Regulatory Mechanism Of Oxidative Stress Response Mediated By NcRNA OsiR In Deinococcus Radiodurans

Reactive oxygen species produced by oxidative stress can cause oxidative damage to proteins,lipids,DNA and other biological macromolecules.The antioxidation protective system?such as catalase KatE?plays an important role in scavenging reactive oxygen species and protecting cells from oxidative damage,however,its expression regulation mechanism is still unclear.Recent reports show that bacterial non-coding RNAs?ncRNAs?are widely involved in various stress responses,but there are few reports on whether ncRNAs from D.radiodurans with strong oxidative stress resistence are involved in oxidative stress response.In this study,an ncRNA specifically responding to oxidative stress signal was identified from D.radiodurans and named as OsiR?Oxidative stress induced regulatory RNA?.This study focused on the function of OsiR and its regulatory mechanism in oxidative stress response.The main results were as follows:1.Sequence alignment analysis showed that OsiR is a unique non-coding RNA of Deinococcus genus.The results of 5'RACE showed that osiR was reverse-transcripted on chromosome I,and the transcription starting site was adenine?A?at 1,029,200.The results of Northern blot and qRT-PCR showed that osiR was significantly induced under oxidative stress?H₂O₂?.The promoter regulatory element prediction and qRT-PCR analyses showed that the expression of osiR was positively regulated by transcription factors Sig1 and OxyR2.2.Under oxidative stress condition,the deletion of osiR resulted in increased sensitivity of the strain to H₂O₂,decreased total antioxidant capacity and catalase activity,and down-regulated expression of genes related to oxidative stress resistance,while the complementary strain could recover the phenotypes.Under normal growth and other stresses?heat and UV radiation?condition,there was no significant difference in viability and sensitivity to heat and UV radiation between the osiR mutant and the wild-type strain.It was suggested that OsiR specifically responds to oxidative stress signals and plays an important role in oxidative stress resistance.3.Further studies showed that the deletion of osiR resulted in a significant decrease in the abundance of katE2 mRNA and KatE2 protein,as well as the half-life of katE2 mRNA.Bioinformatics analysis showed that there was a potential binding site between the stem-loop structure of OsiR and katE2 mRNA,which was confirmed by MST experiment.At the same time,the absence of katE2 also led to a significant decrease in oxidative stress resistance,total antioxidant capacity and catalase activity.The above results indicated that OsiR positively regulates the expression of its target katE2 at the post-transcriptional level,thus improving the oxidative stress resistance of D.radiodurans.4.There was an overlap region of 79 bp between osiR and its adjacent gene trmE,suggesting that trmE may be a target gene of OsiR.MST experiment proved the direct binding of OsiR and trmE mRNA.Under oxidative stress condition,the deletion of osiR led to the significant down-regulation of trmE transcription and protein levels,and the stability of trmE mRNA.The absence of trmE also reduced the oxidative stress resistance of D.radiodurans.It was suggested that OsiR could participate in the regulation of oxidative stress response by combining with different target genes.5.Transcriptome results showed that under oxidative stress,the deletion of osiR resulted in the up-regulation and down-regulation of 25 and 44 differentially expressed genes?including trmE?,which were significantly enriched in oxidative phosphorylation,biological regulation,cell activity and macromolecule metabolism.The absence of osiR has no significant effect on katE2 transcription,which may be caused by the fact that OsiR mainly plays a regulatory role at the post-transcriptional level through its binding to katE2 mRNA.It was suggested that OsiR is involved in the global regulation of the oxidative stress response in D.radiodurans through a variety of biological processes.In conclusion,OsiR is a non-coding RNA of Deinococcus genus specifically induced by oxidative stress,as a gobal regulatory factor,plays an important role in the regulation of oxidative stress resistance in D.radiodurans.