Transcriptomic changes associated with the depletion of RNA degradosome components PNPase, RNase E and RNase J in Mycobacterium tuberculosis.
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ABSTRACT:
PROVIDER: PRJNA521480 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8550310_1.fastq.gz | Fastqsanger.gz | |||
| SRR8550310_2.fastq.gz | Fastqsanger.gz | |||
| SRR8550311_1.fastq.gz | Fastqsanger.gz | |||
| SRR8550311_2.fastq.gz | Fastqsanger.gz | |||
| SRR8550302_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA-Seq results accompanying submission of a manuscript describing roles of RNA degradosome components PNPase, RNase E and RNase J in shaping the transcriptome of the H37Rv Mycobacterium tuberculosis. Next generation sequenicing results are provided for three independent biological replicates for the wild type control, genetic knockout of RNase J and CRISPR/Cas9 (DNA cleavage defiecient) regulated knockdowns of PNPase and RNase E along with a control strain expressing solely the Cas9 enzyme, not the target sgRNA. We have found that removal of RNase J had little effect on the transcriptional profile of investigated strain, while depletion of PNPase or RNase E altered about a quarter of the entire transcriptome, including changes to non-coding RNA.
2019-02-09 | GSE126286 | GEO
Project description:The dataset accompanying a publication describing the RNA degradosome components in Mycobacterium tuberculosis (Mtb). Individual RNA degradosome components from Mtb (PNPase, RNase E, RNase J, RhlE and Enolase) were tagged with eGFP and used as baits to pull-down their interaction partners. The protein-complexes were purified by affinity chromatography, using previously published protocols, and submitted to LC-MS analysis.
2019-04-01 | PXD012662 | Pride
Project description:To identify mRNA substrates for the human RNase MRP complex, we examined the effects of siRNA-mediated depletion of RNase MRP (and RNase P) on the transcriptome of HEp-2 cells. The expression of two RNase MRP protein components, hPop1 and Rpp40, was knocked-down and after 48 hours mRNA was isolated from these cells as well as from cells transfected with an siRNA targeting the GFP mRNA (siEGFP), which was used as a control. The expression levels of mRNAs were analyzed on a genome-wide scale using 21K microarrays.
2011-04-01 | E-TABM-981 | biostudies-arrayexpress
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:The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the degradosome, the PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay since it acts as a 3’- to 5’-exoribonuclease. Furthermore PNPase can catalyze the reverse reaction by elongating RNA molecules in 5’- to 3’-end direction which finally has a destabilizing effect on the prolonged RNA molecule. RNA degradation can be initiated by an endonucleolytic cleavage and then by further promoted by exoribonucleolytic decay. In this study, we analyzed the importance of the Rhodobacter sphaeroides PNPase regarding the physiology and sRNA homeostasis. Moreover, we developed a pipeline to globally identify differential RNA 3’-ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants. The Rhodobacter sphaeroides PNPase mutant exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. Furthermore, the transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, the PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. The RNA 3’-end analysis reveals that 82% of all differential 3’-ends are enriched in the pnp mutant and are mostly located in coding sequences or untranslated regions (UTR). A fair percentage of these ends was also identified as differential RNA 3’-end in RNase E and RNase III mutant strains. The PNPase has a major influence in RNA processing and maturation and thus modulates the transcriptome. This includes regulatory sRNAs, stressing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The 3’-end analysis indicates a sequential processing of UTR- and non-UTR derived sRNAs by several ribonucleases. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5’/3’-ends. It is publicly available on GitHub and is distributed under ICS licence.
2021-02-16 | GSE156818 | GEO
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: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:In Helicobacter pylori, the major actor in post-transcriptional regulation is an RNA degradosome assembly composed of the essential ribonuclease RNase J and the DEAD-box RNA helicase RhpA. Here, we describe the oligomeric state and post-translational modifications of this protein complex. Our crystal structure of RNase J at 2.75 Å resolution reveals a homotetrameric quaternary state, and solution studies show that purified RNase J forms monomers and dimers, that RhpA is a monomer, and that the two proteins interact to form a stable complex with a 1:1 stoichiometry. Using mass spectrometry, we observed that RNase J is acetylated on multiple residues, one of which, K649, is important for RNase J oligomerization. Mutations targeting this residue and others affect the dimerization and in vitro activity of RNase J, suggesting that the dimer is the active form. In H. pylori, we observed that several of the identified acetylated residues impact on cell morphology, suggesting their importance for RNase J cellular function. Using H. pylori mutants, we found that several pathways contribute to RNase J acetylation. We propose acetylation as a regulatory level controlling RNase J properties and as a consequence the activity of the H. pylori RNA degradosome.
2023-12-12 | PXD027670 | Pride
Project description:RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we report that of 11 tested potential RNases of the intestinal pathogen Yersinia pseudotuberculosis, two, the endoribonuclease RNase III and the exoribonuclease PNPase, repress the synthesis of the master virulence regulator LcrF. LcrF activates the expression of virulence plasmid genes encoding the type III secretion system (Ysc-T3SS) and its substrates (Yop proteins), that are employed to inhibit immune cell functions during infection. Loss of both RNases led to an increase in lcrF mRNA levels and stability. Our work indicates that PNPase exerts its influence through YopD, known to accelerate lcrF mRNA degradation. Loss of RNase III results in the downregulation of the CsrB and CsrC RNAs, leading to increased availability of active CsrA, which has previously been shown to enhance lcrF mRNA translation and stability. Other factors that influence the translation process and were found to be differentially expressed in the RNase III-deficient mutant could support this process. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal towards virulence plasmid-encoded genes. A similar extensive transcriptional reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. This illustrated that RNase III enables immediate coordination of virulence traits, such as Ysc-T3SS/Yops, with other functions required for host-pathogen interactions and survival in the host.
2024-01-23 | GSE249386 | GEO
Project description:Abstract: Ribonuclease E (RNase E) has a key role in mRNA degradation and the processing of catalytic and structural RNAs in E. coli. We report the discovery of an evolutionarily conserved 17.4 kDa protein, here named RraA (regulator of ribonuclease activity A) that binds to RNase E and inhibits RNase E endonucleolytic cleavages without altering cleavage site specificity or interacting detectably with substrate RNAs. Overexpression of RraA circumvents the effects of an autoregulatory mechanism that normally maintains the RNase E cellular level within a narrow range, resulting in the genome-wide accumulation of RNase E-targeted transcripts. While not required for RraA action, the C-terminal RNase E region that serves as a scaffold for formation of a multiprotein degradosome complex modulates the inhibition of RNase E catalytic activity by RraA. Our results reveal a possible mechanism for the dynamic regulation of RNA decay and processing by inhibitory RNase binding proteins. This SuperSeries is composed of the SubSeries listed below.
2006-03-08 | GSE4383 | GEO