rRNA Levels Research Articles

Mechanism of Dcp2/RNCR3/Dkc1/Snora62 axis regulating neuronal apoptosis in chronic cerebral ischemia.

RNA-binding proteins (RBPs), long non-coding RNAs (lncRNAs), and small nucleolar RNAs (snoRNAs) were found to play crucial regulatory roles in ischemic injury. Based on GEO databases and our experimental results, we selected Dcp2, lncRNA-RNCR3, Dkc1, and Snora62 and Foxh1 as research candidates. We found that expression levels of Dcp2, RNCR3, Dkc1, Snora62, and Foxh1 were upregulated in oxygen glucose deprivation-treated HT22 cells and hippocampal tissues subject to chronic cerebral ischemia (CCI). Silencing of Dcp2, RNCR3, Dkc1, Snora62, and Foxh1 all inhibited apoptosis of oxygen glucose deprivation-treated HT22 cells. Moreover, Dcp2 promoted RNCR3 expression by increasing its stability. Importantly, RNCR3 may act as a molecular skeleton to bind to Dkc1 and recruit Dck1 to promote snoRNP assembly. Snora62 was responsible for pseudouridylation at 28S rRNA U3507 and U3509 sites. Pseudouridylation levels of 28S rRNA were reduced after knockdown of Snora62. Decreased pseudouridylation levels inhibited the translational activity of its downstream target, Foxh1. Our study further confirmed that Foxh1 transcriptionally promoted the expression of Bax and Fam162a. Notably, experiments in vivo showed that Dcp2 knockdown combined with RNCR3 knockdown and Snora62 knockdown resulted in an anti-apoptosis effect. In conclusion, this study suggests that the axis Dcp2/RNCR3/Dkc1/Snora621 is important for the regulation of neuronal apoptosis induced by CCI.

Seasonal Changes in the Biochemical Composition of Dominant Macroalgal Species along the Egyptian Red Sea Shore

Macroalgae are significant biological resources in coastal marine ecosystems. Seasonality influences macroalgae biochemical characteristics, which consequentially affect their ecological and economic values. Here, macroalgae were surveyed from summer 2017 to spring 2018 at three sites at 7 km (south) from El Qusier, 52 km (north) from Marsa Alam and 70 km (south) from Safaga along the Red Sea coast, Egypt. Across all the macroalgae collected, Caulerpa prolifera (green macroalgae), Acanthophora spicifera (red macroalgae) and Cystoseira myrica, Cystoseira trinodis and Turbinaria ornata (brown macroalgae) were the most dominant macroalgal species. These macroalgae were identified at morphological and molecular (18s rRNA) levels. Then, the seasonal variations in macroalgal minerals and biochemical composition were quantified to determine the apt period for harvesting based on the nutritional requirements for commercial utilizations. The chemical composition of macroalgae proved the species and seasonal variation. For instance, minerals were more accumulated in macroalgae C. prolifera, A. spicifera and T. ornata in the winter season, but they were accumulated in both C. myrica and C. trinodis in the summer season. Total sugars, amino acids, fatty acids and phenolic contents were higher in the summer season. Accordingly, macroalgae collected during the summer can be used as food and animal feed. Overall, we suggest the harvesting of macroalgae for different nutrients and metabolites in the respective seasons.

Ampicillin treated German cockroach extract leads to reduced inflammation in human lung cells and a mouse model of Asthma.

