High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation with CsdA or PNPase in E.coli
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE315071 | GEO | 2026/07/16
REPOSITORIES: GEO
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Project description:Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and several human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. PKO resulted in loss of mitochondrial DNA (mtDNA) and a transcriptional profile similar to rho0 mtDNA deleted cells, with perturbations in cholesterol, lipid, and secondary alcohol metabolic pathways. PKO cells also showed growth and cell cycle profiles similar to rho0 cells. PKO in mouse inner ear hair cells cause progressive hearing loss that parallel human familial hearing loss previously linked to mutations in PNPase. Combined, our data suggest that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase.
2018-05-01 | GSE111668 | GEO
Project description:Background The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome. Results In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. Conclusions In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.
2015-03-17 | GSE60107 | GEO
Project description:Transcriptional profiling of PNPase KO liver hepatocytes (HepKO) generated from Albumin-Cre/wt (liver-specific) Pnpt1 fl/fl C57BL/6J mice. Samples comparing liver hepatocyte PNPase KO (HepKO) cells to litter-, age-, and sex-matched wildtype hepatocyte control cells. The goal of this experiment was to determine effects of PNPase loss on the total RNA transcriptome under physiologic in vivo conditions.
2018-05-03 | GSE109210 | GEO
Project description:In many gram-negative and some gram-positive bacteria small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism, and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA-mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase uniquely contributes to the degradation of specific mRNA cleavage products, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of a sRNA eliminated the requirement of PNPase for sRNA stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing mediated decay.
2019-07-08 | GSE125368 | GEO
Project description:Production of secondary metabolites is controlled by a complicated regulatory network in eukaryotic cells. Several layers of regulators are involved in this process, ranging from pathway-specific regulation, to epigenetic control, to global regulation. Unprecedentedly, we have discovered that interaction of an RNA-binding protein CsdA with a regulator RsdA coordinates fungal secondary metabolism. Employing a genetic deletion approach and transcriptome analysis as well as metabolomics analysis, we revealed that CsdA and RsdA synergistically regulate fungal secondary metabolism comprehensively. Mechanistically, comprehensive genetic and biochemical studies proved that RsdA and CsdA co-localize in the nucleus and physically interact to achieve their function. In particular, we have demonstrated that CsdA mediates rsdA expression by binding specific motif “GUCGGUAU” of its pre-mRNA at a post-transcriptional level. We thus uncover a general mechanism in which RNA-binding protein physically interacts with, and controls the expression level of, the RsdA to coordinate fungal secondary metabolism.
2023-10-23 | GSE241019 | GEO
Project description:Fungal secondary metabolism plays a critical role in pathogen-host interactions, yet the regulatory networks linking metabolic reprogramming to virulence remain poorly understood. This study identifies a conserved regulatory hub in the human pathogen Aspergillus fumigatus, where the RNA-binding protein (RBP) CsdA interacts with the global regulator LaeB in the nucleus to control biosynthesis of the secondary metabolite fumiquinazoline C (FqC). Disruption of the CsdA-LaeB interaction hyperactivates FqC production, enhancing fungal colonization and lethality in murine invasive aspergillosis models. Integrative metabolomic and transcriptomic analyses reveal that CsdA and LaeB function as co-regulators of a broader secondary metabolic gene cluster network, with FqC emerging as an effector that mediates virulence in vivo. Genetic validation confirms that FqC is strictly required for the increased virulence phenotype of CsdA- or LaeB-deficient strains, while analyses of clinical isolates demonstrate a striking inverse correlation: reduced CsdA and LaeB expression coincides with elevated FqC production, showing consistency with the infection outcomes of the deletion mutants. This work identifies the RBP-based interaction that coordinates fungal metabolic virulence, shedding new light on the post-transcriptional regulatory logic linking secondary metabolism to pathogenicity and offering alternative strategies for diagnostic development and therapeutic intervention in invasive fungal diseases.
