rRNA Transcription Research Articles

Significance and Prospect of Brf1 Overexpression

Brf1 (TFIIB-related factor 1) is a transcription factor, which specifically modulates the transcription of RNA polymerase III-dependent genes (RNA Pol III genes), such as tRNAs and 5S rRNA. The products of tRNAs and 5S rRNA transcription will be changed with the alteration of Brf1 expression. Whereas deregulation of Brf1 and RNA Pol III genes are tightly associated with cell proliferation and transformation, and tumorigenesis. In recent years, emerging studies indicate that Brf1 expression is increased in patients with cancers. In this review, we summarize the progress of the abnormal expression of Brf1 in different human cancers to explore an underlying mechanism and its clinical implication, as well as to prompt its application prospect. With the depth of the Brf1 study and the progress of biotechnology, the status of Brf1 expression may be used as a universal indicator of the early detection and prognosis observation of human cancers.

The stress sensor GCN2 differentially controls ribosome biogenesis in colon cancer according to the nutritional context.

Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor general control non-derepressible 2 (GCN2; also known as eIF-2-alpha kinase 4) represses the expression of the precursor of ribosomal RNA (rRNA), 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, a cell-autonomous GCN2 activity supports cell proliferation by stimulating 47S rRNA transcription, independently of the canonical integrated stress response (ISR) axis. Impairment of GCN2 activity prevents nuclear translocation of methionyl-tRNA synthetase (MetRS), resulting in nucleolar stress, mTORC1 inhibition and, ultimately, autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA polymerase I (RNA pol I) inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls ribosome biogenesis according to the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically stressed COAD cells.

Inhibitory Effects of Ammonia on Archaeal 16S rRNA Transcripts in Thermophilic Anaerobic Digester Sludge

High temperatures exacerbate the ammonia inhibition of anaerobic digestion coupled with methanogenesis. The inhibition of methane production by ammonia has been observed in other studies. However, the underlying mechanism is not well understood and requires further investigation. This study explored the effect of ammonia stress on archaeal 16S rRNA transcripts in thermophilic anaerobic digester sludge. Different ammonium concentrations were checked for their influence on the methanogenic rate and hydrogen accumulation. Quantitative PCR was used to compare the changes in total archaeal 16S rRNA expression. A Monte Carlo permutation test within redundancy analysis (RDA) was adopted for exploring the relationship between environmental variables and archaeal 16S rRNA and their transcripts. The results showed that with the increase in ammonium concentration, the methanogenic rate decreased and hydrogen accumulation occurred. The total archaeal 16S rRNA genes and transcripts copy numbers decreased significantly in treatments with higher ammonium concentrations (7 and 10 g NH4+-N/L), but did not change much at lower ammonia concentrations (3 g NH4+-N/L) compared with the 0 g NH4+-N/L treatment. The RDA analysis further revealed that most environmental variables, including ammonia and methane, except for formate, were significantly correlated with the community structure activity of archaeal 16S rRNA transcripts rather than the community structure of their genes. The composition of archaeal 16S rRNA transcripts showed that the hydrogenotrophic methanogen Methanothermobacter dominated the methanogenic community activity in all incubations. It exhibited sensitivity to ammonia stress and should be responsible for the methanogenic inhibition under thermophilic conditions. Our findings suggested that archaeal 16S rRNA transcripts, rather than 16S rRNA genes, are key indicators of ammonia stress and methanogenic activity.

RRNA intermediates coordinate the formation of nucleolar vacuoles in C. elegans

The nucleolus is the most prominent membraneless organelle within the nucleus. How the nucleolar structure is regulated is poorly understood. Here, we identified two types of nucleoli in C.elegans. Type I nucleoli are spherical and do not have visible nucleolar vacuoles (NoVs), and rRNA transcription and processing factors are evenly distributed throughout the nucleolus. Type II nucleoli contain vacuoles, and rRNA transcription and processing factors exclusively accumulate in the periphery rim. The NoV contains nucleoplasmic proteins and is capable of exchanging contents with the nucleoplasm. The high-order structure of the nucleolus is dynamically regulated in C.elegans. Faithful rRNA processing is important to prohibit NoVs. The depletion of 27SA2 rRNA processing factors resulted in NoV formation. The inhibition of RNA polymerase I (RNAPI) transcription and depletion of two conserved nucleolar factors, nucleolin and fibrillarin, prohibits the formation of NoVs. This finding provides a mechanism to coordinate structure maintenance and gene expression.