Cockroaches can cause allergic sensitization in humans via contact with their feces or frass. Antibiotics can affect concentration of major allergen and total bacteria production in German cockroaches (Blattella germanica). This study examined the ability of antibiotic-treated German cockroaches to induce allergic airway inflammation and the effect of antibiotics on their lipopolysaccharide and Bla g1, 2, and 5 expression levels. Specifically, we measured the ability of German cockroach extract (with or without prior antibiotic exposure) to induce allergic inflammation in human bronchial epithelial cells and a mouse model of asthma. Bacterial 16S rRNA and lipopolysaccharide levels were lower in ampicillin-treated cockroaches than in the control group. The Bla g1, Bla g2, and Bla g5 expression in ampicillin-treated cockroaches decreased at both the protein and RNA levels. In human bronchial epithelial cell lines BEAS-2B exposed to the ampicillin-treated extract, expression levels of interleukin-6 and interleukin-8 were lower than that in the control group. The total cell count and eosinophil count in bronchoalveolar lavage fluid was also lower in mice exposed to the ampicillin-treated extract than in those exposed to normal cockroach extract. Mouse lung histopathology showed reduced immune cell infiltration and mucus production in the ampicillin group. Our results showed that ampicillin treatment reduced the symbiont bacterial population and major allergen levels in German cockroaches, leading to reduced airway inflammation in mice. These results can facilitate the preparation of protein extracts for immunotherapy or diagnostics applications.

High production of transfer RNAs identifies the presence of developing oocytes in ovaries and intersex testes of teleost fish

5S rRNA is highly transcribed in fish oocytes and this transcription levels can be used to identify the presence of oocytes in the intersex testes of fish exposed to xenoestrogens. Similar to 5S rRNA, tRNAs are transcribed by RNA polymerase III (Pol-III) in eukaryotes, so this study focuses in the analysis of the levels of expression of tRNAs in the gonads (ovaries and testes) of eight teleost species as a possible new oocyte molecular marker. Total RNA extracted from gonads of six commercial teleost species in the Biscay Bay, from the pollution sentinel species thicklip grey mullet (Chelon labrosus) known present intersex testes in response to xenoestrogens in Gernika estuary and from the laboratory model species Danio rerio were analysed through capillary electrophoresis. Bioanalyzer electropherograms were used to quantify the concentrations of tRNAs, 5S and 5.8S rRNA. All studied ovaries expressed significantly higher levels of tRNAs and 5S rRNA than testes. A tRNA to 5.8S rRNA index was calculated which differentiates ovaries from testes, and identifies some intersex testes in between testes and ovaries in mullets. The tRNA/5.8S ratio was highest in ovaries in previtellogenic stage, decreasing towards maturity. Thus, strong oocyte expression of tRNAs is an additional proof of high activity levels of Pol-III during early stages of oocyte development in teleost ovaries. Incidentally, we observed that miRNA concentrations were always higher in testes than ovaries. The indexing approach developed in the present study could have multiple applications in teleost reproduction research and in the development of early molecular markers of intersex condition.

Chidamide Combined with Cytarabine Plays a Role in the Treatment of AML By Down-Regulating the MYC-RRP9 Pathway

