Ribosomal DNA Locus Research Articles

Release 4.0 of the Plant rDNA Database: A Database on Plant Ribosomal DNA Loci Number, Their Position, and Organization: An Information Source for Comparative Cytogenetics.

This paper presents the latest update to the Plant rDNA database (Release 4.0), a valuable resource for researchers in the field of plant cytogenetics. The database provides information on the number, position, and arrangement of ribosomal DNA loci in plants, including angiosperms, gymnosperms, bryophytes, and pteridophytes. The new release includes new data for 820 species coming from additional 173 papers. In the updated version of the Plant rDNA database, 4948 entries comprising 2760 organisms can be found. A brief guide on how to navigate the database and obtain the desired information is also provided. The regular updating of the database is important for ensuring the information it contains is accurate, up-to-date, and useful for the research community. The Plant rDNA database continues to be beneficial for phylogenetic and cytogenetic studies in a wide range of taxa including angiosperms, gymnosperms, and early diverging groups, such as bryophytes and lycophytes.

Ribosomal DNA Instability as a Potential Cause of Karyotype Evolution.

Karyotype refers to the configuration of the genome into a set of chromosomes. The karyotype difference between species is expected to impede various biological processes, such as chromosome segregation and meiotic chromosome pairing, potentially contributing to incompatibility. Karyotypes can rapidly change between closely related species and even among populations of the same species. However, the forces driving karyotype evolution are poorly understood. Here we describe a unique karyotype of a Drosophila melanogaster strain isolated from the Seychelles archipelago. This strain has lost the ribosomal DNA (rDNA) locus on the X chromosome. Because the Y chromosome is the only other rDNA-bearing chromosome, all females carry at least one Y chromosome as the source of rDNA. Interestingly, we found that the strain also carries a truncated Y chromosome (YS) that is stably maintained in the population despite its inability to support male fertility. Our modeling and cytological analysis suggest that the Y chromosome has a larger negative impact on female fitness than the YS chromosome. Moreover, we generated an independent strain that lacks X rDNA and has a karyotype of XXY females and XY males. This strain quickly evolved multiple karyotypes: two new truncated Y chromosomes (similar to YS), as well as two independent X chromosome fusions that contain the Y-derived rDNA fragment, eliminating females' dependence on the Y chromosome. Considering that Robertsonian fusions frequently occur at rDNA loci in humans, we propose that rDNA loci instability may be one of driving forces of karyotype evolution.

Mobile group I introns at nuclear rDNA position L2066 harbor sense and antisense homing endonuclease genes intervened by spliceosomal introns

BackgroundMobile group I introns encode homing endonucleases that confer intron mobility initiated by a double-strand break in the intron-lacking allele at the site of insertion. Nuclear ribosomal DNA of some fungi and protists contain mobile group I introns harboring His-Cys homing endonuclease genes (HEGs). An intriguing question is how protein-coding genes embedded in nuclear ribosomal DNA become expressed. To address this gap of knowledge we analyzed nuclear L2066 group I introns from myxomycetes and ascomycetes.ResultsA total of 34 introns were investigated, including two identified mobile-type introns in myxomycetes with HEGs oriented in sense or antisense directions. Intriguingly, both HEGs are interrupted by spliceosomal introns. The intron in Didymium squamulosum, which harbors an antisense oriented HEG, was investigated in more detail. The group I intron RNA self-splices in vitro, thus generating ligated exons and full-length intron circles. The intron HEG is expressed in vivo in Didymium cells, which involves removal of a 47-nt spliceosomal intron (I-47) and 3′ polyadenylation of the mRNA. The D. squamulosum HEG (lacking the I-47 intron) was over-expressed in E. coli, and the corresponding protein was purified and shown to confer endonuclease activity. The homing endonuclease was shown to cleave an intron-lacking DNA and to produce a pentanucleotide 3′ overhang at the intron insertion site.ConclusionsThe L2066 family of nuclear group I introns all belong to the group IE subclass. The D. squamulosum L2066 intron contains major hallmarks of a true mobile group I intron by encoding a His-Cys homing endonuclease that generates a double-strand break at the DNA insertion site. We propose a potential model to explain how an antisense HEG becomes expressed from a nuclear ribosomal DNA locus.