Perfect Complementarity Mechanism for Aphid Control: Oligonucleotide Insecticide Macsan-11 Selectively Causes High Mortality Rate for Macrosiphoniella sanborni Gillette.

Macrosiphoniella sanborni is a widespread pest of Chrysanthemum morifolium that causes significant damage to world floriculture. Chemical insecticides and biological methods of control have a number of disadvantages that can be improved by using oligonucleotide insecticides. In this article, we present, for the first time, the results of using oligonucleotide insecticides, for which the target sequence is an internal transcribed spacer (ITS) in a polycistronic rRNA transcript. The mortality of wingless aphid individuals after a Macsan-11 treatment was recorded at a level of 67.15 ± 3.32% 7 days after a single treatment with a solution at a concentration of 100 ng/μL and 97.38 ± 2.49% 7 days after a double treatment with a solution of the same concentration and a daily interval. The contact use of the control oligonucleotide (ACTG)2ACT-11. as well as the oligonucleotide insecticides Macsan-11(3') and Macsan-11(5') was not accompanied by insect mortality. Given the high variability in the internal transcribed spacer, which has proven to be a promising target for the action of oligonucleotide insecticides, it is possible to create selective preparations. This study showed the prospects of ribosomal insect pest genes as targets for the action of olinscides, and also demonstrated the high specificity of such insecticidal agents.

Inhibition of nucleolar transcription by oxaliplatin involves ATM/ATR kinase signaling

Platinum (Pt) compounds are an important class of anti-cancer therapeutics, but outstanding questions remain regarding their mechanism of action. Here, we demonstrate that oxaliplatin, a Pt drug used to treat colorectal cancer, inhibits rRNA transcription through ATM and ATR signaling, and induces DNA damage and nucleolar disruption. We show that oxaliplatin causes nucleolar accumulation of the nucleolar DNA damage response proteins (n-DDR) NBS1 and TOPBP1; however transcriptional inhibition does not depend upon NBS1 or TOPBP1, nor does oxaliplatin induce substantial amounts of nucleolar DNA damage, distinguishing the nucleolar response from previously characterized n-DDR pathways. Taken together, our work indicates that oxaliplatin induces a distinct ATM and ATR signaling pathway that functions to inhibit Pol I transcription in the absence of direct nucleolar DNA damage, demonstrating how nucleolar stress and transcriptional silencing can be linked to DNA damage signaling and highlighting an important mechanism of Pt drug cytotoxicity.

RNA polymerase redistribution supports growth in E. coli strains with a minimal number of rRNA operons.

Bacterial transcription by RNA polymerase (RNAP) is spatially organized. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich medium, with several studies linking RNAP clustering and transcription of rRNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons (Δrrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting five or six of the seven chromosomal rrn operons. Spatial analysis in Δrrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at cell endcaps, where the remaining rrn operons reside. These results support a model where RNAPs in Δrrn strains relocate to copies of the remaining rrn operons. In rich medium, Δrrn strains redistribute RNAP to minimize growth defects due to rrn deletions, with very high RNAP densities on rrn genes leading to genomic instability. Our study links RNAP clusters and rrn transcription, and offers insight into how bacteria maintain growth in the presence of only 1-2 rrn operons.

Ribosomal intergenic spacers are filled with transposon remnants.

Eukaryotic ribosomal DNA (rDNA) comprises tandem units of highly-conserved coding genes separated by rapidly-evolving spacer DNA. The spacers of all 12 species examined were filled with short direct repeats (DRs) and multiple long tandem repeats (TRs), completing the rDNA maps that previously contained unannotated and inadequately studied sequences. The external transcribed spacers also were filled with DRs and some contained TRs. We infer that the spacers arose from transposon insertion, followed by their imprecise excision, leaving short DRs characteristic of transposon visitation. The spacers provided a favored location for transposon insertion because they occupy loci containing hundreds to thousands of gene repeats. The spacers' primary cellular function may be to link one rRNA transcription unit to the next, whereas transposons flourish here because they have colonized the most frequently-used part of the genome.

