Identification and characterization of novel regulatory small RNAs in Deinococcus radiodurans

Date

2017-12

Authors

Tsai, Chen-Hsun

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Abstract

Gene expression regulation mechanisms during stress recovery are ubiquitous across all organisms. Some organisms have evolved more robust and distinct regulatory systems to survive exposures of extreme conditions. For instance, Deinococcus radiodurans is one of the most radioresistive organisms that have been identified. It can withstand 15,000 Gray of gamma radiation, which is 5,000 times of human and 250 times of E. coli. Many studies have been done to decipher the radioresistance mechanisms of D. radiodurans. However, most analysis with the genome and proteome are not conclusive. Although a recent study suggested that the small molecules that protect the proteome of D. radiodurans under irradiation are a key to its radioresistance, the mechanisms of gene regulation under irradiation are still unclear. In this study, we have introduced small non-coding RNAs (sRNA) as potential regulators in the radioresistance mechanisms. In bacteria, sRNAs have been identified in multiple species and have been shown to play essential roles in responsive mechanisms to environmental stresses. A key property of sRNAs is their ability to up or down regulate global metabolic pathways in response to lethal environmental signals. The versatility and specificity of sRNA regulation make it highly relevant for developing engineering applications. Since very little about sRNAs is known or discovered in D. radiodurans, we have used bioinformatics algorithms to analyze genomics patterns of sRNAs coding regions in 13 bacterial species to develop a pipeline and have identified novel transcripts in bacteria based on the conservation level and size of the intergenic regions. For example, alone with computation predictions, we found 40 novel transcripts in Mycobacteria smegmatis and Mycobacteria bovis. The combination of this bioinformatics approach and experimental verification has led us to identify 41 sRNAs in D. radiodurans, and many of them show differential expression during recovery from exposures to various environmental stresses. Among them, one transcript Dsr2 showed a unique expression pattern under ionizing radiation and the deletion mutant has a reduced survival rate under irradiation. We designed multiple experiments to verify the regulatory networks of Dsr2, including HITS-CLIP analysis, transcriptome analysis and electrophoresis mobility shift assay. We have found 14 mRNAs that have direct interaction with Dsr2 sRNA and many of them have important functions related stress response. For example, PprM protein has been verified as a modulator of PprA protein, which activates DNA end-repair under stresses. This pathway is also regulated by PprI but the regulatory mechanism of PprM was previously unclear. We found Dsr2 is able to bind with pprM and the transcriptome analysis suggested its expression level is correlated with Dsr2 expression. Dsr2 was also shown to have interactions with many ribosomal proteins and translation initiation factors. In summary, our work provides insight and evidence of how non-coding transcripts regulate radioresistance mechanisms in D. radiodurans, and how bioinformatics analysis can lead us to the discovery of more regulatory transcripts in bacteria of interest

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