Purpose: To explore the therapeutic effect and mechanism of Chidamide combined with Cytarabine in acute myeloid leukemia (AML), and to provide a new treatment strategy for AML patients. Methods: The effects of Chidamide and Cytarabine alone or in combination on the phenotype of AML cell lines were detected by MTT, flow cytometry and Western blot. Transcriptome sequencing and GSEA software were used to explore the mechanism of Chidamide combined with Cytarabine in the treatment of AML. RRP9 overexpression and knockdown AML cell lines were constructed by lentivirus to explore the effects of RRP9 on the proliferation, drug sensitivity, precursor ribosomal RNA (rRNA) process and protein production capacity of AML cell lines. Mature rRNA was detected by qPCR, nascent RNA was detected by EU kit, nucleolar area was observed by electron microscope, and nascent protein was detected by protein synthesis kit. Lentivirus and chromatin immunoprecipitation (CHIP) were used to explore the upstream regulatory genes of RRP9. The effects of Chidamide combined with Cytarabine on the survival rate and molecular level of primary AML cells were detected by CCK-8 and qPCR, respectively. An AML mouse model was constructed to explore the effects of Chidamide and Cytarabine on tumor burden and survival time in AML mice. Results: Chidamide combined with Cytarabine was more effective in killing AML cell lines than single drug, and the combination index was less than 1. The apoptosis rate (P<0.01) and the apoptotic protein level (P<0.05) induced by double drug were both higher than those induced by single drug. Chidamide combined with Cytarabine significantly down-regulated MYC pathway RRP9. In this pathway, RRP9 was selected for further research. An AML cell line overexpressing RRP9 was constructed. RRP9 overexpression promoted the proliferation of AML cell line (p<0.01) and induced the AML cell line to be resistant to the dual-drug combination (P<0.01). Construction of RRP9 knockdown AML cell line. The mature rRNA (P<0.01), nascent RNA (P<0.05), nucleolar area (P<0.0001) and nascent protein level (P<0.05) of cells in the RRP9 knockdown group were significantly lower than those in the control group. In addition, Chidamide combined with Cytarabine reduced the levels of mature rRNA (P<0.05), nascent RNA, nucleolar area (P<0.05), and nascent protein (P<0.05) in AML cell lines. The expression levels of RRP9 in MYC-overexpressing cells and knockdown cells were higher and lower than those in the control group, respectively (P<0.05). CHIP showed that the enrichment abundance of MYC at the RRP9 promoter site was significantly higher than that of IgG (P<0.05). The downregulation of MYC by dual-drug combination was stronger than that of single-drug(P<0.05). Chidamide combined with Cytarabine had stronger killing effect on AML primary cells than single drug, and the combination index was less than 1. Chidamide combined with Cytarabine down-regulated MYC (P<0.05) and RRP9 (P<0.01) mRNA levels in AML primary cells. The results of in vivo imaging of small animals showed that Chidamide alone could significantly reduce the tumor burden in mice (P<0.01), and the combination with Cytarabine further reduced the tumor burden (P<0.01). The survival time of mice in the Chidamide combined with Cytarabine treatment group was significantly longer than that in the control group (median survival time 28 days (28-31 days) vs 23 days (19-26 days)) (P<0.001), and the difference was still statistically significant compared with the single drug (P<0.05). Conclusion: Chidamide combined with cytarabine has a synergistic therapeutic effect on AML cell lines, primary cells and AML mice, which inhibits mature rRNA, total RNA and protein synthesis in AML cells caused AML cell death by synergistically down-regulating the MYC-RRP9 pathway.

Transcription Factor Profiling Reveals That CEBPA Promotes Ribosomal RNA Synthesis in Myeloid Progenitors

Hematopoiesis involves orchestrated differentiation of hematopoietic stem cells (HSCs) and lineage-restricted progenitors to mature blood cells. Different populations in this hierarchy vary in function, size, proliferation, and protein synthesis rates. It is also known that different normal hematopoietic cell types differ in the rates of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been known to have enlarged nucleoli, indicating aberrantly increased rRNA transcription and ribosome biogenesis. Ribosome biogenesis is an energy-intensive process, beginning with the transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA), which is further processed into mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig A). Because rRNA transcription is often considered a housekeeping process, the variations in its cell-type and disease-specific rates have not been studied, leading to a lack of understanding of the mechanisms underlying its regulation. To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we compiled a list of 249 hematopoietic transcription factors (TFs) and epigenetic factors, and mapped 2200 publicly available ChIP-Seq datasets for these factors to rDNA, creating the first atlas of hematopoietic TF-rDNA binding. Through this atlas, we identified that CEBPA shows consistent and abundant binding to human and mouse rDNA at a conserved, previously unknown motif (Fig B). CEBPA is an essential myeloid lineage-specific TF whose knockout leads to complete loss of granulocyte-monocyte progenitors (GMPs) and all downstream myeloid lineage cells. CEBPA also shows hotspot mutations in 10% of AML patients. To characterize the role of CEBPA in rRNA transcription, we used a mouse HoxA9-ER cell line (which closely resembles GMPs), and biallelically fused FKBP degron into endogenous loci of the Cebpa gene, enabling rapid degradation of CEBPA protein using small molecule ligand dTAGV-1 (Fig C, D). To quantify effects on rRNA transcription, we developed a novel assay named '47S-FISH-Flow’ that involves flowcytometric quantification of fluorescent probes hybridized to nascent rRNA in the nucleolus (Fig E, F). We found that depleting CEBPA caused a rapid reduction in 47S rRNA level, plateauing at 35% reduction within hours (Fig G). Decrease in rRNA production was followed by reduction in nucleolar size, reduction in mature ribosome subunit abundance, and reduced growth (Fig H, I). To understand the molecular function of CEBPA on rDNA machinery, we performed time course ChIP-Seq on key rDNA transcriptional machinery, and found that CEBPA degradation specifically reduced occupancy of Pol I along with its initiation factor RRN3 on rDNA (Fig J). In summary, we have found a novel role for the extensively studied myeloid lineage-specific TF CEBPA in directly binding rDNA and regulating rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Microbial molecule ingress promotes neuroinflammation and brain CCR5 expression in persons with HIV-associated neurocognitive disorders

