Isolation of endophytes from Dioscorea nipponica Makino for stimulating diosgenin production and plant growth.

KEY MESSAGE: Both bacterial and fungal endophytes exhibited one or more plant growth-promoting (PGP) traits. Among these strains, the Paenibacillus peoriae SYbr421 strain demonstrated the greatest activity in the direct biotransformation of tuber powder from D. nipponica into diosgenin. Endophytes play crucial roles in shaping active metabolites within plants, significantly influencing both the quality and yield of host plants. Dioscorea nipponica Makino accumulates abundant steroidal saponins, which can be hydrolyzed to produce diosgenin. However, our understanding of the associated endophytes and their contributions to plant growth and diosgenin production is limited. The present study aimed to assess the PGP ability and potential of diosgenin biotransformation by endophytes isolates associated with D. nipponica for the efficient improvement of plant growth and development of a clean and effective approach for producing the valuable drug diosgenin. Eighteen bacterial endophytes were classified into six genera through sequencing and phylogenetic analysis of the 16S rDNA gene. Similarly, 12 fungal endophytes were categorized into 5 genera based on sequencing and phylogenetic analysis of the ITS rDNA gene. Pure culture experiments revealed that 30 isolated endophytic strains exhibited one or more PGP traits, such as nitrogen fixation, phosphate solubilization, siderophore synthesis, and IAA production. One strain of endophytic bacteria, P. peoriae SYbr421, effectively directly biotransformed the saponin components in D. nipponica. Moreover, a high yield of diosgenin (3.50%) was obtained at an inoculum size of 4% after 6 days of fermentation. Thus, SYbr421 could be used for a cleaner and more eco-friendly diosgenin production process. In addition, based on the assessment of growth-promoting isolates and seed germination results, the strains SYbr421, SYfr1321, and SYfl221 were selected for greenhouse experiments. The results revealed that the inoculation of these promising isolates significantly increased the plant height and fresh weight of the leaves and roots compared to the control plants. These findings underscore the importance of preparing PGP bioinoculants from selected isolates as an additional option for sustainable diosgenin production.

Hmo1 Promotes Efficient Transcription Elongation by RNA Polymerase I in Saccharomyces cerevisiae.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

rDNA transcription, replication and stability in Saccharomyces cerevisiae.

The ribosomal DNA locus (rDNA) is central for the functioning of cells because it encodes ribosomal RNAs, key components of ribosomes, and also because of its links to fundamental metabolic processes, with significant impact on genome integrity and aging. The repetitive nature of the rDNA gene units forces the locus to maintain sequence homogeneity through recombination processes that are closely related to genomic stability. The co-presence of basic DNA transactions, such as replication, transcription by major RNA polymerases, and recombination, in a defined and restricted area of the genome is of particular relevance as it affects the stability of the rDNA locus by both direct and indirect mechanisms. This condition is well exemplified by the rDNA of Saccharomyces cerevisiae. In this review we summarize essential knowledge on how the complexity and overlap of different processes contribute to the control of rDNA and genomic stability in this model organism.

Improved application of immobilized Enterobacter cloacae into a bio-based polymer for Reactive Blue 19 removal, an eco-friendly advancement in potential decolorizing systems.

The widespread use of highly complex synthetic dyes like reactive dyes in the textile industry has some adverse environmental impacts and deserves close attention. Biological treatment of these effluents utilizing various species of bacteria with remarkable efficiency in dye removal is still considered promising. Our current study deals with immobilizing an isolated bacterial strain into calcium alginate (Ca/Alg) gel beads and using it to treat pernicious pollutants like synthetic dyes. A potential Reactive Blue 19 (RB19)-degrading Enterobacter cloacae strain A1 was isolated from the Kashan textile industry and was characterized by 16S rDNA gene sequencing. The decolorization ability of strain A1 was assessed by time-based studies using free bacterial cells/immobilized in Ca/Alg. Based on the results of the 16S rDNA gene sequencing, it appears that strain A1 belonged to E. cloacae, with a 99.74% similarity. The findings suggest that immobilized strain A1 accomplished maximum decolorization activity compared with the free cells. The immobilized strain could utterly decompose and decolorize 0.05 mg/mL of RB19 within 48 h, while the free bacterial strain decolorized RB19 within 5 days. Moreover, Ca/Alg gel beads can maintain their efficiency for over three decolorization cycles. Further infrared spectroscopy (FTIR) and gas chromatograph mass spectrometer (GC/MS) investigation declared complete RB19 decomposition on reaction products. Artemia salina was used to investigate the toxicity of dye and its degraded metabolites. The LC50 values signified the pure dye as very toxic with 0.01 mg/mL concentration, while after-treatment products showed no toxic effect on larvae. This immobilization technique increased the applicability of bacterial strain for dye removal. It was beneficial for the decolorization of RB19 from textile wastewater due to a remarkable reduction in time. Notably, strain A1-immobilized beads can maintain their activity for three consecutive decolorization cycles without a considerable decrease in efficiency. PRACTITIONER POINTS: The remarkable capacity of immobilized Enterobacter cloacae strain A1 for Reactive Blue 19 (RB19) removal Immobilized A1 strain showed two-fold higher removal than free one over 48 h A promising method for enhancing RB19 decolorization Decolorization was due to degradation based on UV-Vis, FTIR, and GC/MS analysis Non-toxic posttreatment products for Artemia.