Sequencing and Genomic Analysis of Sorghum DNA Introgression Variant Line R21 and Recipient Rice Jin Hui 1 Revealed Repetitive Element Variation.

Transferring the genome of distant species to crops is an efficient way to create new germplasms. However, the molecular mechanisms involved are unclear. In this study, a new rice restorer line R21 with heat tolerance was created by introgressing the genomic DNA of sorghum into the recipient restorer line Jin Hui 1. Assembly of rice R21 and Jin Hui 1 genomes was performed using PacBio sequencing technology. Comparative genome analysis and coverage statistics showed that the repetitive sequence atr0026 was a candidate introgression fragment of sorghum DNA. Sequence similarity analysis revealed that atr0026 was distributed at different copy numbers on the telomeric position of chromosomes 9 or 10 in R21, Jin Hui 1, and several rice varieties, indicating that the repetitive sequence from sorghum was highly conserved in rice. The repeat annotation in Gramineae indicated that ribosomal DNA loci that existed in atr0026 may be cause a rearrangement of chromosomes 9 and 10 of the R21 genome, resulting in a copy number variation at the 5′ end of it. Our study lays the foundation for further elucidation of the molecular mechanisms underlying the heat tolerance of sorghum DNA introgression variant line R21, which is of great significance for guiding crop genetic breeding.

The Chromosome Number and rDNA Loci Evolution in Onobrychis (Fabaceae).

The evolution of chromosome number and ribosomal DNA (rDNA) loci number and localisation were studied in Onobrychis Mill. Diploid and tetraploid species, as well as two basic chromosome numbers, x = 7 and x = 8, were observed among analysed taxa. The chromosomal distribution of rDNA loci was presented here for the first time using fluorescence in situ hybridisation (FISH) with 5S and 35S rDNA probes. Onobrychis species showed a high polymorphism in the number and localisation of rDNA loci among diploids, whereas the rDNA loci pattern was very similar in polyploids. Phylogenetic relationships among the species, inferred from nrITS sequences, were used as a framework to reconstruct the patterns of basic chromosome number and rDNA loci evolution. Analysis of the evolution of the basic chromosome numbers allowed the inference of x = 8 as the ancestral number and the descending dysploidy and polyploidisation as the major mechanisms of the chromosome number evolution. Analyses of chromosomal patterns of rRNA gene loci in a phylogenetic context resulted in the reconstruction of one locus of 5S rDNA and one locus of 35S rDNA in the interstitial chromosomal position as the ancestral state in this genus.

Physical mapping of 45S and 5S rDNA and telomeric repeat loci in eight diploid hyacinth cultivars

Hyacinth, one of the five best-selling bulbous flowers in the world, is a favored flower of garden landscapes in the spring. Hyacinth has a single origin, but there are many horticultural varieties. Physical mapping of ribosomal DNA (rDNA) and telomeric repeat loci by fluorescence in situ hybridization (FISH) is helpful to understand the genetic relationship and chromosome inheritance in hyacinth breeding. In this study, 45S rDNA, 5S rDNA and telomeric repeats were used as probes, the mitotic metaphase chromosomes of 8 diploid hyacinth cultivars were analyzed by FISH location, and their karyotype parameters were analyzed by Q-type cluster analysis. The results showed that two 45S rDNA loci were detected in all eight hyacinth cultivars, four 5S rDNA loci were detected in ‘Gypsy Queen’, and three were detected in other cultivars. In addition to the presence of telomere loci at both ends of chromosomes, seven telomere loci were detected in ‘Gypsy Princess’ and ‘Yellow Stone’, and eight telomere loci were detected in other cultivars. At the same time, when the euclidean distance between groups was 20, the eight hyacinth cultivars were clustered into three groups (‘Yellow Stone’, ‘Red Pearl’ and ‘Pink Pearl’), and ‘Gypsy Prince’ was a single group; the other four cultivars were in one group. The distribution of 45S rDNA loci in diploid hyacinth cultivars was relatively conservative, while the locus number and distribution of 5S rDNA and telomeric repeats were highly polymorphic. The FISH physical mapping in this study provides a basic understanding of the karyotype evolution of hyacinth species, which could increase the understanding of the evolutionary history and phylogenetic relationships of hyacinths.

Nonrandom sister chromatid segregation mediates rDNA copy number maintenance in Drosophila.

