporidium infection (96.0%), 2-week-old calves (80.0%) had the second highest, and calves with watery diarrhea also had a higher occurrence of infection (92.3%). Four Cryptosporidium species, C. parvum, C. bovis, C. ryanae, and C. andersoni, were identified, with C. parvum being the most common. Forty-eight C. parvum isolates were further subtyped via nested PCR amplification of the 60-kDa glycoprotein gene, and all were identified as subtype IIdA15G1. The results demonstrated that C. parvum mainly infects dairy calves which are younger than 3 weeks old.

Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota.

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies-ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies-ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies-ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.

Mushroom DNA barcoding project: Sequencing a segment of the 28S rRNA gene.

DNA barcoding is an important molecular methodology for species identification that was developed over the last two decades and it should be covered in the biology bachelor curriculum. Here, we present an example of DNA barcoding by sequencing a segment of the 28S nuclear ribosomal large subunit rRNA gene of wild mushrooms and framing the education in a project form for undergraduate students in biology. Students perform this project in 6-8 weeks, which also includes preparing a poster, writing a report and presenting a paper related to the work in a journal club format. First, fieldwork in the Netherlands was carried out, during which students collected mushrooms under supervision of a professional mycologist with the goal to (a) verify morphologically based identifications with a molecular method and (b) assess phylogenetic relationships of the different species collected. Next, DNA extractions and quantitation were performed, PCR amplification was done, and samples were sent out for Sanger sequencing. Students aligned and analyzed the sequences using BLAST and Geneious and subsequently created a phylogenetic tree. In case of collecting DNA barcodes of an earlier sequenced species, students could upload the data to a repository established for facilitation of future research projects. The method described is very robust, reagents and equipment are readily available, and costs are relatively low. In addition, the results can be compared to published fungal phylogenetic trees.

RIC-seq for global in situ profiling of RNA-RNA spatial interactions.

Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L-a super-enhancer hub RNA-interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA.

The fate of 35S rRNA genes in the allotetraploid grass Brachypodium hybridum.

Nucleolar dominance (ND) consists of the reversible silencing of 35S/45S rDNA loci inherited from one of the ancestors of an allopolyploid. The molecular mechanisms by which one ancestral rDNA set is selected for silencing remain unclear. We applied a combination of molecular (Southern blot hybridization and reverse-transcription cleaved amplified polymorphic sequence analysis), genomic (analysis of variants) and cytogenetic (fluorescence in situ hybridization) approaches to study the structure, expression and epigenetic landscape of 35S rDNA in an allotetraploid grass that exhibits ND, Brachypodium hybridum (genome composition DDSS), and its putative progenitors, Brachypodium distachyon (DD) and Brachypodium stacei (SS). In progenitor genomes, B. stacei showed a higher intragenomic heterogeneity of rDNA compared with B. distachyon. In all studied accessions of B. hybridum, there was a reduction in the copy number of S homoeologues, which was accompanied by their inactive transcriptional status. The involvement of DNA methylation in CG and CHG contexts in the silencing of the S-genome rDNA loci was revealed. In the B. hybridum allotetraploid, ND is stabilized towards the D-genome units, irrespective of the polyphyletic origin of the species, and does not seem to be influenced by homoeologous 35S rDNA ratios and developmental stage.