Daxx knockdown promoted rDNA transcription without impairing H3.3 expression in mouse preimplantation embryos.

During fertilization, DAXX (death domain-associated protein) mediates histone variant H3.3 incorporation into heterochromatin, which plays an important role in the maintenance of genomic integrity. rDNA, the ribosomal gene, is included in the first wave of gene activation after fertilization. Our and other studies indicated that loss of Daxx disturbs rDNA heterochromatinization and promotes rDNA transcription without change in protein expression of H3.3. However, maternal and zygotic deletion of Daxx impairs blastocyst development. Whether Daxx knockdown affects H3.3 expression and improves the rDNA transcription in preimplantation development has not been reported. In the present study, we injected HA-labelled H3.3 (H3.3-HA) into oocytes during ICSI procedure, and detected H3.3 and DAXX by immunofluorescent staining. Then, we knockdowned Daxx and detected the gene expression levels of Daxx, H3.3, 18s and 47s rRNA. We also performed immunofluorescent staining of B23, γH2A and EdU incorporation to demonstrate nuclear structure, DNA damage and replication. We found injection of H3.3-HA did not impair preimplantation development. Daxx siRNA did not change expression of H3.3 mRNA, and the development of two-cell embryos and blastocysts, but the overall replication and expression levels of rRNA were increased compared with that in the control group. Finally, knockdown of DAXX did not aggravate the DNA damage but loosened the nucleolus. We concluded that Daxx knockdown promoted DNA replication and rDNA transcription, but did not affect H3.3 expression and subsequent preimplantation development.

Microaerobic methane-driven denitrification in a biotrickle bed - Investigating the active microbial biofilm community composition using RNA-stable isotope probing.

A microaerobic (2% O2 v/v) biotrickle bed reactor supplied continuously with 2% methane to drive nitrate removal (MAME-D) was investigated using 16S rDNA and rRNA amplicon sequencing in combination with RNA-stable isotope probing (RNA-SIP) to identify the active microorganisms. Methane removal rates varied from 500 to 1000 mmol m-3h-1 and nitrate removal rates from 25 to 58 mmol m-3h-1 over 55 days of operation. Biofilm samples from the column were incubated in serum bottles supplemented with 13CH4. 16S rDNA analysis indicated a simple community structure in which four taxa accounted for 45% of the total relative abundance (RA). Dominant genera included the methanotroph Methylosinus and known denitrifiers Nubsella and Pseudoxanthomonas; along with a probable denitrifier assigned to the order Obscuribacterales. The 16S rRNA results revealed the methanotrophs Methylocystis (15% RA) and Methylosinus (10% RA) and the denitrifiers Arenimonas (10% RA) and Pseudoxanthomonas (7% RA) were the most active genera. Obscuribacterales was the most active taxa in the community at 22% RA. Activity was confirmed by the Δ buoyant density changes with time for the taxa, indicating most of the community activity was associated with methane oxidation and subsequent consumption of methanotrophic metabolic intermediates by the denitrifiers. This is the first report of RNA stable isotope probing within a microaerobic methane driven denitrification system and the active community was markedly different from the full community identified via 16S-rDNA analysis.

TOR inactivation triggers heterochromatin formation in rDNA during glucose starvation.

