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A small RNA regulates pprM, a modulator of pleiotropic proteins promoting DNA repair, in Deinococcus radiodurans under ionizing radiation.

ABSTRACT: Networks of transcriptional and post-transcriptional regulators are critical for bacterial survival and adaptation to environmental stressors. While transcriptional regulators provide rapid activation and/or repression of a wide-network of genes, post-transcriptional regulators, such as small RNAs (sRNAs), are also important to fine-tune gene expression. However, the mechanisms of sRNAs remain poorly understood, especially in less-studied bacteria. Deinococcus radiodurans is a gram-positive bacterium resistant to extreme levels of ionizing radiation (IR). Although multiple unique regulatory systems (e.g., the Radiation and Desiccation Response (RDR)) have been identified in this organism, the role of post-transcriptional regulators has not been characterized within the IR response. In this study, we have characterized an sRNA, PprS (formerly Dsr2), as a post-transcriptional coordinator of IR recovery in D. radiodurans. PprS showed differential expression specifically under IR and knockdown of PprS resulted in reduced survival and growth under IR, suggesting its importance in regulating post-radiation recovery. We determined a number of potential RNA targets involved in several pathways including translation and DNA repair. Specifically, we confirmed that PprS binds within the coding region to stabilize the pprM (DR_0907) transcript, a RDR modulator. Overall, these results are the first to present an additional layer of sRNA-based control in DNA repair pathways associated with bacterial radioresistance.

SUBMITTER: Villa JK

PROVIDER: S-EPMC8217566 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A small RNA regulates pprM, a modulator of pleiotropic proteins promoting DNA repair, in Deinococcus radiodurans under ionizing radiation

Project description:This study explores the regulatory network of a sRNA named PprS (previously Dsr2) in Deinococcus radiodurans R1. We compared the transcriptomes of two strains with and without exposure to 10 kGy acute ionizing radiation (IR): D. radiodurans R1 (wild-type) and a PprS knockdown mutant (PprSKD) that expresses ~2-fold less PprS compared to WT levels. Analysis of this RNAseq dataset demonstrated significant differential expression of several transcripts in both WT (34) and PprSKD (61) strains during recovery from IR. However, we did not observe any significantly differentially expressed transcripts between the two strains during recovery from IR. 31 transcripts were signficantly differentially expressed when comparing the two strains under unirradiated conditions. To determine the regulatory network that this sRNA, PprS could regulate we performed a MAPS (MS2-affinity purification followed by RNAsequencing) experiment in which a MS2-binding domain tagged PprS was expressed from a constutive promoter from a plasmid (pRADgro) in D. radiodurans R1. A control plasmid (containing only the MS2-binding domain) was also expressed and differential enrichment was compared to this control. MAPS suggested ~130 potential interacting transcripts with PprS and we were able to validate the binding and stabilization of one specific transcript, pprM, for PprS.

2021-06-18 | GSE176207 | GEO

A small RNA regulates pprM, a modulator of pleiotropic proteins promoting DNA repair, in Deinococcus radiodurans under ionizing radiation

Project description:This study tracks the proteome during recovery from 10 kGy acute ionizing radiation (IR) in Deinococcus radiodurans R1 (WT). After 1 hour of recovery post-IR exposure, we observed 37 proteins significantly differentially expressed, including several within the Radiation and Dessication Response (RDR) pathway. Additionally, we also explored the regulatory network of a sRNA named PprS (previously Dsr2) in Deinococcus radiodurans by comparing the proteome of a sRNA knockdown strain (PprSKD, which demonstrates a ~2-fold decrease in PprS expression) to WT D. radiodurans during unirradiated conditions at late-exponential phase. Comparison between these two strains demonstrated decreased levels of one of PprS's targets, PprM, in the PprSKD strain which validated the activation mechanism we propose for PprS on pprM.

