Comparison of global mRNA decay at single-gene resolution in E. coli Δrnb Δrnr versus E. coli Δrnb Δrnr Δfbp
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE315070 | GEO | 2026/07/16
REPOSITORIES: GEO
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Project description:Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives compared to wild type (stabilome) in the stationary phase. The stabilome is an original dynamic transcriptome-based analysis to measure the rates of mRNA degradation at the genome scale. We have combined the analysis of stabilome with the steady state concentrations of mRNAs (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. The stabilome demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. This work allowed the roles of RNase R and PNPase in coordinating E. coli RNA metabolism to be discussed and PNPase to be identified as a central player of the degradation machinery in stationary phase.
2018-12-10 | GSE116576 | GEO
Project description:Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives compared to wild type (stabilome) in the stationary phase. The stabilome is an original dynamic transcriptome-based analysis to measure the rates of mRNA degradation at the genome scale. We have combined the analysis of stabilome with the steady state concentrations of mRNAs (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. The stabilome demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. This work allowed the roles of RNase R and PNPase in coordinating E. coli RNA metabolism to be discussed and PNPase to be identified as a central player of the degradation machinery in stationary phase.
2018-12-10 | GSE116577 | GEO
Project description:PNPase, one of the major enzymes with 3ʹ-to-5ʹ single-stranded RNA (ssRNA) degradation and processing activities, can interact with the RNA helicase RhlB independent of RNA degradosome formation in E. coli. Here we report that loss of interaction between RhlB and PNPase impacts cysteine homeostasis in E. coli. By random mutagenesis, we identified a mutant RhlBP238L that loses 75% of its ability to interact with PNPase, but retains normal interaction with RNase E and RNA in addition to exhibiting normal helicase activity. Applying microarray analyses to an E. coli strain with impaired RNA degradosome formation, we investigated the biological consequences of a weakened interaction between RhlB and PNPase.
2015-11-10 | GSE57784 | 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: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:Our results show the RNA transcripts in complex with the helicase CsdA or the exoribonuclease PNPase. We used CsdA or a catalytically inactive variant of PNPase (PNPase D492G) as bait to capture enriched substrates bound by the enzymes. Sequencing of RNA isolated by crosslinking immunoprecipitation with these enzymes identified numerous transcripts bound to CsdA or PNPase.
2026-07-16 | GSE315071 | GEO
Project description:In all bacterial species examined thusfar, small regulatory RNAs (sRNAs) contribute to intricate patterns of genetic regulation. Many of the actions of these nucleic acids are mediated by chaperones such as the Hfq protein, and genetic screens have identified the exoribonuclease polynucleotide phosphorylase (PNPase) as a stabilizer and facilitator of sRNAs in vivo. We observe that the protective and facilitating effects of PNPase in vivo require the RNA-binding KH and S1 domains and the catalytic site, suggesting a requirement for physical interation of the ribonuclease with either the sRNA itself or other RNAs acting upstream of the process. Although purified PNPase can readily degrade sRNAs in vitro, those same substrates, as well as numerous other sRNAs and transcripts, can be co-purified from cells by immunoprecipitation with neither degradation nor modification to the 3â end. Our results indicate that PNPase can bind RNA in two modes in vivo â either destructive or stabilizing, and that there is active flux of RNAs on PNPase so that the stable molecules do not accumulate. In the presence of Hfq, PNPase and sRNA form a ternary complex in which the enzyme plays a non-destructive, structural role, but the complex does not confer protection against the action of RNase E in vitro. Taken together, our results indicate that PNPase, Hfq and additional factors form a protective ribonucleoprotein assembly that stabilizes certain sRNAs and facilitates their activities. Using cells from the same original culture, immunoprecipitiations using the anti-FLAG M2 resin were performed in the presence or absence of tungstate. Total RNA and immunoprecipitated RNA were sequenced from two independent biological replicates. The number of normalized reads (RPKM) were compared between the total RNA (input) and the immunoprecipitated RNA (output).
2016-01-13 | E-GEOD-69856 | biostudies-arrayexpress
Project description:Glycerol is an attractive feedstock for biofuels since it accumulates as a byproduct during biodiesel operations; hence, it is interesting to consider converting glycerol to hydrogen using the formate hydrogen lyase system of Escherichia coli which converts pyruvate to hydrogen. Starting with Escherichia coli BW25113 frdC that lacks fumarate reductase to eliminate the negative effect of accumulated hydrogen on glycerol fermentation and by using both adaptive evolution and chemical mutagenesis combined with a selection method based on increased growth on glycerol, we obtained an improved strain, HW2, that produces 20-fold more hydrogen in glycerol medium (0.68 mmol/L/h) compared to that of frdC mutant. HW2 also grows 5-fold faster (0.25 1/h) than BW25113 frdC on glycerol, so it achieves a reasonable growth rate. Corroborating the increase in hydrogen production, glycerol dehydrogenase activity in HW2 increased 4-fold compared to BW25113 frdC. In addition, a whole-transcriptome study revealed that several pathways that would decrease hydrogen yields were repressed in HW2 (fbp, focA, and gatYZ) while a beneficial pathway, eno which encodes enolase was induced.
2010-10-31 | GSE18972 | GEO
Project description:We measured steady-state E. coli transcript levels in 1) a pykF(C8Y) mutant, 2) a rpoB(T1037P) mutant, and 3) five rifampicin-resistant rpoB mutant strains selected from each of these single mutants, in the presence and absence of rifampicin.
2020-10-23 | GSE136977 | GEO
Project description:In this study, we performed RNA-sequencing on an E. coli model system to confirm known sites, identify novel targets, and determine the impact of RNase III cleavage events on transcript degradation and metabolic phenotypes. To find cleavage sites, we compared the abundance of sequencing reads across the transcriptome of a wild-type E. coli and an rnc- deletion mutant. The RNA-sequencing approach provided wider coverage and unprecedented resolution of mRNA abundance at each position in the transcriptome compared to prior studies that used qPCR, Northern blots, or microarrays. In addition to data collected from exponentially growing cells, we observed the effects of RNase III cleavage on transcript degradation by collecting samples in a time course after stopping nascent transcription with rifampicin. This work was supported by the Department of Energy Grant DE-SC0010329
2017-02-24 | GSE95318 | GEO