Although considered to be exact copies of each other, sister chromatids can segregate nonrandomly in some cases. For example, sister chromatids of the X and Y chromosomes segregate nonrandomly during asymmetric division of male germline stem cells (GSCs) in Drosophila melanogaster. Here, we demonstrate that the ribosomal DNA (rDNA) loci, which are located on the X and Y chromosomes, and an rDNA binding protein Indra are required for nonrandom sister chromatid segregation (NRSS). We provide the evidence that NRSS, following unequal sister chromatid exchange, is a mechanism by which GSCs recover rDNA copy number, counteracting the spontaneous copy number loss that occurs during aging. Our study reveals an unexpected role for NRSS in maintaining germline immortality through maintenance of a vulnerable genomic element, rDNA.

First Report of Anthracnose on Passiflora edulis Caused by Colletotrichum siamense in Guangdong Province in China.

Passion fruit (Passiflora edulis Sims) is an important tropical and subtropical species with high commercial value due to its wide uses in juice production and fresh fruit consumption. A disease affecting the leaves was observed on passion fruit vines in the South Subtropical Botanical Garden in Zhanjiang city in April and September 2020 with an incidence of 30 to 60%. Affected leaves usually developed small circular reddish-brown spots, which enlarged to oval spots and large irregular spots. The spots were brown and surrounded by a yellow halo with sunken cavities, which are typical symptoms of anthracnose. Diseased cultivars HuangjinBaiXiangGuo were collected in Zhanjiang city (21.2N110.3E), Suixi city (21.4N110.2E) and Leizhou city (20.9N110.1E) in Guangdong province. Leaf samples were disinfested with 1% NaOCl for 5 min, and cultured on potato dextrose agar (PDA) at 28 °C for one week. Eight single-spore isolates were recovered from PSA medium. Colonies developing on PSA were grayish white with a regular border. Conidia weretraight, hyaline with rounded ends, 5.3 to 6.8×15.2 to 21.3μm (n = 50 conidia of each isolate). Fungal mycelia were hyaline, septate, and branched. Conidia were born on a long conidiogenous cell, appressoria were dark, smooth-walled, oval, 15.5 to 21.3×4.0 to 5.8 μm (n=50).The isolates were morphologically identified as Colletotrichum sp. (Weir et al. 2012). Tests of pathogenicity were performed according to Koch's postulates using three isolates. Five plants with 2-3 vines were inoculated with each isolate separately and 3-4 leaves of each plant were inoculated. Fresh wounds were made with a sterile needle on the healthy surface of leaves of P. edulis HuangjinBaiXiangGuo and each leaf was covered with a piece of cotton drenched with 200 μL of conidial suspension (106 conidia/ml) from each isolate. Control seedlings were inoculated identiclly except sterile water was used to drench the cotton. Inoculated plants were placed in a moisturizing light incubator at 25℃ and 80% humidity under a 12-h light/dark cycle for 14 days and examined daily to monitor disease symptom development. Small round brown spots were observed at the inoculation sites 3 days after the inoculation and eventually became large brown lesions. There were no symptoms observed in the water-inoculated plants. A Colletotrichum sp. strain based on morphology was consistently reisolated from leaves lesions fulfilling Koch's postulates. For molecular identification, the internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (Cal), tubulin (Tub) and Apmat loci of three isolates were amplified using primer pairs of ITS4/ITS5, CL1C/CL2C, T1/T2 and AM-F/AM-R (Sharma et al. 2015). A phylogenetic tree derived from a neighbor-joining analysis of a concatenated alignment of ITS, CAL, Tub and ApMAT sequences was created. The accession numbers of three isolates BXGZJ 1-1, BXGSX 2-2 and BXGLZ 3-2 used in this study were MZ605383- MZ605385 for ITS, MZ605389- MZ605391 for CAL, MZ605392- MZ605394 for Tub and MZ605386- MZ605388 for ApMAT. The sequences of the three isolates were aligned with related species of Colletotrichum (Sharma et al. 2015). Analyses based on concatenated data sets of four genes showed that the sequences of the 3 isolates had the highest identity of 99% to those of the type strain C. siamense ICMP18578. According to both morphological and sequence analyses, the P. edulia pathogen was identified as C. siamense. There is a report of foliar diseases on P. edulia caused by C. brevisporum in Hainan province in China (Qiu et al., 2021). To our knowledge, this is the first report of anthracnose on P. edulia caused by C. siamense in China. Identification of pathogens could provide valuable information for disease diagnosis and effective chemicals to control the disease, so as to reduce yield loss and improve fruit quality.