In response to environmental cues, such as nutrient starvation, living organisms modulate gene expression through mechanisms involving histone modifications. Specifically, nutrient depletion inactivates the TOR (target of rapamycin) pathway, leading to reduced expression of ribosomal genes. While these regulatory mechanisms are well elucidated in budding yeast Saccharomyces cerevisiae, their conservation across diverse organisms remains unclear. In this study, we demonstrate that fission yeast Schizosaccharomyces pombe cells repress ribosomal gene transcription through a different mechanism. TORC1, which accumulates in the rDNA region, dissociates upon starvation, resulting in enhanced methylation of H3K9 and heterochromatin formation, facilitated by dissociation of the stress-responsive transcription factor Atf1 and accumulation of the histone chaperone FACT. We propose that this mechanism might be adapted in mammals that possess Suv39H1 and HP1, which are absent in budding yeast.

Integration of microbiota and metabolomics reveals the analgesic mechanisms of emodin against neuropathic pain.

BACKGROUND AND OBJECTIVE: Neuropathic pain (NeP) induced dysbiosis of intestinal microbiota in chronic constriction injury (CCI) rats. Emodin has analgesic effect but the detailed mechanism is not clear at the present time. This study aims to explore the underling mechanism of action of emodin against NeP with in CCI model. METHODS: Male SD rats (180-220 g) were randomly divided into three groups: sham group, CCI group, and emodin group. Behavioral tests were performed to evaluate the therapeutic effects of emodin on CCI model. Feces and spinal cords of all rats were collected 15 days after surgery. 16S rDNA sequencing, untargeted metabolomics, qPCR and ELISA were performed. RESULTS: Mechanical withdrawal thresholds (MWT), thermal withdrawal latency (TWL) and Sciatic functional index (SFI) in emodin group were significantly higher than CCI group (P < 0.05). Emodin not only inhibited the expression of pro-inflammatory cytokines in the spinal cords and colonic tissue, but also increased the expression of tight junction protein in colonic tissue. 16S rDNA sequencing showed that emodin treatment changed the community structure of intestinal microbiota in CCI rats. Untargeted metabolomics analysis showed that 33 differential metabolites were screened out between CCI group and emodin group. After verification, we found that emodin increased the level of S-adenosylmethionine (SAM) and Histamine in the spinal cord of CCI rats. CONCLUSION: Emodin was effective in relieving neuropathic pain, which is linked to inhibition inflammatory response, increasing the proportion of beneficial bacteria and beneficial metabolites.

rDNA magnification is a unique feature of germline stem cells.

Ribosomal DNA (rDNA) encodes ribosomal RNA and exists as tandem repeats of hundreds of copies in the eukaryotic genome to meet the high demand of ribosome biogenesis. Tandemly repeated DNA elements are inherently unstable; thus, mechanisms must exist to maintain rDNA copy number (CN), in particular in the germline that continues through generations. A phenomenon called rDNA magnification was discovered over 50 y ago in Drosophila as a process that recovers the rDNA CN on chromosomes that harbor minimal CN. Our recent studies indicated that rDNA magnification is the mechanism to maintain rDNA CN under physiological conditions to counteract spontaneous CN loss that occurs during aging. Our previous studies that explored the mechanism of rDNA magnification implied that asymmetric division of germline stem cells (GSCs) may be particularly suited to achieve rDNA magnification. However, it remains elusive whether GSCs are the unique cell type that undergoes rDNA magnification or differentiating germ cells are also capable of magnification. In this study, we provide empirical evidence that suggests that rDNA magnification operates uniquely in GSCs, but not in differentiating germ cells. We further provide computer simulation that suggests that rDNA magnification is only achievable through asymmetric GSC divisions. We propose that despite known plasticity and transcriptomic similarity between GSCs and differentiating germ cells, GSCs' unique ability to divide asymmetrically serves a critical role of maintaining rDNA CN through generations, supporting germline immortality.

Insights into microbial communities and metabolic profiles in the traditional production of the two representative Hongqu rice wines fermented with Gutian Qu and Wuyi Qu based on single-molecule real-time sequencing.

