Functional Characterization Of Non-coding SRNA DrrA Involved In Oxidative Resistance In Deinococcus Radiodurans

Non-coding RNA,mostly in size with 20-400 nucleotides,usually dos not encode its functional protein and are widely involved in multiple regulation of physiological processes,such as metabolism,stress response and pathogenicity,treating as a kind of post-transcriptionally regulatory factors.Deinococcus radiodurans has a strong adversity stress adaptability,showing not only radiation resistance but super strong oxidative-stress resistance.Currently,there are few reports on non-coding RNA in D.radiodurans and only some potential sRNA has been predicted in D.radiodurans through sRNA deep sequencing.Although the studies suggest that sRNA in D.radiodurans play an significantly important role in stress-resistant responses such as radiation and oxidation,there is no report on the function and characteristics of sRNA in D.radiodurans.In this study,we verify a non-coding RNA?drrA?,whose gene is located in the intergenic region in D.radiodurans,and construct drrA deletion mutant in the D.radiodurans,explore the function of this gene faced with the oxidative stress environment using stress phenotype assay,real-time quantitative PCR and?-galatocidase activity measurement,and finally obtain some results as follows:1.drrA,251bp in length,is located in the intergenic region?IGR?between dr1016 and dr1017 inchromosome I of D.radiodurans.Rfam database suggests that drrA tend to be a class of new unidentified non-coding RNA family.Northern blot assay turns out that drrA is indeed the non-coding RNA with its relative expression significantly increasing under oxidative stress.Meanwhile,5‘RACE assay demonstrates that drrA is reverse transcribed,treating an adenine?A?as its transcription start site.Sequence analysis of the promoter region upstream of the transcription start site viewed a conserved-35/-10 region?AGGAAA-N17-TAAAAT?recognized by the?70?RpoD/SigA?.Bioinformatics analysis showed that drrA is likely to be specific and conversed in Deinococcus ssp.and the secondary structure prediction viewed that sRNA drrA consists of multiple stem-loop structures,speculated to be the target mRNA binding site.2.Real-time quantitative PCR assay displays that drrA express to different degrees under different stress condations,especially varies significantly on the condition of oxidative stress.On the circumstance of 80mM H₂O₂ stress,the expression of drrA rises up to 51 times in the transcription levels,and up-regulates 3 folds and down-regulate 5 folds under heat and UV radiation stress,separately.The expression analysis of drrA under different oxidative stress intensity reveals drrA is quite sensitive to oxidative stress in consideration of its expression obviously increasing to 10 times under low concentration of H₂O₂.3.In this study,drrA deletion mutant??drrA?is constructed by fusion PCR and homologous recombination.We carry out the phenotype analysis of D.radiodurans R1 wild type and drrA mutant under oxidative stress,heat stress and UV radiation stress,finding that the growth ability of?drrA is markedly reduced by three orders of magnitude compared with D.radiodurans R1 wild type under the oxidative and heat stress,respectively,and nevertheless reduced by an order of magnitude under the UV radiation stress.The results proved out that the deletion of drrA could significantly reduce the oxidative stress resistance of D.radiodurans and consequently result in the cell more sensitive to oxidative stress.4.We predict the direct mRNA target for drrA with online software called TargetRNA2,finding 8candidates related to oxidative resistance,namely dr1016?trmE?,dr0435,dr0841,dra0010,dra0233,dra0232,dra0013 and drb0092.In order to further oxidative stress regulatory mechanism study of drrA gene,we conduct?-galatocidase activity assay to analyze the promoter activities of oxidative-related gen katE,oxyR1,oxyR2 and global regulatory factors irrE.The results view that deletion of drrA gene inhibits the expression of katE,oxyR2,irrE and promote oxyR1 expression,and thus speculate drrA is likely to participate in expression regulation of the related gene mentioned above.This study identify drrA,specific in Deinococcus,to be a non-coding RNA in D.radiodurans,analyze its function under oxidative stress and preliminarily explore the regulation of drrA to oxidative stress.Ultimately,we speculate drrA play an important role in oxidative stress adversity.Nevertheless,the mechanism of sRNA drrA in the stress-resistant regulatory network is still unclear and requires further study.