A chromosome-level reference genome of Ensete glaucum gives insight into diversity and chromosomal and repetitive sequence evolution in the Musaceae.

Background Ensete glaucum (2n = 2x = 18) is a giant herbaceous monocotyledonous plant in the small Musaceae family along with banana (Musa). A high-quality reference genome sequence assembly of E. glaucum is a resource for functional and evolutionary studies of Ensete, Musaceae, and the Zingiberales.FindingsUsing Oxford Nanopore Technologies, chromosome conformation capture (Hi-C), Illumina and RNA survey sequence, supported by molecular cytogenetics, we report a high-quality 481.5 Mb genome assembly with 9 pseudo-chromosomes and 36,836 genes. A total of 55% of the genome is composed of repetitive sequences with predominantly LTR-retroelements (37%) and DNA transposons (7%). The single 5S ribosomal DNA locus had an exceptionally long monomer length of 1,056 bp, more than twice that of the monomers at multiple loci in Musa. A tandemly repeated satellite (1.1% of the genome, with no similar sequence in Musa) was present around all centromeres, together with a few copies of a long interspersed nuclear element (LINE) retroelement. The assembly enabled us to characterize in detail the chromosomal rearrangements occurring between E. glaucum and the x = 11 species of Musa. One E. glaucum chromosome has the same gene content as Musa acuminata, while others show multiple, complex, but clearly defined evolutionary rearrangements in the change between x= 9 and 11.ConclusionsThe advance towards a Musaceae pangenome including E. glaucum, tolerant of extreme environments, makes a complete set of gene alleles, copy number variation, and a reference for structural variation available for crop breeding and understanding environmental responses. The chromosome-scale genome assembly shows the nature of chromosomal fusion and translocation events during speciation, and features of rapid repetitive DNA change in terms of copy number, sequence, and genomic location, critical to understanding its role in diversity and evolution.

Site-Specific Integration of TRAIL in iPSC-Derived Mesenchymal Stem Cells for Targeted Cancer Therapy.

Mesenchymal stem cells (MSCs) are a promising cellular vehicle for transferring anti-cancer factors to malignant tumors. Currently, a variety of anti-cancer agents, including the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been loaded into MSCs derived from a range of sources through different engineering methods. These engineered MSCs exhibit enormous therapeutic potential for various cancers. To avoid the intrinsic defects of MSCs derived from tissues and the potential risk of viral vectors, TRAIL was site-specifically integrated into the ribosomal DNA (rDNA) locus of human-induced pluripotent stem cells (iPSCs) using a non-viral rDNA-targeting vector and transcription activator-like effector nickases (TALENickases). These genetically modified human iPSCs were differentiated into an unlimited number of homogeneous induced MSCs (TRAIL-iMSCs) that overexpressed TRAIL in both culture supernatants and cell lysates while maintaining MSC-like characteristics over continuous passages. We found that TRAIL-iMSCs significantly induced apoptosis in A375, A549, HepG2, and MCF-7 cells in vitro. After intravenous infusion, TRAIL-iMSCs had a prominent tissue tropism for A549 or MCF-7 xenografts and significantly inhibited tumor growth through the activation of apoptotic signaling pathways without obvious side effects in tumor-bearing mice models. Altogether, our results showed that TRAIL-iMSCs have strong anti-tumor effects in vitro and in vivo on a range of cancers. This study allows for the development of an unlimited number of therapeutic gene-targeted MSCs with stable quality and high homogeneity for cancer therapy, thus highlighting a universal and safe strategy for stem cell-based gene therapy with high potential for clinical applications.

Fine structure and transcription dynamics of bread wheat ribosomal DNA loci deciphered by a multi‐omics approach

Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head-to-tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and gene copy number, sequence composition, and expression status of particular arrays remain elusive, especially in complex genomes harboring multiple loci. Here we conducted a multi-omics study to decipher the 45S rDNA loci in hexaploid bread wheat. Coupling chromosomal genomics with optical mapping, we reconstructed individual rDNA arrays, enabling locus-specific analyses of transcription activity and methylation status from RNA- and bisulfite-sequencing data. We estimated a total of 6,650 rDNA units in the bread wheat genome, with approximately 2,321, 3,910, 253, and 50 gene copies located in short arms of chromosomes 1B, 6B, 5D, and 1A, respectively. Only 1B and 6B loci contributed substantially to rRNA transcription at a roughly 2:1 ratio. The ratio varied among five tissues analyzed (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG context in the silenced 5D locus as compared with the active 1B and 6B loci. In conclusion, a fine genomic organization and tissue-specific expression of rDNA loci were deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of wheat evolution and transcription regulation.