2021-06-25 | PXD026633 | Pride

A small RNA regulates pprM, a modulator of pleiotropic proteins promoting DNA repair, in Deinococcus radiodurans under ionizing radiation

Project description:A small RNA regulates pprM, a modulator of pleiotropic proteins promoting DNA repair, in Deinococcus radiodurans under ionizing radiation

| PRJNA735381 | ENA

Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation.

Project description:Deinococcus radiodurans R1 (DEIRA) is a bacterium best known for its extreme resistance to the lethal effects of ionizing radiation, but the molecular mechanisms underlying this phenotype remain poorly understood. To define the repertoire of DEIRA genes responding to acute irradiation (15 kGy), transcriptome dynamics were examined in cells representing early, middle, and late phases of recovery by using DNA microarrays covering approximately 94% of its predicted genes. At least at one time point during DEIRA recovery, 832 genes (28% of the genome) were induced and 451 genes (15%) were repressed 2-fold or more. The expression patterns of the majority of the induced genes resemble the previously characterized expression profile of recA after irradiation. DEIRA recA, which is central to genomic restoration after irradiation, is substantially up-regulated on DNA damage (early phase) and down-regulated before the onset of exponential growth (late phase). Many other genes were expressed later in recovery, displaying a growth-related pattern of induction. Genes induced in the early phase of recovery included those involved in DNA replication, repair, and recombination, cell wall metabolism, cellular transport, and many encoding uncharacterized proteins. Collectively, the microarray data suggest that DEIRA cells efficiently coordinate their recovery by a complex network, within which both DNA repair and metabolic functions play critical roles. Components of this network include a predicted distinct ATP-dependent DNA ligase and metabolic pathway switching that could prevent additional genomic damage elicited by metabolism-induced free radicals.

| S-EPMC153069 | biostudies-literature

Transcriptional analysis of Deinococcus radiodurans reveals novel small RNAs that are differentially expressed under ionizing radiation.

Project description:Small noncoding RNAs (sRNAs) are posttranscriptional regulators that have been identified in multiple species and shown to play essential roles in responsive mechanisms to environmental stresses. The natural ability of specific bacteria to resist high levels of radiation has been of high interest to mechanistic studies of DNA repair and biomolecular protection. Deinococcus radiodurans is a model extremophile for radiation studies that can survive doses of ionizing radiation of >12,000 Gy, 3,000 times higher than for most vertebrates. Few studies have investigated posttranscriptional regulatory mechanisms of this organism that could be relevant in its general gene regulatory patterns. In this study, we identified 199 potential sRNA candidates in D. radiodurans by whole-transcriptome deep sequencing analysis and confirmed the expression of 41 sRNAs by Northern blotting and reverse transcriptase PCR (RT-PCR). A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy). We have also found and confirmed 7 sRNAs in Deinococcus geothermalis, a closely related radioresistant species. The identification of several novel sRNAs in Deinococcus bacteria raises important questions about the evolution and nature of global gene regulation in radioresistance.

| S-EPMC4325154 | biostudies-literature

DdrB protein, an alternative Deinococcus radiodurans SSB induced by ionizing radiation.

Project description:Deinococcus radiodurans exhibits an extraordinary resistance to the effects of exposure to ionizing radiation (IR). DdrB is one of five proteins induced to high levels in Deinococcus following extreme IR exposure and that play a demonstrable role in genome reconstitution. Although homology is limited, DdrB is a bacterial single-stranded DNA-binding protein. DdrB features a stable core with a putative OB-fold, and a C-terminal segment with properties consistent with other bacterial SSBs. In solution, the protein functions as a pentamer. The protein binds single-stranded DNA but not duplex DNA. Electron microscopy and assays with two RecA proteins provide further structural and functional identification with bacterial SSB. Overall, the results establish DdrB as the prototype of a new bacterial SSB family. Given the role of SSB as a mobilization scaffold for many processes in DNA metabolism, the induction of an alternative and quite novel SSB following irradiation has potentially broad significance for the organization of genome reconstitution functions.

| S-EPMC2755865 | biostudies-literature

Transcriptional Analysis of Deinococcus radiodurans for Novel sRNAs expression that are Differentially Expressed under Ionizing Radiation

Project description:In this study, we identified potential sRNA candidates in D. radiodurans by a whole transcriptome deep sequencing analysis, and confirm the expression of 31 sRNAs in D. radiodurans with Northern blotting analysis and RT-PCR. A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy).