BackgroundSystemic inflammation accompanies HIV-1 infection, resulting in microbial translocation from different tissues. We investigated interactions between lentivirus infections, neuroinflammation and microbial molecule presence in the brain. MethodsBrain tissues from adult humans with (n = 22) and without HIV-1 (n = 11) infection as well as adult nonhuman primates (NHPs) with (n = 11) and without (n = 4) SIVmac251 infection were investigated by RT-PCR/ddPCR, immunofluorescence and western blotting. Studies of viral infectivity, host immune gene expression and viability were performed in primary human neural cells. FindingsAmong NHPs, SIV DNA quantitation in brain showed increased levels among animals with SIV encephalitis (n = 5) that was associated with bacterial genomic copy number as well as CCR5 and CASP1 expression in brain. Microbial DnaK and peptidoglycan were immunodetected in brains from uninfected and SIV-infected animals, chiefly in glial cells. Human microglia infected by HIV-1 showed increased p24 production after exposure to peptidoglycan that was associated CCR5 induction. HIV-1 Vpr application to human neurons followed by peptidoglycan exposure resulted in reduced mitochondrial function and diminished beta-III tubulin expression. In human brains, bacterial genome copies (250–550 copies/gm of tissue), were correlated with increased bacterial rRNA and GroEL transcript levels in patients with HIV-associated neurocognitive disorders (HAND). Glial cells displayed microbial GroEL and peptidoglycan immunoreactivity accompanied by CCR5 induction in brains from patients with HAND. InterpretationIncreased microbial genomes and proteins were evident in brain tissues from lentivirus-infected humans and animals and associated with neurological disease. Microbial molecule translocation into the brain might exacerbate neuroinflammatory disease severity and represent a driver of lentivirus-associated brain disease.

The P-type pentatricopeptide repeat protein DWEORG1 is a non-previously reported rPPR protein of Arabidopsis mitochondria

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein—DWEORG1—from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes.

Quantification of polyhydroxyalkanoate accumulated in waste activated sludge

Polyhydroxyalkanoate accumulation experiments at pilot scale were performed with fullscale municipal waste activated sludge. Development of biomass PHA content was quantified by thermogravimetric analysis. Over 48 h the biomass reached up to 0.49 ± 0.03 gPHA/gVSS (n=4). Samples were processed in parallel to characterise the distribution of PHA in the biomass. Selective staining methods and image analysis were performed by Confocal Laser Scanning Microscopy. The image analysis indicated that nominally 55% of this waste activated sludge was engaged in PHA storage activity. Thus even if the biomass PHA content reached 0.49gPHA/gVSS, the accumulating fraction of the biomass was estimated to have attained about 0.64gPHA/gVSS. The combination of quantitative microscopy and polymer mass assessment enabled to distinguish the effect of level of enrichment in PHA storing bacteria and the average PHA storage capacity of the accumulating bacteria. The distribution of microbial 16S rRNA levels did not follow a measurable trend during PHA accumulation.

