维氏气单胞菌tmRNA以sRNA形式特异调控的下游靶标筛选和鉴定

Screening and identification of downstream targets specifically regulated by Aeromonas veronii tmRNA in the form of sRNA

摘要:

转移-信使RNA (transfer-message RNA, tmRNA)是细菌中普遍存在的稳定非编码sRNA，同时具备tRNA和mRNA双重特性，主要介导反式翻译核糖体拯救机制，对病原菌致病性和胁迫响应具有重要影响。【目的】从tmRNA功能研究入手，揭示对水产养殖和人类公共安全造成严重危害的维氏气单胞菌(Aeromonas veronii)的致病分子机制。【方法】采用生物学信息学工具IntaRNA 2.0对维氏气单胞菌tmRNA可能结合的下游靶标进行预测，通过基因本体论(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)富集分析，鉴定预测靶标参与的生物学过程及信号通路，并使用qPCR技术验证候选靶标基因在维氏气单胞菌野生型、tmRNA敲除株和smpB敲除株中的表达情况，以初步鉴定维氏气单胞菌tmRNA以sRNA形式调控的潜在下游靶标。【结果】tmRNA可以sRNA形式与下游100个潜在特异性靶标结合，这些靶标基因主要结合于tmRNA的3′端tRNA样结构域(tRNA-like domain, TLD)、H2域及PK3、PK4域，参与细菌基本代谢过程。qPCR结果表明，在特异性结合的靶标基因中，基因WP_201994931.1以SmpB非依赖的方式受到tmRNA调节；WP_201954220.1、WP_005335875.1、WP_265062582.1、WP_265061484.1和WP_265061494.1等基因的表达则受到SmpB蛋白调控。【结论】本研究初步鉴定基因WP_201994931.1有可能是tmRNA作为sRNA调控的下游靶标，对拓展tmRNA功能研究领域有一定指导作用，有助于后续进一步探究维氏气单胞菌的致病和环境适应等重要生命活动的分子机制。

Abstract:

Transfer-message RNA (tmRNA) is a ubiquitous and stable non-coding small RNA in bacteria, with characteristic regions similar with both tRNA and mRNA. tmRNA is known to mediate the ribosome rescue mechanism called trans-translation and has effects on the pathogenicity and stress responses of pathogenic bacteria. [Objective] To study the function of tmRNA in Aeromonas veronii causing threats to aquaculture and human public safety, so as to reveal the molecular mechanism for the pathogenicity of A. veronii. [Methods] IntaRNA 2.0 was used to predict the downstream targets binding to tmRNA. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted to predict the biological processes and signaling pathways involving the predicted targets. Real-time qPCR was employed to compare the expression levels of the candidate target genes among the wild type, tmRNA knockout strain, and smpB knockout strain of A. veronii, thus identifying the potential targets regulated by tmRNA in the form of sRNA. [Results] One hundred potential specific downstream targets might bind to tmRNA at the 3′-end tRNA-like domain (tRNA-like domain, TLD), H2 domain, and PK3 and PK4 regions, thus participating in the general metabolic pathways of the pathogen. The results of qPCR indicated that the expression of WP_201994931.1 was regulated by tmRNA in an SmpB-independent manner, while the expression of WP_201954220.1, WP_005335875.1, WP_265062582.1, WP_265061484.1, and WP_265061494.1 was regulated by SmpB. [Conclusion] We preliminarily identified that WP_201994931.1 might constitute a downstream target regulated by tmRNA as an sRNA. This study provides basic information for expanding the functions of tmRNA in the form of sRNA and facilitates the further studies regarding the molecular mechanisms of the pathogenicity and environmental adaptation of A. veronii.