Detection of Plasmodium simium gametocytes in non-human primates from the Brazilian Atlantic Forest

BackgroundPlasmodium species of non-human primates (NHP) are of great interest because they can naturally infect humans. Plasmodium simium, a parasite restricted to the Brazilian Atlantic Forest, was recently shown to cause a zoonotic outbreak in the state of Rio de Janeiro. The potential of NHP to act as reservoirs of Plasmodium infection presents a challenge for malaria elimination, as NHP will contribute to the persistence of the parasite. The aim of the current study was to identify and quantify gametocytes in NHP naturally-infected by P. simium.MethodsWhole blood samples from 35 NHP were used in quantitative reverse transcription PCR (RT-qPCR) assays targeting 18S rRNA, Pss25 and Pss48/45 malaria parasite transcripts. Absolute quantification was performed in positive samples for 18S rRNA and Pss25 targets. Linear regression was used to compare the quantification cycle (Cq) and the Spearman's rank correlation coefficient was used to assess the correlation between the copy numbers of 18S rRNA and Pss25 transcripts. The number of gametocytes/µL was calculated by applying a conversion factor of 4.17 Pss25 transcript copies per gametocyte.ResultsOverall, 87.5% of the 26 samples, previously diagnosed as P. simium, were positive for 18S rRNA transcript amplification, of which 13 samples (62%) were positive for Pss25 transcript amplification and 7 samples (54%) were also positive for Pss48/45 transcript. A strong positive correlation was identified between the Cq of the 18S rRNA and Pss25 and between the Pss25 and Pss48/45 transcripts. The 18S rRNA and Pss25 transcripts had an average of 1665.88 and 3.07 copies/µL, respectively. A positive correlation was observed between the copy number of Pss25 and 18S rRNA transcripts. Almost all gametocyte carriers exhibited low numbers of gametocytes (< 1/µL), with only one howler monkey having 5.8 gametocytes/µL.ConclusionsFor the first time, a molecular detection of P. simium gametocytes in the blood of naturally-infected brown howler monkeys (Alouatta guariba clamitans) was reported here, providing evidence that they are likely to be infectious and transmit P. simium infection, and, therefore, may act as a reservoir of malaria infection for humans in the Brazilian Atlantic Forest.

Microbial insights towards understanding the role of hydrochar in enhancing phenol degradation in anaerobic digestion

Anaerobic digestion (AD) of wastewater is the most promising bioprocess for organic conversion, however, phenol is toxic and resistant to anaerobic degradation. The current study compared the effect of hydrochar and granular activated carbon (GAC) on AD of phenol at four concentrations (100 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L). Results demonstrated that hydrochar significantly improved the methane production rate and reduced the lag phase at all concentrations of phenol. The methane production rate was improved by about 50% at both 100 mg/L and 250 mg/L phenol, while it was raised by >160% at 500 mg/L and 1000 mg/L phenol by hydrochar. The GAC only increased the methane production rate at 500 mg/L and 1000 mg/L due to high adsorption capacity. Further, the adsorption of phenol by hydrochar had no apparent impact on the methane production rate, even though certain amounts of phenol were adsorbed. At 500 mg/L, the amount of methane produced significantly increased, so 16S rRNA transcripts sequencing and metabolomic analysis were conducted. 16S rRNA transcripts sequencing analysis indicated that hydrochar resulted in the enrichment of syntrophic bacteria (e.g., Syntrophorhabdus &Syntrophobacter) and Methanosaeta, which might be related with direct interspecies electron transfer. Further, it was noticed that the growth of Methanobacterium was repressed at 500 mg/L phenol, while hydrochar promoted its growth. Phenol was degraded into L-tyrosine and then followed the benzoate degradation pathway for methane production as revealed by metabolomic analysis. In addition, metabolomic analysis also revealed that hydrochar promoted the degradation of all metabolites and enhanced the phenol degradation into methane.