Hongqu rice wine, a famous traditional fermented alcoholic beverage, is brewed with traditional Hongqu (mainly including Gutian Qu and Wuyi Qu). This study aimed to compare the microbial communities and metabolic profiles in the traditional brewing of Hongqu rice wines fermented with Gutian Qu and Wuyi Qu. Compared with Hongqu rice wine fermented with Wuyi Qu (WY), Hongqu rice wine fermented with Gutian Qu (GT) exhibited higher levels of biogenic amines. The composition of volatile flavor components of Hongqu rice wine brewed by different fermentation starters (Gutian Qu and Wuyi Qu) was obviously different. Among them, ethyl acetate, isobutanol, 3-methylbutan-1-ol, ethyl decanoate, ethyl palmitate, ethyl oleate, nonanoic acid, 4-ethylguaiacol, 5-pentyldihydro-2(3H)-furanone, ethyl acetate, n-decanoic acid etc. were identified as the characteristic aroma-active compounds between GT and WY. Microbiome analysis based on high-throughput sequencing of full-length 16S rDNA/ITS-5.8S rDNA amplicons revealed that Lactococcus, Leuconostoc, Pseudomonas, Serratia, Enterobacter, Weissella, Saccharomyces, Monascus and Candida were the predominant microbial genera during the traditional production of GT, while Lactococcus, Lactobacillus, Leuconostoc, Enterobacter, Kozakia, Weissella, Klebsiella, Cronobacter, Saccharomyces, Millerozyma, Monascus, Talaromyces and Meyerozyma were the predominant microbial genera in the traditional fermentation of WY. Correlation analysis revealed that Lactobacillus showed significant positive correlations with most of the characteristic volatile flavor components and biogenic amines. Furthermore, bioinformatical analysis based on PICRUSt revealed that microbial enzymes related to biogenic amines synthesis were more abundant in GT than those in WY, and the enzymes responsible for the degradation of biogenic amines were less abundant in GT than those in WY. Collectively, this study provides important scientific data for enhancing the flavor quality of Hongqu rice wine, and lays a solid foundation for the healthy and sustainable development of Hongqu rice wine industry.

Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops.

Hypoxia is a common feature of solid tumors and is associated with poor patient prognosis, therapy resistance and metastasis. Radiobiological hypoxia (<0.1% O2) is one of the few physiologically relevant stresses that activates both the replication stress/DNA damage response and the unfolded protein response. Recently, we found that hypoxia also leads to the robust accumulation of R-loops, which led us to question here both the mechanism and consequence of hypoxia-induced R-loops. Interestingly, we found that the mechanism of R-loop accumulation in hypoxia is dependent on non-DNA damaging levels of reactive oxygen species. We show that hypoxia-induced R-loops play a critical role in the transcriptional stress response, evidenced by the repression of ribosomal RNA synthesis and the translocation of nucleolin from the nucleolus into the nucleoplasm. Upon depletion of R-loops, we observed a rescue of both rRNA transcription and nucleolin translocation in hypoxia. Mechanistically, R-loops accumulate on the rDNA in hypoxia and promote the deposition of heterochromatic H3K9me2 which leads to the inhibition of Pol I-mediated transcription of rRNA. These data highlight a novel mechanistic insight into the hypoxia-induced transcriptional stress response through the ROS-R-loop-H3K9me2 axis. Overall, this study highlights the contribution of transcriptional stress to hypoxia-mediated tumorigenesis.

Downregulation of ribosomal RNA (rRNA) genes in human head and neck squamous cell carcinoma (HNSCC) cells correlates with rDNA promoter hypermethylation.

Eukaryotes carry hundreds of ribosomal RNA (rRNA) genes as tandem arrays, which generate rRNA for protein synthesis. Humans carry âˆ¼ 400 rRNA gene copies and their expression is epigenetically regulated. Dysregulation of rRNA synthesis and ribosome biogenesis are characteristic features of cancers. Targeting aberrant rRNA expression for cancer therapy is being explored. Head and neck squamous cell carcinoma (HNSCC) is among the most prevalent cancers globally. Using quantitative PCR and bisulfite sequencing, we show that rRNA genes are downregulated and their promoters are hypermethylated in HNSCC cell lines. These findings may have relevance for prognosis and diagnosis of HNSCC.

Saccharomyces cerevisiae deficient in the early anaphase release of Cdc14 can traverse anaphase I without ribosomal DNA disjunction and successfully complete meiosis.

Eukaryotic meiosis is a specialized cell cycle of two nuclear divisions that give rise to haploid gametes. The phosphatase Cdc14 is essential for meiosis in the yeast Saccharomyces cerevisiae. Cdc14 is sequestered in the nucleolus, a nuclear domain containing the ribosomal DNA, by its binding partner Net1, and released in two distinct waves, first in early anaphase I, then in anaphase II. Current models posit that the meiosis I release is required for ribosomal DNA disjunction, disassembly of the anaphase spindle, spindle pole re-duplication and counteraction of cyclin-dependent kinase, all of which are essential events. We examined Cdc14 release in net1-6cdk mutant cells, which lack six key Net1 CDK phosphorylation sites. Cdc14 release in early anaphase I was partially inhibited, and disjunction of the rDNA was fully inhibited. Failure to disjoin the rDNA is lethal in mitosis, and we expected the same to be true for meiosis I. However, the cells reliably completed both meiotic divisions to produce four viable spores. Therefore, segregation of the rDNA into all four meiotic products can be postponed until meiosis II without decreasing the fidelity of chromosome inheritance.

