Cooperation of GRSF1 and the mitochondrial degradosome (hSuv3-PNPase complex) in degradation of mitochondrial RNA
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ABSTRACT:
PROVIDER: PRJNA416454 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Vertebrate mitochondrial genomes show extraordinary GC skew which results in the synthesis of RNAs prone to form G-quadruplexes (G4). Such RNAs, although mostly non-coding, are transcribed at high rate and swiftly degraded by an unknown mechanism. Comprehensive proteomic and transcriptomic approaches revealed that quasi-RRM protein GRSF1 together with the mitochondrial degradosome composed of RNA helicase hSuv3 and PNPase control the levels of G4 containing RNAs in mitochondria. When this machinery is inactivated non-coding mtRNAs are upregulated and novel, previously undescribed transcript, which we named tRNA-like, strongly accumulates. In vitro reconstitution experiments showed that GRSF is responsible for G4 RNA melting essential for degradosome-mediated decay.
2018-06-17 | GSE106368 | GEO
Project description:Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts capable of forming long double-stranded RNA structures. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here, we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single cell level and identify keyroles for the degradosome components, mitochondrial dsRNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms evolved to protect vertebrates against microbial and viral attack.
2018-05-03 | GSE94957 | GEO
Project description:The human mitochondrial degradosome is a complex of the ribonuclease PNPase (Q8TCS8, encoded by PNPT1 gene) and RNA helicase SUV3 (Q8IYB8, encoded by SUPV3L1 gene). The aim of the project was to identify proteins which co-purify with both subunits of the degradosome. To this end we used human 293 cells stably expressing hSuv3 or PNPase with a C-terminal TAP tag or TAP tag fused to a mitochondria targeting sequence (control bait) (cell lines are described in details in (PMID: 19864255). Mitochondria were isolated from the cells, lysed and the protein extracts were subjected to affinity chromatography. Co-purified proteins were identified by mass spectrometry and their amounts were quantified by a label-free approach (MaxQuant). These experiments are part of the project “Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria”.
2018-07-06 | PXD009826 | Pride
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: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: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:The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.
2019-06-28 | GSE131742 | GEO
Project description:Localization of RNase E to the inner membrane in Escherichia coli is well documented, but the functional consequences of this localization are largely unknown. Here we characterize the rne∆MTS strain, which expresses cytoplasmic RNase E (cRNase E). CsrB and CsrC regulatory RNAs are stabilized in the rne∆MTS strain resulting in leaky glycogen expression. There is a small but significant global slowdown in mRNA degradation with no bias considering function or localization of encoded proteins. RNase E is a stable protein, but cRNase E is unstable with a half-life equal to the doubling time of exponentially growing cells. cRNase E instability is compensated by increased synthesis. Co-purification experiments show that cRNase E associates with RhlB, enolase and PNPase to form a cytoplasmic RNA degradosome. Measurements in multiple turnover assays show that there is no difference in Km or kcat between cRNase E and RNase E. In contrast to the global slowdown of mRNA degradation, the inactivation of a ribosome-free lacZ transcript is faster in the rne∆MTS strain. We discuss how the association of RNase E with the inner cytoplasmic membrane is important for carbon storage regulation, degradation of polyribosomal mRNA, protection of ribosome-free transcripts from inactivation and stability of RNase E.
2019-11-25 | GSE118058 | 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: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