Abstract A45: UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus

Abstract Pediatric acute myeloid leukemia (AML) is often refractory to chemotherapy and remains a devastating disease with poor outcomes. Consequently, there is an urgent need to characterize molecular drivers of pediatric AML with the goal of developing more effective molecular therapies. Our group recently identified somatic tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) as a subtype-defining genomic alteration in pediatric AML that is associated with poor outcomes, high relapse rate, and measurable residual disease (MRD) positivity. However, how these alterations drive leukemogenesis is yet to be investigated. Wild-type UBTF regulates rRNA transcription, ribosome biogenesis, and nucleolar formation. Our recent work suggests that UBTF-TD retains these functions. However, we found that expression of UBTF-TD in cord blood CD34+ cells, but not wild-type UBTF, leads to their increased proliferation, self-renewal, and CD117/CD33 surface marker expression, suggesting a potential gain of function as well. Furthermore, we also observed increased expression of HOXB locus genes, PRDM16, MEIS1, and NKX2-3 in the CD34+ UBTF-TD overexpression model relative to our WT control. These expression patterns are similar to what we see in patients, collectively implicating UBTF-TD driving leukemic phenotypes. Based on our localization studies of UBTF-TD to DNA, we hypothesized that UBTF-TD protein may gain the ability to interact with genomic loci that characterize UBTF-TD AML, like the HOXB locus. We therefore wanted to test if UBTF-TD localized to the HOXB cluster and if this lead direct transcriptional upregulation. To test this, we expressed HA-tagged UBTF-TD in cord-blood CD34+ cells and mapped UBTF-TD genomic occupancy using CUT&amp;RUN. We found that UBTF-TD localized to the promoters and gene bodies of key genes, including PRDM16, MEIS1, NKX2-3, and the HOXB cluster. These sites were accompanied by H3K4me3 and H3K27ac active marks without the H3K27me3 repressive mark. To validate these as bone-fide UBTF-TD-occupied regions, we utilized a expressed FKBP12F36V-HA-UBTF-TD fusion in CD34+ cells. This degradation tag (dTAG) system allows for rapid degradation of UBTF-TD protein upon dTAG-13 treatment. We treated our FKBP12F36V-HA-UBTF-TD expressing cord blood CD34+ cells with 1uM of dTAG-13 or DMSO. We found that a 3-day treatment with dTAG-13 led to downregulation of HOXB gene locus expression, but loss of CD117 expression or cell growth required prolonged treatment. Furthermore, our CUT&amp;RUN data showed that dTAG-13 treatment resulted in loss of UBTF-TD protein at the HOXB gene cluster. Collectively, these data show that UBTF-TD interacts with genomic loci at the HOXB gene cluster to directly upregulate their expression. This loss of HOX gene expression leads then leads to loss of a leukemic phenotype. Our data highlights a potential mechanism for UBTF-TD leukemogenesis and sets a foundation where inhibiting the UBTF-TD molecular signature may be a viable therapeutic option. Citation Format: Juan M Barajas, Masayuki Umeda, Ryan Hiltenbrand, Reethu Krishnan, Tamara Westover, Michael P Walsh, Jing Ma, Sherif Abdelhamed, Jeffery M Klco. UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A45.

Novel rRNA transcriptional activity of NhaR revealed by its growth recovery for the bipA-deleted Escherichia coli at low temperature.

The BipA protein is a universally conserved GTPase in bacterial species and is structurally similar to translational GTPases. Despite its wide distribution, BipA is dispensable for growth under optimal growth conditions but is required under stress conditions. In particular, bipA-deleted cells (ESC19) have been shown to display a variety of phenotypic changes in ribosome assembly, capsule production, lipopolysaccharide (LPS) synthesis, biofilm formation, and motility at low temperature, suggesting its global regulatory roles in cold adaptation. Here, through genomic library screening, we found a suppressor clone containing nhaR, which encodes a Na+-responsive LysR-type transcriptional regulator and whose gene product partially restored the growth of strain ESC19 at 20°C. The suppressed cells showed slightly reduced capsule production and improved biofilm-forming ability at 20°C, whereas the defects in the LPS core and swimming motility were not restored but aggravated by overexpression of nhaR. Notably, the overexpression partially alleviated the defects in 50S ribosomal subunit assembly and rRNA processing of ESC19 cells by enhancing the overall transcription of rRNA. Electrophoretic mobility shift assay revealed the association of NhaR with the promoter of seven rrn operons, suggesting that NhaR directly regulates rRNA transcription in ESC19 at 20°C. The suppressive effects of NhaR on ribosomes, capsules, and LPS were dependent on its DNA-binding activity, implying that NhaR might be a transcriptional factor involved in regulating these genes at 20°C. Furthermore, we found that BipA may be involved in adaptation to salt stress, designating BipA as a global stress-responsive regulator, as the deletion of bipA led to growth defects at 37°C and high Na+ concentrations without ribosomal defects.