Ability of a selfish B chromosome to evade genome elimination in the jewel wasp, Nasonia vitripennis.

B chromosomes are non-essential, extra chromosomes that can exhibit transmission-enhancing behaviors, including meiotic drive, mitotic drive, and induction of genome elimination, in plants and animals. A fundamental but poorly understood question is what characteristics allow B chromosomes to exhibit these extraordinary behaviors. The jewel wasp, Nasonia vitripennis, harbors a heterochromatic, paternally transmitted B chromosome known as paternal sex ratio (PSR), which causes complete elimination of the sperm-contributed half of the genome during the first mitotic division of fertilized embryos. This genome elimination event may result from specific, previously observed alterations of the paternal chromatin. Due to the haplo-diploid reproduction of the wasp, genome elimination by PSR causes female-destined embryos to develop as haploid males that transmit PSR. PSR does not undergo self-elimination despite its presence with the paternal chromatin until the elimination event. Here we performed fluorescence microscopic analyses aimed at understanding this unexplained property. Our results show that PSR, like the rest of the genome, participates in the histone-to-protamine transition, arguing that PSR does not avoid this transition to escape self-elimination. In addition, PSR partially escapes the chromatin-altering activity of the intracellular bacterium, Wolbachia, demonstrating that this ability to evade chromatin alteration is not limited to PSR's own activity. Finally, we observed that the rDNA locus and other unidentified heterochromatic regions of the wasp's genome also seem to evade chromatin disruption by PSR, suggesting that PSR's genome-eliminating activity does not affect heterochromatin. Thus, PSR may target an aspect of euchromatin to cause genome elimination.

Isolation of Pseudomonas oleovorans Carrying Multidrug Resistance Proteins MdtA and MdtB from Wastewater.

The pollution of industrial wastewater has become a global issue in terms of economic development and ecological protection. Pseudomonas oleovorans has been studied as a bacterium involved in the treatment of petroleum pollutants. Our study aimed to investigate the physicochemical properties and drug resistance of Pseudomonas oleovorans isolated from industrial wastewater with a high concentration of sulfate compounds. Firstly, Pseudomonas oleovorans was isolated and then identified using matrix-assisted flight mass spectrometry and 16S rDNA sequencing. Then, biochemical and antibiotic resistance analyses were performed on the Pseudomonas oleovorans, and a microbial high-throughput growth detector was used to assess the growth of the strain. Finally, PCR and proteomics analyses were conducted to determine drug-resistance-related genes/proteins. Based on the results of the spectrum diagram and sequencing, the isolated bacteria were identified as Pseudomonas oleovorans and were positive to reactions of ADH, MTE, CIT, MLT, ONPG, and ACE. Pseudomonas oleovorans was sensitive to most of the tested antibiotics, and its resistance to SXT and CHL and MIN and TIM was intermediate. The growth experiment showed that Pseudomonas oleovorans had a good growth rate in nutrient broth. Additionally, gyrB was the resistance gene, and mdtA2, mdtA3, mdtB2, mdaB, and emrK1 were the proteins that were closely associated with the drug resistance of Pseudomonas oleovorans. Our results show the biochemical properties of Pseudomonas oleovorans from industrial wastewater with a high concentration of sulfate compounds and provide a new perspective for Pseudomonas oleovorans to participate in biological removal of chemical pollutants in industrial wastewater.

Induction of the Erythroid Differentiation of K562 Cells Is Coupled with Changes in the Inter-Chromosomal Contacts of rDNA Clusters.