Mitochondrial Ribosome Dysfunction in Human Alveolar Type II Cells in Emphysema.

Pulmonary emphysema is characterized by airspace enlargement and the destruction of alveoli. Alveolar type II (ATII) cells are very abundant in mitochondria. OXPHOS complexes are composed of proteins encoded by the mitochondrial and nuclear genomes. Mitochondrial 12S and 16S rRNAs are required to assemble the small and large subunits of the mitoribosome, respectively. We aimed to determine the mechanism of mitoribosome dysfunction in ATII cells in emphysema. ATII cells were isolated from control nonsmokers and smokers, and emphysema patients. Mitochondrial transcription and translation were analyzed. We also determined the miRNA expression. Decreases in ND1 and UQCRC2 expression levels were found in ATII cells in emphysema. Moreover, nuclear NDUFS1 and SDHB levels increased, and mitochondrial transcribed ND1 protein expression decreased. These results suggest an impairment of the nuclear and mitochondrial stoichiometry in this disease. We also detected low levels of the mitoribosome structural protein MRPL48 in ATII cells in emphysema. Decreased 16S rRNA expression and increased 12S rRNA levels were observed. Moreover, we analyzed miR4485-3p levels in this disease. Our results suggest a negative feedback loop between miR-4485-3p and 16S rRNA. The obtained results provide molecular mechanisms of mitoribosome dysfunction in ATII cells in emphysema.

A clinically-relevant residue of POLR1D is required for Drosophila development.

POLR1D is a subunit of RNA Polymerases I and III, which synthesize ribosomal RNAs. Dysregulation of these polymerases cause several types of diseases, including ribosomopathies. The craniofacial disorder Treacher Collins Syndrome (TCS) is a ribosomopathy caused by mutations in several subunits of RNA Polymerase I, including POLR1D. Here, we characterized the effect of a missense mutation in POLR1D and RNAi knockdownof POLR1D on Drosophila development. We found that a missense mutation in Drosophila POLR1D (G30R) reduced larval rRNA levels, slowed larval growth, and arrested larval development. Remarkably, the G30R substitution is at an orthologous glycine in POLR1D that is mutated in a TCS patient (G52E). We showed that the G52E mutation in human POLR1D, and the comparable substitution (G30E) in Drosophila POLR1D, reduced their ability to heterodimerize with POLR1C in vitro. We also found that POLR1D is required early in the development of Drosophila neural cells. Furthermore, an RNAi screen revealed that POLR1D is also required for development of non-neural Drosophila cells, suggesting the possibility of defects in other cell types. These results establish a role for POLR1D in Drosophila development, and present Drosophila as an attractive model to evaluate the molecular defects of TCS mutations in POLR1D.

Cryo-EM structure and rRNA modification sites of a plant ribosome

Protein synthesis in crop plants contributes to the balance of food and fuel on our planet, which influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via the deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and a quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins and 71 plant-specific rRNA modifications, and it re-annotates 30 rRNA residues in the available sequence. At the protein level, isoAsp is found in position 137 of uS11, and a zinc finger previously believed to be universal is missing from eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. At the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation, highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.

Nucleolar function is regulated by the mitochondrial protein sulfite oxidase (SUOX)