TELOMERE BIOLOGY AND RIBOSOME BIOGENESIS: STRUCTURAL AND FUNCTIONAL INTERCONNECTIONS.

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

Functional and Taxonomic Diversity of Anaerobes in Supraglacial Microbial Communities.

Cryoconite holes are small ponds present on the surface of most glaciers filled with meltwater and sediment at the bottom. Although they are characterized by extreme conditions, they host bacterial communities with high taxonomic and functional biodiversity. Despite that evidence for a potential niche for anaerobic microorganisms and anaerobic processes has recently emerged, the composition of the microbial communities of the cryoconite reported so far has not shown the relevant presence of anaerobic taxa. We hypothesize that this is due to the lower growth yield of anaerobes compared to aerobic microorganisms. In this work, we aim at evaluating whether the anaerobic bacterial community represents a relevant fraction of the biodiversity of the cryoconite and at describing its structure and functions. We collected sediment samples from cryoconite holes on the Forni Glacier (Italy) and sequenced both 16S rRNA amplicon genes and 16S rRNA amplicon transcripts at different times of the day along a clear summer day. Results showed that a relevant fraction of taxa has been detected only by 16S rRNA transcripts and was undetectable in 16S rRNA gene amplicons. Furthermore, in the transcript approach, anaerobic taxa were overrepresented compared with DNA sequencing. The metatranscriptomics approach was used also to investigate the expression of the main metabolic functions. Results showed the occurrence of syntrophic and commensalism relationships among fermentative bacteria, hydrogenothrophs, and consumers of fermentation end products, which have never been reported so far in cryoconite. IMPORTANCE Recent evidence disclosed the presence of a potential niche for anaerobic microorganisms and anaerobic processes in supraglacial sediments (cryoconite), but a detailed description of the structure and functions of the anaerobic population is still lacking. This work used rRNA and mRNA sequencing and demonstrated that anaerobes are very active in these environments and represent a relevant albeit neglected part of the ecosystem functions in these environments.

Linking Transcriptional Dynamics of Peat Microbiomes to Methane Fluxes during a Summer Drought in Two Rewetted Fens.

Rewetted peatlands are reestablished hot spots for CH4 emissions, which are subject to increased drought events in the course of climate change. However, the dynamics of soil methane-cycling microbiomes in rewetted peatlands during summer drought are still poorly characterized. Using a quantitative metatranscriptomic approach, we investigated the changes in the transcript abundances of methanogen and methanotroph rRNA, as well as mcrA and pmoA mRNA before, during, and after the 2018 summer drought in a coastal and a percolation fen in northern Germany. Drought changed the community structure of methane-cycling microbiomes and decreased the CH4 fluxes as well as the rRNA and mRNA transcript abundances of methanogens and methanotrophs, but they showed no recovery or increase after the drought ended. The rRNA transcript abundance of methanogens was not correlated with CH4 fluxes in both fens. In the percolation fen, however, the mcrA transcript abundance showed a positive and significant correlation with CH4 fluxes. Importantly, when integrating pmoA abundance, a stronger correlation was observed between CH4 fluxes and mcrA/pmoA, suggesting that relationships between methanogens and methanotrophs are the key determinant of CH4 turnover. Our study provides a comprehensive understanding of the methane-cycling microbiome feedbacks to drought events in rewetted peatlands.

Comparison of genomic and transcriptional microbiome analysis in gastric cancer patients and healthy individuals.