The expression of clusters of rDNA genes influences pluripotency; however, the underlying mechanisms are not yet known. These clusters shape inter-chromosomal contacts with numerous genes controlling differentiation in human and Drosophila cells. This suggests a possible role of these contacts in the formation of 3D chromosomal structures and the regulation of gene expression in development. However, it has not yet been demonstrated whether inter-chromosomal rDNA contacts are changed during differentiation. In this study, we used human leukemia K562 cells and induced their erythroid differentiation in order to study both the changes in rDNA contacts and the expression of genes. We observed that approximately 200 sets of rDNA-contacting genes are co-expressed in different combinations in both untreated and differentiated K562 cells. rDNA contacts are changed during differentiation and coupled with the upregulation of genes whose products are mainly located in the nucleus and are highly associated with DNA- and RNA-binding, along with the downregulation of genes whose products mainly reside in the cytoplasm or intra- or extracellular vesicles. The most downregulated gene is ID3, which is known as an inhibitor of differentiation, and thus should be switched off to allow for differentiation. Our data suggest that the differentiation of K562 cells leads to alterations in the inter-chromosomal contacts of rDNA clusters and 3D structures in particular chromosomal regions as well as to changes in the expression of genes located in the corresponding chromosomal domains. We conclude that approximately half of the rDNA-contacting genes are co-expressed in human cells and that rDNA clusters are involved in the global regulation of gene expression.

Nuclear envelope-remodeling events as models to assess the potential role of membranes on genome stability.

The nuclear envelope (NE) encloses the genetic material and functions in chromatin organization and stability. In Saccharomyces cerevisiae, the NE is bound to the ribosomal DNA (rDNA), highly repeated and transcribed, thus prone to genetic instability. While tethering limits instability, it simultaneously triggers notable NE remodeling. We posit here that NE remodeling may contribute to genome integrity maintenance. The NE importance in genome expression, structure, and integrity is well recognized, yet studies mostly focus on peripheral proteins and nuclear pores, not on the membrane itself. We recently characterized a NE invagination drastically obliterating the rDNA, which we propose here as a model to probe if and how membranes play an active role in genome stability preservation.

PAF49: An RNA Polymerase I subunit essential for rDNA transcription and stabilization of PAF53.

The application of genetic and biochemical techniques in yeast has informed our knowledge of transcription in mammalian cells. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in the nature of the transcription factors essential for transcription by Pol I in yeast and mammalian cells, and yeast RNA polymerase I contains 14 subunits while mammalian polymerase contains 13 subunits. We previously reported the adaptation of the auxin-dependent degron that enabled a combination of a "genetics-like" approach and biochemistry to study mammalian rDNA transcription. Using this system, we studied the mammalian orthologue of yeast RPA34.5, PAF49, and found that it is essential for rDNA transcription and cell division. The auxin-induced degradation of PAF49 induced nucleolar stress and the accumulation of P53. Interestingly, the auxin-induced degradation of AID-tagged PAF49 led to the degradation of its binding partner, PAF53, but not vice versa. A similar pattern of co-dependent expression was also found when we studied the non-essential, yeast orthologues. An analysis of the domains of PAF49 that are essential for rDNA transcription demonstrated a requirement for both the dimerization domain and an "arm" of PAF49 that interacts with PolR1B. Further, we demonstrate this interaction can be disrupted to inhibit Pol I transcription in normal and cancer cells which leads to the arrest of normal cells and cancer cell death. In summary, we have shown that both PAF53 and PAF49 are necessary for rDNA transcription and cell growth.

New insights on the evolution of nucleolar dominance in newly resynthesized hexaploid wheat Triticum zhukovskyi.

Nucleolar dominance (ND) is a widespread epigenetic phenomenon in hybridizations where nucleolus transcription fails at the nucleolus organizer region (NOR). However, the dynamics of NORs during the formation of Triticum zhukovskyi (GGAu Au Am Am ), another evolutionary branch of allohexaploid wheat, remains poorly understood. Here, we elucidated genetic and epigenetic changes occurring at the NOR loci within the Am , G, and D subgenomes during allopolyploidization by synthesizing hexaploid wheat GGAu Au Am Am and GGAu Au DD. In T. zhukovskyi, Au genome NORs from T. timopheevii (GGAu Au ) were lost, while the second incoming NORs from T. monococcum (Am Am ) were retained. Analysis of the synthesized T. zhukovskyi revealed that rRNA genes from the Am genome were silenced in F1 hybrids (GAu Am ) and remained inactive after genome doubling and subsequent self-pollinations. We observed increased DNA methylation accompanying the inactivation of NORs in the Am genome and found that silencing of NORs in the S1 generation could be reversed by a cytidine methylase inhibitor. Our findings provide insights into the ND process during the evolutionary period of T. zhukovskyi and highlight that inactive rDNA units may serve as a 'first reserve' in the form of R-loops, contributing to the successful evolution of T. zhukovskyi.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.