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Identification of PNPase and RhlB binding proteins in Deinococcus radiodurans

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Deinococcus Radiodurans Atcc 13939

SUBMITTER: RUNHUA HAN

LAB HEAD: Lydia M. Contreras

PROVIDER: PXD028866 | Pride | 2022-07-09

REPOSITORIES: pride

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			Action	DRS
	8040_RH_1.msf	Msf
	8040_RH_10.msf	Msf
	8040_RH_11.msf	Msf
	8040_RH_12.msf	Msf
	8040_RH_2.msf	Msf

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Identification of 8-oxoG binding proteins in Deinococcus radiodurans

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.

2022-07-10 | PXD028861 | Pride

Immunoprecipitation from rat kidney with 8-oxoguanine

Project description:8-oxoguanine (8-oxoG) is one of the most common DNA lesions generated by reactive oxygen species. Here we show the genome-wide distribution of 8-oxoG by coupling immunoprecipitation by antibodies specific for the DNA fragments containing 8-oxoG with microarray that covers rat genome. Genome-wide mapping of 8-oxoG in rat kidney reveals that 8-oxoG preferentially located at gene deserts and depleted in genic regions. We did not observe the difference in 8-oxoG level between highly- and lowly-expressed groups of genes, may be because of the low level of 8-oxoG in genic regions in general comparing to gene deserts. Rather, the distribution of 8-oxoG highly correlates with the distribution of lamina associated domains (LADs), suggesting that the spatial location of the genomic regions in the nucleus is the major determinant for the susceptibility to oxidative modifications. One possible explanation for the enrichment of 8-oxoG in LADs is that the nuclear periphery is more susceptible to the oxidative damage coming from outside the nucleus. Another explanation is that LADs take rather compact conformation, which might prevent the recruitment of the repairing complex to the modified bases. Normal condition. Biological replicate 2

2014-01-13 | E-GEOD-54007 | biostudies-arrayexpress

Using 14mer affinity pulldown to identify sRNA associating proteins in the bacterium Deinococcus radiodurans

Project description:We employed a 14-mer pulldown protocol that was originally developed for eukaryotic miRNA interacting RBPs. In this assay, the sRNA (Dsr2) and a scrambled RNA with the same length were in vitro transcribed with a 5 located, 14 nt long tag and subsequently incubated with D. radiodurans lysates. The resulting co-purified proteins were subjected to LC-MS/MS. Using this alternative approach (hereinafter referred to as 14 mer pull-down), we successfully enriched a total of 66 proteins in the 14 mer Dsr2 samples compared to the control sample. Except for several metabolic and hypothetical proteins, many of these proteins have potential nucleic acid binding functions were enriched, including three ribosomal proteins, two translation initiation factors, two TetR transcriptional regulators, five tRNA modification or processing proteins, DNA polymerase I, and five DNA repair proteins (e.g., RuvB, SbcD, and RecR). DR_2281 is one of the most enriched RBPs which contains a double stranded RNA binding motif (DSBM) but its function is unknown. In contrast, five other proteins (PNPase, RhlB, Ffh, DR_2281, KhpA and KhpB) were known as RBPs previously or contain known RNA binding domains. PNPase, RhlB, and Ffh have been revealed to play crucial roles in oxidative stress response in D. radiodurans by interacting with either oxidized RNA or Signal Recognition Particle RNA (Qpr6). PNPase also plays an indispensable role in paradoxically stabilizing sRNAs bound to other RBPs and promoting their function in gene regulation in E. coli (indicating a similar role of PNPase in D. radiodurans).

2024-05-23 | MSV000094855 | MassIVE

Proteins associated with Cdc48A identified by LC-MS\MS data

Project description:His6-Cdc48a and associated proteins purified from H. volcanii. A) SDS- PAGE of proteins purified by Ni2+ -chromatography. H. volcanii carrying empty vector control (H1209-pJAM202c, lane 1) and ectopically expressing His6-Cdc48a (H1209- pJAM1409, lane 2). Red bars indicate regions of gel excised. B) Proteins identified in gel slices by LC-MS/MS analysis. Criteria for protein inclusion: FDR < 0.01%, normalized total spectra minus control > 50, protein threshold > 99% and 2 peptide minimum. Based on spectral counting, Cdc48a and DUF111 were most abundant in regions (a) and (b), respectively, as indicated. DHS1, deoxyhypusine synthase. DUF111, Cdc48a gene neighbor of unknown function. TnaA, tryptophanase; Rps3, ribosomal protein S3; n.d., not detected.

2023-06-22 | PXD019906 | Pride

Promoters Active in Interphase are Bookmarked during Mitosis by Ubiquitination

Project description:We analyzed genome-wide modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle using modified ChIP-seq technique (ChAP-seq). Chromatin affinity purification was based on a standard ChIP method with modification of a two-step affinity purification.

2013-04-04 | E-GEOD-45771 | biostudies-arrayexpress

Polynucleotide phosphorylase can interact with small noncoding RNAs in both stabilizing and degradative modes

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

Immunoprecipitation from rat kidney with 8-oxoguanine

2014-01-13 | GSE54007 | GEO

Ubiquitination of DEAD-box RNA helicase DDX3X by TRIM25

Project description:Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade and plays both RING (really interesting new gene)-dependent and RING-independent roles in RIG-I-mediated IFN induction. We report that the PRY-SPRY domain of TRIM25 interacts with the N-terminal extension (NTE) of DEAD-box helicase 3X (DDX3X), a host protein with multiple roles in RLR signalling. Gene reporter assays and knockdown studies reveal DDX3X and TRIM25 cooperate to activate the IFN- promoter following RIG-I activation independent of DDX3X’s catalytic activity. We also show that TRIM25 ubiquitinates DDX3X at several lysine residues in vitro and in cells.

2021-09-08 | PXD028122 | Pride

RIP-seq of RNA associated in vivo with the PNPase and PNPaseD493A proteins, performed in Bacillus subtilis 9407

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

8-oxoG RNA IP data set 2: Air pollution mixture derived from the reaction of 100 ppb acrolein, 97 ppb methacrolein, 44 ppb alpha-pinene and 100 ppb O3 for 1.5 h

Project description:While environmental agents such as air pollution have been shown to be causative to diseases, yet limited knowledge exist on their mechanism of action. Here we used RNA-sequencing in combination with specific immunoprecipitation of 8-oxo-7,8-dihydroguanosine (8-oxoG) to identify transcripts accumulating oxidation after exposure to air pollution (derived from the reaction of acrolein, methacrolein, alpha-pinene and ozone) in bronchial epithelial BEAS-2B cells. Results from this analysis suggest a functional role of the 8-oxoG modified transcripts after air pollution exposure in pathways that are central regulators of cell metabolism, growth, proliferation and maintenance of cellular structure. This study highlights a critical role for a RNA epitranscriptomics modification that can be used to further characterize biological responses to external stressors once considered indistinguishable.

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