2025-12-31 | GSE312095 | GEO
Project description:In the present study we applied NAD captureSeq to charecterize the 5'-NAD-RNA profile of E. coli BW25113 wt strain as well as nudC and csdA knockouts. The data obtained showed that both knockouts resulted in an increased number of identified 5’-NAD-RNA species, thus suggesting that CsdA might add a potentially new layer of control on NAD-epitranscriptomic landscape.
2023-08-22 | GSE224605 | GEO
Project description:PNPase is the primary RNA turnover enzyme in Bacillus subtilis and plays a crucial role in regulating gene expression. To investigate the life activities that PNPase participated in Bacillus subtilis 9407, RNA immunoprecipitation-sequencing (RIP-seq) analysis was performed to pinpoint the direct RNA targets of PNPase using 6×his-tagged PNPase (PNPase group) and PNPaseD493A (D493A group) constructs that were driven by the IPTG-induced Pgrac promoter.
2023-09-20 | GSE243342 | GEO
Project description:Biofilm-related diseases are typically persistent infections, and a challenge for medical treatment. Biofilms are communities of bacteria that attach to surfaces and are enclosed in an extracellular matrix. These sessile microorganisms can endure external stresses like nutrient deprivation, antibiotic treatments, and immune defences. Therefore, biofilms create conditions favourable for bacterial pathogenesis. The knowledge of novel biofilm regulators may contribute to develop new strategies to fight microbial infections. In this work we study the role of the RNA-binding protein and RNA-degradative enzyme polynucleotide phosphorylase (PNPase) from the human pathogen Listeria monocytogenes. We show that inactivation of Listeria PNPase not only leads to strong defects in biofilm production, but also affects biofilm morphology. RNA-seq analysis of the RNA extracted from biofilms of the wild-type and the PNPase mutant strains revealed major changes in the expression of genes affecting the metabolism of carbon. Lastly, infection assays in eukaryotic cell lines confirmed that PNPase deletion leads to the severe attenuation of Listeria monocytogenes pathogenicity. Overall, our results show that PNPase is a novel regulator of biofilm formation and human cellular invasion of a bacterial pathogen. This work presents PNPase as a new and attractive target for the control of bacterial infection and highlights the expanding role of RNA-binding proteins as critical players in pathogenicity.
2023-06-05 | GSE210097 | GEO
Project description:8-oxo-7,8-dihydroguanine (8-oxoG) is a common RNA oxidation that has been implicated in carcinogenesis, neurodegeneration, and aging. Some? Many? RNA-binding proteins have shown binding preference for 8-oxoG modified RNA; such binding? can protect cells from oxidative stress. To date, the presence of specific 8-oxoG-recognizing proteins is only known in model organisms, but, how these proteins cooperate is still unclear. Here, we use RNA affinity chromatography and mass spectrometry to search for proteins that specifically bind 8-oxoG-modified RNA in Deinococcus radiodurans, an extremophilic bacterium with extraordinary resistance to oxidative stresses. We identified four proteins that selectively bind to oxidized RNA: polynucleotide phosphorylase (DR_2063/PNPase), ATP-dependent DEAD-box RNA helicase (DR_0335/RhlB), ribosomal protein S1 (DR_1983/RpsA), and transcriptional termination factor (DR_1338/Rho). The knockout of PNPase and RhlB in D. radiodurans caused increased sensitivity to hydrogen peroxide and a significant accumulation of 8-oxoG modified RNA. Importantly, PNPase directly interacts with RhlB in D. radiodurans; however, no additional phenotypic effect was observed for the double deletion of pnp and rhlB compared to the individual single deletions. Overall, our findings suggest a role of the PNPase-RhlB complex in targeting 8-oxoG-modified RNAs for rapid degradation and thereby constitutes an important component of D. radiodurans resistance to oxidative stress.
2022-07-09 | PXD028866 | Pride