2015-01-22 | GSE64952 | GEO

Transcriptional Analysis of Deinococcus radiodurans for Novel sRNAs expression that are Differentially Expressed under Ionizing Radiation

Project description:In this study, we identified potential sRNA candidates inÂ D. radiodurans by a whole transcriptome deep sequencing analysis, and confirm the expression of 31 sRNAs in D. radiodurans with Northern blotting analysis and RT-PCR. A total of 8 confirmed sRNAs showed differential expression during recovery after acute ionizing radiation (15 kGy). Two samples (sham and 15kGy irradiated) were sequenced.

2015-01-22 | E-GEOD-64952 | biostudies-arrayexpress

Untargeted metabolite profiling reveals that nitric oxide bioynthesis is an endogenous modulator of carotenoid biosynthesis in Deinococcus radiodurans and is required for extreme ionizing radiation resistance.

Project description:Deinococcus radiodurans (Drad) is the most radioresistant organism known. Although mechanisms that underlie the extreme radioresistance of Drad are incompletely defined, resistance to UV irradiation-induced killing was found to be greatly attenuated in an NO synthase (NOS) knockout strain of Drad (Δnos). We now show that endogenous NO production is also critical for protection of Drad against γ-irradiation (3000 Gy), a result of accelerated growth recovery, not protection against killing. NO-donor treatment rescued radiosensitization in Δnos Drad but did not influence radiosensitivity in wild type Drad. To discover molecular mechanisms by which endogenous NO confers radioresistance, metabolite profiling studies were performed. Untargeted LC-MS-based metabolite profiling in Drad quantified relative abundances of 1425 molecules and levels of 294 of these were altered by >5-fold (p < 0.01). Unexpectedly, these studies identified a dramatic perturbation in carotenoid biosynthetic intermediates in Δnos Drad, including a reciprocal switch in the pathway end-products from deoxydeinoxanthin to deinoxanthin. NO supplementation rescued these nos deletion-associated changes in carotenoid biosynthesis, and fully-restored radioresistance to wildtype levels. Because carotenoids were shown to be important contributors to radioprotection in Drad, our findings suggest that endogenously-produced NO serves to maintain a spectrum of carotenoids critical for Drad's ability to withstand radiation insult.

| S-EPMC4698065 | biostudies-literature

Deletion of the rnl gene encoding a nick-sealing RNA ligase sensitizes Deinococcus radiodurans to ionizing radiation.

Project description:Deinococcus radiodurans RNA ligase (DraRnl) seals 3΄-OH/5΄-PO4 nicks in duplex nucleic acids in which the 3΄-OH nick terminus consists of two or more ribonucleotides. DraRnl exemplifies a widely distributed Rnl5 family of nick-sealing RNA ligases, the physiological functions of which are uncharted. Here we show via gene knockout that whereas DraRnl is inessential for growth of D. radiodurans, its absence sensitizes the bacterium to killing by ionizing radiation (IR). DraRnl protein is present in exponentially growing and stationary phase cells, but is depleted during the early stages of recovery from 10 kGy of IR and subsequently replenished during the late phase of post-IR genome reassembly. Absence of DraRnl elicts a delay in reconstitution of the 10 kGy IR-shattered D. radiodurans replicons that correlates with the timing of DraRnl replenishment in wild-type cells. Complementation with a catalytically dead mutant highlights that nick sealing activity is important for the radioprotective function of DraRnl. Our findings suggest a scenario in which DraRnl acts at genomic nicks resulting from gap-filling by a ribonucleotide-incorporating repair polymerase.

| S-EPMC5397189 | biostudies-literature

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