Ectopic Expression of FVIII in HPCs and MSCs Derived from hiPSCs with Site-Specific Integration of ITGA2B Promoter-Driven BDDF8 Gene in Hemophilia A.

Hemophilia A (HA) is caused by mutations in the coagulation factor VIII (FVIII) gene (F8). Gene therapy is a hopeful cure for HA; however, FVIII inhibitors formation hinders its clinical application. Given that platelets promote coagulation via locally releasing α-granule, FVIII ectopically expressed in platelets has been attempted, with promising results for HA treatment. The B-domain-deleted F8 (BDDF8), driven by a truncated ITGA2B promoter, was targeted at the ribosomal DNA (rDNA) locus of HA patient-specific induced pluripotent stem cells (HA-iPSCs). The F8-modified, human induced pluripotent stem cells (2bF8-iPSCs) were differentiated into induced hematopoietic progenitor cells (iHPCs), induced megakaryocytes (iMKs), and mesenchymal stem cells (iMSCs), and the FVIII expression was detected. The ITGA2B promoter-driven BDDF8 was site-specifically integrated into the rDNA locus of HA-iPSCs. The 2bF8-iPSCs were efficiently differentiated into 2bF8-iHPCs, 2bF8-iMKs, and 2bF8-iMSCs. FVIII was 10.31 ng/106 cells in lysates of 2bF8-iHPCs, compared to 1.56 ng/106 cells in HA-iHPCs, and FVIII was 3.64 ng/106 cells in 2bF8-iMSCs lysates, while 1.31 ng/106 cells in iMSCs with CMV-driven BDDF8. Our results demonstrated a high expression of FVIII in iHPCs and iMSCs derived from hiPSCs with site-specific integration of ITGA2B promoter-driven BDDF8, indicating potential clinical prospects of this platelet-targeted strategy for HA gene therapy.

Top1p targeting by Fob1p at the ribosomal Replication Fork Barrier does not account for camptothecin sensitivity in Saccharomyces cerevisiae cells.

Camptothecin (CPT) is a specific inhibitor of the DNA topoisomerase I (Top1p), currently used in cancer therapy, which induces DNA damage and cell death. Top1p is highly active at the repeated ribosomal DNA locus (rDNA) to relax DNA supercoiling caused by elevated transcription and replication occurring in opposite directions. Fob1p interacts with, and stabilizes, Top1p at the rDNA Replication Fork Barrier (rRFB), where replication and transcription converge. Here, we have investigated if the absence of Fob1p and the consequent loss of Top1p specific targeting to the rRFB impact the sensitivity and the cell cycle progression of wild-type cells to CPT. We have also investigated the consequences of the absence of Fob1p in rad52∆ mutants, which are affected in the repair of CPT-induced DNA damage by homologous recombination. The results show that CPT sensitivity and the global cell cycle progression in cells exposed to CPT is not changed in the absence of Fob1p. Moreover, we have observed in fob1∆ cells treated with CPT that the homologous recombination factor Rad52p still congregates in the shape of foci in the nucleolus, which hosts the rDNA. This suggests that, in the absence of Fob1p, Top1p is still recruited to the rDNA, presumably at sequences other than the rRFB, and its inhibition by CPT leads to recombination events.

First Report of Anthracnose on Hymenocallis littoralis Caused by Colletotrichum siamense in China.

