RIP-seq of RNA associated in vivo with the PNPase and PNPaseD493A proteins, performed in Bacillus subtilis 9407
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ABSTRACT:
PROVIDER: PRJNA1017853 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26071600_1.fastq.gz | Fastqsanger.gz | |||
| SRR26071600_2.fastq.gz | Fastqsanger.gz | |||
| SRR26071601_1.fastq.gz | Fastqsanger.gz | |||
| SRR26071601_2.fastq.gz | Fastqsanger.gz | |||
| SRR26071602_1.fastq.gz | Fastqsanger.gz |
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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:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.
2012-05-25 | GSE34505 | 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:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.7) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three or four independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.
2012-05-24 | E-GEOD-34505 | biostudies-arrayexpress
Project description:The Bacillus subtilis genome encodes four 3’ exoribonucleases: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Previous work showed that PNPase, encoded by the pnpA gene, is the major 3’ exonuclease involved in mRNA turnover; in a pnpA deletion strain, numerous mRNA decay intermediates accumulate. Whether B. subtilis mRNA decay occurs in the context of a degradosome complex is controversial. In this study, global mapping of mRNA decay intermediate 3’ ends within coding sequences was performed in strains that were either deleted for, or had an inactivating point mutation in, the pnpA gene. The pattern of 3’ end accumulation in these strains was highly similar, suggesting that mRNA decay was not occurring in the context of a degradosome, whose structure would be affected by the absence of PNPase. A comparison with mapped 3’ ends in a strain lacking CshA, the major RNA helicase, indicated that different mRNAs may require both PNPase and CshA for efficient decay. RNA-seq analysis of strains lacking RNase R suggested that this enzyme did not play a major role in mRNA turnover in the wild-type strain. Strains were constructed that contained only one of the four known 3’ exoribonucleases. When RNase R was the only 3’ exonuclease present, it was able to degrade a model mRNA efficiently, showing processive decay even through a strong stem-loop structure that inhibits PNPase processivity. Strains containing only RNase PH or only YhaM were also insensitive to this RNA secondary structure, suggesting the existence of another, as-yet unidentified, 3’ exoribonuclease.
2022-01-03 | GSE192670 | 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
Project description:In order to assess the prevalence of cotranslational assembly of protein complexes we performed RIp-chip experiments with many proteins that do not conatin RNA-binding motifs
2012-05-20 | E-TABM-1158 | biostudies-arrayexpress
Project description:Messenger RNA decay in Bacillus subtilis is accomplished by a combination of exoribonucleases and endoribonucleases. Intermediates in the decay process have not been readily detectable, and previous studies on mRNA decay have used a handful of highly expressed transcripts as models. Here, we use RNA‐Seq analysis to probe mRNA turnover globally. A significant fraction of messages showed differential accumulation of RNA fragments that mapped near the 5′ or 3′ end of the coding sequence, consistent with initiation of decay from either the 5′ end or from an internal cleavage site. Patterns of mRNA decay in the wild type were compared with patterns in a mutant strain lacking polynucleotide phosphorylase (PNPase), which is considered the major 3′ exonuclease activity in mRNA decay and which is one of four known 3′ exonucleases in B. subtilis. The results showed a striking dependence on PNPase for mRNA turnover in many cases, suggesting specificity in the ability of 3′ exonucleases to degrade from 3′‐hydroxyl termini. RNA‐Seq data demonstrated a sharp decrease in expression of Sigma D in the PNPase‐deletion strain. Reduction in sigD regulon expression explained the chain growth phenotype of the PNPase mutant and also predicted a defect in swarming motility.
2018-09-18 | GSE120050 | 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:We have used RIp-chip to identify mRNAs that coprecipitae with specific RNA-binding proteins
2014-11-06 | E-MTAB-2709 | biostudies-arrayexpress