Helicobacter pylori and the stomach microbiome play a crucial role in gastric carcinogenesis, and detailed characterization of the microbiome is necessary for a better understanding of the pathophysiology of the disease. There are two common modalities for microbiome analysis: DNA (16S rRNA gene) and RNA (16S rRNA transcript) sequencing. The implications from the use of one or another sequencing approach on the characterization and comparability of the mucosal microbiome in gastric cancer (GC) are poorly studied. To characterize the microbiota of GC using 16S rRNA gene and its transcript and determine difference in the bacterial composition. In this study, 316 DNA and RNA samples extracted from 105 individual stomach biopsies were included. The study cohort consisted of 29 healthy control individuals and 76 patients with GC. Gastric tissue biopsy samples were collected from damaged mucosa and healthy mucosa at least 5 cm from the tumor tissue. From the controls, healthy stomach mucosa biopsies were collected. From all biopsies RNA and DNA were extracted. RNA was reverse transcribed into cDNA. V1-V2 region of bacterial 16S rRNA gene from all samples were amplified and sequenced on an Illumina MiSeq platform. Bray-Curtis algorithm was used to construct sample-similarity matrices abundances of taxonomic ranks in each sample type. For significant differences between groups permutational multivariate analysis of variance and Mann-Whitney test followed by false-discovery rate test were used. Microbial analysis revealed that only a portion of phylotypes (18%-30%) overlapped between microbial profiles obtained from DNA and RNA samples. Detailed analysis revealed differences between GC and controls depending on the chosen modality, identifying 17 genera at the DNA level and 27 genera at the RNA level. Ten of those bacteria were found to be different from the control group at both levels. The key taxa showed congruent results in various tests used; however, differences in 7 bacteria taxa were found uniquely only at the DNA level, and 17 uniquely only at the RNA level. Furthermore, RNA sequencing was more sensitive for detecting differences in bacterial richness, as well as differences in the relative abundance of Reyranella and Sediminibacterium according to the type of GC. In each study group (control, tumor, and tumor adjacent) were found differences between DNA and RNA bacterial profiles. Comprehensive microbial study provides evidence for the effect of choice of sequencing modality on the microbiota profile, as well as on the identified differences between case and control.

Elevated atmospheric CO2 concentrations caused a shift of the metabolically active microbiome in vineyard soil

BackgroundElevated carbon dioxide concentrations (eCO2), one of the main causes of climate change, have several consequences for both vine and cover crops in vineyards and potentially also for the soil microbiome. Hence soil samples were taken from a vineyard free-air CO2 enrichment (VineyardFACE) study in Geisenheim and examined for possible changes in the soil active bacterial composition (cDNA of 16S rRNA) using a metabarcoding approach. Soil samples were taken from the areas between the rows of vines with and without cover cropping from plots exposed to either eCO2 or ambient CO2 (aCO2).ResultsDiversity indices and redundancy analysis (RDA) demonstrated that eCO2 changed the active soil bacterial diversity in grapevine soil with cover crops (p-value 0.007). In contrast, the bacterial composition in bare soil was unaffected. In addition, the microbial soil respiration (p-values 0.04—0.003) and the ammonium concentration (p-value 0.003) were significantly different in the samples where cover crops were present and exposed to eCO2. Moreover, under eCO2 conditions, qPCR results showed a significant decrease in 16S rRNA copy numbers and transcripts for enzymes involved in N2 fixation and NO2− reduction were observed using qPCR. Co-occurrence analysis revealed a shift in the number, strength, and patterns of microbial interactions under eCO2 conditions, mainly represented by a reduction in the number of interacting ASVs and the number of interactions.ConclusionsThe results of this study demonstrate that eCO2 concentrations changed the active soil bacterial composition, which could have future influence on both soil properties and wine quality.

Prostaglandins limit nuclear actin to control nucleolar function during oogenesis.