Spider lily (Hymenocallis littoralis (Jacq.) Salisb.) is a widely cultivated horticultural plant worldwide and has ornamental and medicinal value. Spider lily plants were seriously affected by a leaf spot disease in the campus of Guangdong Ocean University and gardens in Zhanjiang city in February 2018 with an incidence of 30 to 100%. Affected leaves usually developed small circular purple spots, which enlarged to oval spots and large irregular spots. The spots were brown at the center, deep purple at the border and surrounded by a yellow halo. Diseased cultivars were collected in Zhanjiang city, Gangzhou city in Guangdong province and and Zhangping city in Fujian province. Symptomatic leaf samples were disinfested with 1% NaOCl, and cultured on sucrose agar (PSA) at 28 °C for one week. Ten single-spore isolates were recovered from PSA medium. Colonies developing on PSA were grayish white with a regular border. Conidia were straight, hyaline with rounded ends, 4.3 to 6.1×12.8 to 32.1μm (n = 50 conidia of each isolate). Fungal mycelia were hyaline, septate, and branched. Conidia were born on a long conidiogenous cell, appressoria were oval, 6.7 to 10.7 × 5.2 to 6.2 μm (n=50). The isolates were morphologically identified as Colletotrichum sp. (Weir et al. 2012). Tests of pathogenicity were performed according to Koch's postulates using three isolates. Fresh wounds were made with a sterile needle on the healthy surface of leaves of H. littoralis at the 4- to 6-leaf stage and each leaf was covered with a piece of cotton drenched with 200 μL of conidial suspension (106 conidia/ml) from each isolate. Control seedlings were inoculated identically except sterile water was used to drench the cotton. Inoculated plants were placed in a moisturizing light incubator at 25℃ and 80% humidity under a 12-h light/dark cycle for 20 days and examined daily to monitor disease symptom development. Small round brown spots were observed at the inoculation sites 3 days after the inoculation. The brown spots developed into large brown lesions 5 days after inoculation. There were no symptoms observed in the water-inoculated plants. A Colletotrichum spp. strain based on morphology was consistently reisolated from leaves lesions fulfilling Koch's postulates. For molecular identification, the internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CAL), Tublin (Tub) and Apmat loci of three isolates were amplified using primer pairs of ITS4/ITS5, CL1C/CL2C, T1/T2 and AM-F/AM-R (Sharma et al. 2015). A phylogenetic tree derived from a neighbor-joining analysis of a concatenated alignment of ITS, CAL, Tub and ApMAT sequences was created. The accession numbers of three isolates GZHLCG, ZJHLCG and FJHLCG used in this study were MW553083, MN540457, MN540458 for ITS, MW553087- MW553089 for CL, MW553090-MW553092 for Tub and MW553084-MW553086 for ApMAT. The sequences of the three isolates were aligned with related species of Colletotrichum (Sharma et al. 2015). Analyses based on concatenated data sets of four genes showed that the sequences had high levels of identity to those of the C. siamense strains. According to both morphological and sequence analyses, the H. littoralis pathogen was identified as C. siamense. There is a report of foliar diseases on H. littoralis caused by Colletotrichum sp. (Tan et al., 2009). To our knowledge, this is the first report of anthracnose on H. littoralis caused by C. siamense in China. Identification of the pathogen provide valuable information for diagnosis and controlling this disease in H. littoralis.

PiggyBac transposase and transposon derivatives for gene transfer targeting the ribosomal DNA loci of CHO cells

Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.

Repetitive Sequence Distribution on Saguinus, Leontocebus and Leontopithecus Tamarins (Platyrrhine, Primates) by Mapping Telomeric (TTAGGG) Motifs and rDNA Loci.

Tamarins are a distinct group of small sized New World monkeys with complex phylogenetic relationships and poorly studied cytogenetic traits. In this study, we applied molecular cytogenetic analyses by fluorescence in situ hybridization with probes specific for telomeric sequences and ribosomal DNA loci after DAPI/CMA3 staining on metaphases from five tamarin species, namely Leontocebus fuscicollis, Leontopithecus rosalia, Saguinus geoffroyi, Saguinus mystax and Saguinus oedipus, with the aim to investigate the distribution of repetitive sequences and their possible role in genome evolution. Our analyses revealed that all five examined species show similar karyotypes, 2n = 46, which differ mainly in the morphology of chromosome pairs 16-17 and 19-22, due to the diverse distribution of rDNA loci, the amplification of telomeric-like sequences, the presence of heterochromatic blocks and/or putative chromosomal rearrangements, such as inversions. The differences in cytogenetic traits between species of tamarins are discussed in a comparative phylogenetic framework, and in addition to data from previous studies, we underline synapomorphies and apomorphisms that appeared during the diversification of this group of New World monkeys.