Ribosome biogenesis occurs in the nucleolus and relies upon RNA Polymerase I (Pol I) transcription of the rDNA into rRNA. This process is critical for normal cells but is dysregulated in diseases such as Alzheimer’s disease and cancer. To date, however, we have not yet explored the potential relationship between nucleoli and mitochondria. Mitochondria are critical for cellular energy and mitochondrial disorders exhibit dysregulation of nutrient and energy homeostasis. Like nucleoli, mitochondria are also dysregulated in Alzheimer’s disease and cancer, suggesting we may have overlooked a regulatory relationship between these two organelles.In pursuing novel regulators of nucleoli number and function, we have identified a mitochondrial protein that when individually depleted by siRNA in breast epithelial cells (MCF10As) causes a change in nucleolar number – sulfite oxidase (SUOX). Upon SUOX depletion, the nucleolar number decreases from the typical 2‐3 nucleoli to 1 nucleolus. SiRNA‐mediated SUOX depletion reduces nucleolar rRNA levels in a high‐throughput 5‐EU assay (Bryant et al. 2021) and reduces global protein translation by a puromycin incorporation assay, indicating defective nucleolar function. Furthermore, combined metabolomic and transcriptomic analyses following siRNA‐mediated SUOX depletion reveal disruption in the activated methyl cycle, consistent with aberrant ribosome biogenesis. Experiments are underway to validate defective methylation experimentally at specific steps in ribosome biogenesis. The glutamate cycle is also disrupted, congruent with the presentation of the recessive nervous system disorder Isolated Sulfite Oxidase Deficiency (ISOD). Thus, we have defined a novel mitochondrial regulator of nucleolar number and function, nucleolar rRNA biogenesis, and protein translation with implications in human disease. Our work will open up a new avenue of nucleolus‐mitochondria relationships and regulation, and potentially connect mitochondrial disorders with ribosome biogenesis as well as identify how mitochondrial homeostasis is critical for nucleolar function.

Suppression of methanogenesis in paddy soil increases dimethylarsenate accumulation and the incidence of straighthead disease in rice

Some soil microbes can methylate arsenic (As) and produce dimethylarsenate (DMA) as a main product. Excessive accumulation of DMA by rice plants can cause the straighthead disease, a physiological disorder leading to substantial yield losses. DMA can also be demethylated in soil, but the mechanism and the microbes involved are not well understood. We investigated the dynamics of methylated As species, including monomethylarsenate (MMA), DMA and dimethyl-monothioarsenate (DMMTA), in three paddy soils that produced the straighthead disease. The soils were incubated under flooded conditions with or without the addition of 2-bromoethanesulfonate (BES), a specific inhibitor of methanogenesis. DMA and DMMTA concentrations in porewater increased initially as soil redox potential decreased, and then decreased rapidly coinciding with the production of methane. BES addition largely suppressed methanogenesis and the disappearance of DMA and DMMTA, but not of MMA. BES addition suppressed the transcript levels of archaeal 16S rRNA and, particularly, mcrA gene encoding methyl-coenzyme reductase subunit A. Among the core genera of archaea, the absolute abundances of Methanomassiliicoccus and Methanosarcina were decreased significantly by BES in the three soils. In a pot experiment with two soils, BES addition significantly increased DMA accumulation in rice husks and the incidence of the straighthead disease in rice. The results suggest that DMA and DMMTA demethylation in paddy soil is coupled to methanogenesis with Methanomassiliicoccus and Methanosarcina likely playing an important role.

Distinct Survival, Growth Lag, and rRNA Degradation Kinetics during Long-Term Starvation for Carbon or Phosphate.