Prostaglandins (PGs), locally acting lipid signals, regulate female reproduction, including oocyte development. However, the cellular mechanisms of PG action remain largely unknown. One cellular target of PG signaling is the nucleolus. Indeed, across organisms, loss of PGs results in misshapen nucleoli, and changes in nucleolar morphology are indicative of altered nucleolar function. A key role of the nucleolus is to transcribe ribosomal RNA (rRNA) to drive ribosomal biogenesis. Here we take advantage of the robust, in vivo system of Drosophila oogenesis to define the roles and downstream mechanisms whereby PGs regulate the nucleolus. We find that the altered nucleolar morphology due to PG loss is not due to reduced rRNA transcription. Instead, loss of PGs results in increased rRNA transcription and overall protein translation. PGs modulate these nucleolar functions by tightly regulating nuclear actin, which is enriched in the nucleolus. Specifically, we find that loss of PGs results in both increased nucleolar actin and changes in its form. Increasing nuclear actin, by either genetic loss of PG signaling or overexpression of nuclear targeted actin (NLS-actin), results in a round nucleolar morphology. Further, loss of PGs, overexpression of NLS-actin or loss of Exportin 6, all manipulations that increase nuclear actin levels, results in increased RNAPI-dependent transcription. Together these data reveal PGs carefully balance the level and forms of nuclear actin to control the level of nucleolar activity required for producing fertilization competent oocytes.

Dynamics of bacterial ribonucleoprotein bodies probed by single-molecule fluorescence imaging of RNase E.

Ribonucleoprotein (RNP) bodies that exhibit liquid-like internal dynamics facilitate processes such as rRNA transcription and processing, while RNP bodies with gel- or solid-like dynamics (e.g., P-bodies and stress granules) facilitate processes such as mRNA storage. Recently, it has been shown that bacterial RNA degradosomes can assemble into phase-separated condensates called bacterial ribonucleoprotein bodies (BR-bodies). In Caulobacter crescentus, BR-bodies are dynamic and exhibit liquid-like properties, consistent with their mRNA degradation activity. However, under cell stress, the number of BR-bodies increases significantly to give C. crescentus cells a fitness advantage. Furthermore, upon entrance into the stationary phase, an increased number of BR-bodies correlates with a slowdown in the mRNA decay rate. These results raise the question of whether BR-bodies can facilitate mRNA storage. Here, we use bulk fluorescence imaging and live-cell single-molecule tracking to probe the dynamics of the ribonuclease RNase E, the central scaffold of the BR-body. We found that during stationary phase, BR-bodies are stable for longer have a longer compared to exponential phase. Consistent with these results, the residence time of RNase E in BR-bodies in stationary phase cells is at least two orders of magnitude longer than in exponential phase cells. Finally, we measured the size and abundance of BR-bodies during both growth stages and found that during stationary phase, BR-bodies are smaller and more abundant, which suggests that the condensate phase is not conducive to fusion. Together, our data shows an increased stability of RNAse E in the stationary phase, which may indicate a gel or solid phase transition. Furthermore, we anticipate our single-molecule measurements will provide a foundation for probing the mechanical properties of subcellular compartments in living bacteria.

Improving an rRNA depletion protocol with statistical design of experiments

<h2>Abstract</h2> In prokaryotic RNA-seq library preparation, rRNA depletion is required to remove highly abundant rRNA transcripts from total RNA. rRNA is so abundant that small improvements in depletion efficiency lead to large increases in mRNA sequencing coverage. The current gold-standard method for rRNA depletion makes rRNA depletion the most expensive step in prokaryotic RNA-seq library preparation. A variety of commercial and home-made methods exist to lower the cost or increase the efficiency of rRNA removal. Many of these techniques are suboptimal when applied to new species of bacteria or when the protocol or reagents need to be changed. Re-optimizing a protocol by trial-and-error is an expensive and laborious process. Systematic frameworks like the statistical design of experiments (DOE) can efficiently improve processes by exploring the quantitative relationship between multiple factors. DOE allows experimenters to find factor interactions that may not be apparent when factors are studied in isolation. We used DOE to optimize an rRNA depletion protocol by updating reagents and identifying factors that maximize rRNA removal and minimize cost. The optimized protocol more efficiently removes rRNA, uses fewer reagents, and is less expensive than the original protocol. Our optimization required only 36 experiments and identified two significant interactions among three protocol factors. Overall, our approach demonstrates the utility of a rational, DOE framework for improving complex molecular biology protocols.