The relevance of chromosome fissions for major ribosomal DNA dispersion in hymenopteran insects

Ribosomal DNA (rDNA) loci are essential for cellular metabolism due to their participation in ribosome biogenesis. Although these genes have been widely cytogenetically mapped, the evolutionary mechanisms behind their variability in number and chromosomal location remain elusive, even in well-known biological groups, such as ants, bees and wasps (Insecta: Hymenoptera). To address this question in Hymenoptera and therefore advance the understanding of rDNA evolution in insects in general, we integrated molecular cytogenetic data, a phylogenomic framework, model-based predictions and genome sequencing. Hence, we assessed the main evolutionary trends shaping the chromosomal distribution of rDNA loci in Hymenoptera. We noticed the conservation of one site of rDNA per haploid genome, suggesting that a single 45S rDNA locus is the putative ancestral pattern for aculeate Hymenoptera. Moreover, our results highlighted a nonrandom distribution of rDNA in Hymenoptera karyotypes, as well as a lineage-specific preferential location. The proximal location of rDNA is favoured in species with multiple loci and in the two families of Hymenoptera that show the highest range of chromosome numbers: Formicidae and Vespidae. We propose that chromosome fissions have played a crucial role in the distribution pattern of rDNA loci through the evolutionary diversification of Hymenoptera. Moreover, our genomic analysis of two species, one with a single locus of rDNA and one with multiple loci, supported that loci multiplication is followed by sequence divergence. Our results provide detailed information about the number and chromosomal position of rDNA in Hymenoptera and, therefore, broaden our knowledge regarding rDNA evolutionary dynamics in insects.

An essential role for the piRNA pathway in regulating the ribosomal RNA pool in C.elegans.

Piwi-interacting RNAs (piRNAs) are RNA effectors with key roles in maintaining genome integrity and promoting fertility in metazoans. In Caenorhabditis elegans loss of piRNAs leads to a transgenerational sterility phenotype. The plethora of piRNAs and their ability to silence transcripts with imperfect complementarity have raised several (non-exclusive) models for the underlying drivers of sterility. Here, we report the extranuclear and transferable nature of the sterility driver, its suppression via mutations disrupting the endogenous RNAi and poly-uridylation machinery, and copy-number amplification at the ribosomal DNA locus. In piRNA-deficient animals, several small interfering RNA (siRNA) populations become increasingly overabundant in the generations preceding loss of germline function, including ribosomal siRNAs (risiRNAs). A concomitant increase in uridylated sense rRNA fragments suggests that poly-uridylation may potentiate RNAi-mediated gene silencing of rRNAs. We conclude that loss of the piRNA machinery allows for unchecked amplification of siRNA populations, originating from abundant highly structured RNAs, to deleterious levels.

A Phylogenetic Approach to Structural Variation in Organization of Nuclear Group I Introns and Their Ribozymes.

Nuclear group I introns are restricted to the ribosomal DNA locus where they interrupt genes for small subunit and large subunit ribosomal RNAs at conserved sites in some eukaryotic microorganisms. Here, the myxomycete protists are a frequent source of nuclear group I introns due to their unique life strategy and a billion years of separate evolution. The ribosomal DNA of the myxomycete Mucilago crustacea was investigated and found to contain seven group I introns, including a direct repeat-containing intron at insertion site S1389 in the small subunit ribosomal RNA gene. We collected, analyzed, and compared 72 S1389 group IC1 introns representing diverse myxomycete taxa. The consensus secondary structure revealed a conserved ribozyme core, but with surprising sequence variations in the guanosine binding site in segment P7. Some S1389 introns harbored large extension sequences in the peripheral region of segment P9 containing direct repeat arrays. These repeats contained up to 52 copies of a putative internal guide sequence motif. Other S1389 introns harbored homing endonuclease genes in segment P1 encoding His-Cys proteins. Homing endonuclease genes were further interrupted by small spliceosomal introns that have to be removed in order to generate the open reading frames. Phylogenetic analyses of S1389 intron and host gene indicated both vertical and horizontal intron transfer during evolution, and revealed sporadic appearances of direct repeats, homing endonuclease genes, and guanosine binding site variants among the myxomycete taxa.

Commuting to Work: Nucleolar Long Non-Coding RNA Control Ribosome Biogenesis from Near and Far.

Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events.