ABSTRACTThe stationary phase is the general term for the state a bacterial culture reaches when no further increase in cell mass occurs due to exhaustion of nutrients in the growth medium. Depending on the type of nutrient that is first depleted, the metabolic state of the stationary phase cells may vary greatly, and the subsistence strategies that best support cell survival may differ. As ribosomes play a central role in bacterial growth and energy expenditure, ribosome preservation is a key element of such strategies. To investigate the degree of ribosome preservation during long-term starvation, we compared the dynamics of rRNA levels of carbon-starved and phosphorus-starved Escherichia coli cultures for up to 28 days. The starved cultures’ contents of full-length 16S and 23S rRNA decreased as the starvation proceeded in both cases, and phosphorus starvation resulted in much more rapid rRNA degradation than carbon starvation. Bacterial survival and regrowth kinetics were also quantified. Upon replenishment of the nutrient in question, carbon-starved cells resumed growth faster than cells starved for phosphate for the equivalent amount of time, and for both conditions, the lag time increased with the starvation time. While these results are in accordance with the hypothesis that cells with a larger ribosome pool recover more readily upon replenishment of nutrients, we also observed that the lag time kept increasing with increasing starvation time, also when the amount of rRNA per viable cell remained constant, highlighting that lag time is not a simple function of ribosome content under long-term starvation conditions.IMPORTANCE The exponential growth of bacterial populations is punctuated by long or short periods of starvation lasting from the point of nutrient exhaustion until nutrients are replenished. To understand the consequences of long-term starvation for Escherichia coli cells, we performed month-long carbon and phosphorus starvation experiments and measured three key phenotypes of the cultures, namely, the survival of the cells, the time needed for them to resume growth after nutrient replenishment, and the levels of intact rRNA preserved in the cultures. The starved cultures’ concentration of rRNA dropped with starvation time, as did cell survival, while the lag time needed for regrowth increased. While all three phenotypes were more severely affected during starvation for phosphorus than for carbon, our results demonstrate that neither survival nor lag time is correlated with ribosome content in a straightforward manner.

Nimbolide attenuates gut dysbiosis and prevents bacterial translocation by improving intestinal barrier integrity and ameliorating inflammation in hepatocellular carcinoma.

Gut microbiota imbalance plays a key pathological role in hepatocellular carcinoma (HCC) progression; however, the mechanism is poorly understood. We previously showed nimbolide impede tumor development by improving hepatic tight junction (TJ) proteins expression and attenuating inflammation in HCC mice. Here, we aimed to study the role of nimbolide in regulating gut microbiota imbalance and bacterial translocation (BT) through modulating intestinal TJ proteins in an experimental hepatocarcinogenesis. Nimbolide (6mg/kg) was administered orally for 4 weeks following induction of HCC in mice at the 28th week. Nimbolide treatment attenuated the gut microbiota imbalance by decreasing 16 s rRNA levels of Escherichia coli, Enterococcus, Bacteroides and increasing Bifidobacterium, and Lactobacillus in the intestinal tissue, which was otherwise altered in HCC mice. Furthermore, nimbolide improved intestinal barrier integrity in HCC mice by upregulating TJ proteins such as occludin and ZO-1 expression and subsequently prevented hepatic BT and decreased BT markers such as LBP, sCD14, and procalcitonin in the plasma of HCC mice. Moreover, nimbolide ameliorated intestinal and hepatic inflammation by downregulating TLR4, MyD88, and NF-κB protein expression in HCC mice. Thus, nimbolide represents a novel therapeutic drug for HCC treatment by targeting the gut-liver axis, which plays an imperative role in HCC pathogenesis.

Maize RNA 3'-terminal phosphate cyclase-like protein promotes 18S pre-rRNA cleavage and is important for kernel development.

Plant ribosomes contain four specialized ribonucleic acids, the 5S, 5.8S, 18S, and 25S ribosomal RNAs (rRNAs). Maturation of the latter three rRNAs requires cooperative processing of a single transcript by several endonucleases and exonucleases at specific sites. In maize (Zea mays), the exact nucleases and components required for rRNA processing remain poorly understood. Here, we characterized a conserved RNA 3′-terminal phosphate cyclase (RCL)-like protein, RCL1, that functions in 18S rRNA maturation. RCL1 is highly expressed in the embryo and endosperm during early seed development. Loss of RCL1 function resulted in lethality due to aborted embryo cell differentiation. We also observed pleiotropic defects in the rcl1 endosperm, including abnormal basal transfer cell layer growth and aleurone cell identity, and reduced storage reserve accumulation. The rcl1 seeds had lower levels of mature 18S rRNA and the related precursors were altered in abundance compared with wild type. Analysis of transcript levels and protein accumulation in rcl1 revealed that the observed lower levels of zein and starch synthesis enzymes mainly resulted from effects at the transcriptional and translational levels, respectively. These results demonstrate that RCL1-mediated 18S pre-rRNA processing is essential for ribosome function and messenger RNA translation during maize seed development.

BMP7 increases protein synthesis in SW1353 cells and determines rRNA levels in a NKX3-2-dependent manner.

BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.

ВКЛАД mTORC1 И GSK-3 В РЕГУЛЯЦИЮ БИОГЕНЕЗА РИБОСОМ В ПОСТУРАЛЬНОЙ МЫШЦЕ КРЫСЫ В УСЛОВИЯХ МОДЕЛИРУЕМОЙ МИКРОГРАВИТАЦИИ

The investigation was aimed to reveal a potential role of mechanistic target of rapamycin (mTORC1) and glycogen synthase kinase (GSK-3) in the regulation of ribosome biogenesis and protein synthesis in rat soleus muscle during simulated gravitational unloading. Wistar rats were subject to 7-day tail-suspension (hindlimb unloading) and treated with rapamycin (mTORC1 inhibitor) or AR-A014418 (GSK-3 inhibitor). Protein synthesis was measured with SUnSET. Phosphorylation of glycogen synthase-1 (GS-1) and ribosomal protein S6 (rpS6) was measured with the use of Western-blotting. Levels of 28S and 18S rRNA were determined using agarose gel electrophoresis. Expression of 47S pre-rRNA and c-Myc was assessed with RT-PCR. Gravitational unloading led to an increase in phosphorylation of GS-1 (a marker of GSK-3 activity) and inhibited phosphorylation of rpS6 (a marker of mTORC1 activity), expression of the ribosome biogenesis markers (total RNA РНК, 28S and 18S rRNA, 47S pre-rRNA, с-Myc) and protein synthesis in soleus muscle. GSK-3 inhibition prevented an increase in GS-1 phosphorylation and, partly, a reduction in the markers of both ribosomal biogenesis and protein synthesis. The mTORC1 inhibitor contributed to the phospho-rpS6 reduction but did not substantially influence the parameters of translational capacity and protein synthesis. To sum up, unlike mTORC1, an elevated GSK-3 activity can contribute to the inhibition of ribosome biogenesis and protein synthesis in rat soleus muscle under 7-day gravitational unloading.

Organization and expression analysis of 5S and 45S ribosomal DNA clusters in autotetraploid Carassius auratus

BackgroundAutotetraploid Carassius auratus (4n = 200, RRRR) (abbreviated as 4nRR) is derived from whole genome duplication of Carassius auratus red var. (2n = 100, RR) (abbreviated as RCC). Ribosome DNA (rDNA) is often used to study molecular evolution of repeated sequences because it has high copy number and special conserved coding regions in genomes. In this study, we analysed the sequences (5S, ITS1-5.8S-ITS2 region), structure, methylation level (NTS and IGS), and expression level (5S and 18S) of 5S and 45S ribosomal RNA (rRNA) genes in 4nRR and RCC in order to elucidate the effects of autotetraploidization on rDNA in fish.ResultsResults showed that there was high sequence similarity of 5S, 5.8S and ITS1 region between 4nRR and RCC. This study also identified two different types of ITS2 region in 4nRR and predicted the secondary structure of ITS2. It turns out that both secondary structures are functional. Compared with RCC, there was no significant difference in NTS (5S rRNA) methylation level, but the expression level of 5S rRNA was lower in 4nRR, indicating that methylation had little effect on the expression level in 4nRR. IGS (45S rRNA) was hypermethylated in 4nRR compared to RCC, but the expression of 18S rRNA gene was no significantly different from that in RCC, indicating that methylation regulation affected gene expression in 4nRR.ConclusionThe above studies initially revealed the effects of autotetraploidization on the structure and function of 5S and 45S rRNA in Carassius auratus, and provided a theoretical support for the systematic study of the evolution pattern and characteristics of rDNA in vertebrates.