分枝杆菌中反义RNA靶向tmRNA介导的反式翻译调控新机制

Bacteria devote a large percentage of their resources to protein synthesis, which therefore must be protected delicately. For example, ribosomes that stall during translation need to be rescued to ensure that the protein synthesis capacity of the cell is maintained. Stalling arises when ribosomes become trapped at the 3’-end of an mRNA, which occurs when a codon is unsuitable, leading to the synthesis arrest during protein elongation or termination. Almost all bacteria use a mechanism known as trans-translation, which is mediated by tmRNA, to rescue stalled ribosomes. During trans-translation, alanine charged tmRNA recognizes the arrested ribosome to drive its translocation. The ribosome then resumes protein translation by utilizing an open reading frame (ORF) within tmRNA to provide a stop codon for translation termination and rescue of trapped ribosomes. In this manner, the nascent polypeptide is attached with a proteolysis tag, and the chimeric polypeptide is released from the ribosome, followed by degradation with specific ATP dependent proteases. Despite its well established importance, the regulation of tmRNA has rarely been investigated. In a recent study, by analyzing the transcriptome data, we found the existence of a cis-acting antisense RNA (hereafter referred to as “astmRNA”) encoded via the opposite strand of tmRNA in many bacteria. We have verified the transcription of astmRNA in M. smegmatis by specific primer-mediated reverse PCR, promoter activity, and RACE assays. In this project, we devote to study how does astmRNA regulates the expression, stability, and biological function of tmRNA in response to various intra/extracellular cues. We also aim to identify the upstream transcription regulators of astmRNA to examine the influence of tmRNA mediated tran-translation when astmRNA is being regulated. Besides, this project will also explore the biological significance of astmRNA and analyze its distribution and evolution among bacteria. We are the first group to identify the astmRNA and will further conduct in-depth research on the regulation mechanism of tmRNA. It will not only profoundly clarify the regulation mechanism of trans-translation rescue system but also provide ideas for the design of new drug targets and development of new vaccines.

蛋白质合成时，很多因素导致核糖体无法及时与mRNA分离而停滞。为此，细菌进化出核糖体拯救系统，对停滞在mRNA上的核糖体进行回收利用。以tmRNA为核心的反式翻译是细菌中主要的核糖体拯救系统。tmRNA类似携带丙氨酰的tRNA，识别受困的核糖体并致其移位，核糖体继续以tmRNA的ORF为模板在缺陷肽C端翻译出降解标签肽，以完成核糖体拯救并促进缺陷肽水解。但目前尚无tmRNA调控机制研究的报道。申请人通过转录组分析，在多种细菌中发现了tmRNA的反义RNA，命名为astmRNA。本项目拟以耻垢分枝杆菌为出发菌株，研究细胞内外信号及转录因子对astmRNA的调控，揭示其受到上游调控后，进一步通过调节tmRNA的转录量、稳定性以影响其生理功能的作用机制。本项目首次发现astmRNA，并对tmRNA的调控机制进行深入研究，不仅深刻阐明了反式翻译拯救系统的调控机制，而且为药物靶标的设计提供新思路。

蛋白质合成时，很多因素导致核糖体无法及时与mRNA分离而停滞。为此，细菌进化出核糖体拯救系统，对停滞在mRNA上的核糖体进行回收利用。以tmRNA为核心的反式翻译是细菌中主要的核糖体拯救系统。tmRNA类似携带丙氨酰的tRNA，识别受困的核糖体并致其移位，核糖体继续以tmRNA的ORF为模板在缺陷肽C端翻译出降解标签肽，以完成核糖体拯救并促进缺陷肽水解。但目前尚无tmRNA调控机制研究的报道。本项目在耻垢分枝杆菌中发现了 tmRNA 的反义 RNA，将其命名为 AtmR，并深入探究了 AtmR 对 tmRNA 的调控机制，具体结果如下：.(1) 通过分析转录组数据我们发现了 tmRNA 的反义 RNA (AtmR)，并通过反转录 PCR、启动子活性测定以及 5’-RACE 实验对其进行了验证。.(2) 构建了 AtmR 过表达 (AtmROE)及敲降 (AtmRKD)菌株，并通过荧光定量PCR、lacZ 报告实验以及Western blot实验证实了 AtmR 通过降低 tmRNA 的稳定性来负调控其表达,并最终抑制反式翻译效率。.(3) 在基因组上将 tmRNA 标签的 C 端序列突变成了 His×6 的编码序列，并通过 Western blot 全局考察了 AtmR 对反式翻译的影响。结果显示，AtmROE 菌株中被 tmRNA 标记的蛋白明显减少，这进一步证实了 AtmR 对反式翻译起抑制作用。.(4) 考察了 AtmROE 菌株对作用于核糖体的抗生素的耐受性，发现 AtmROE 菌株对氨基糖苷类抗生素特异性敏感。.(5) 发现过表达 AtmR 有利于含稀有密码子簇基因 MSMEG_0069 和 MSMEG_4082 合成全长蛋白。我们认为这是由于 MSMEG_0069 和 MSMEG_4082 的正常翻译受到了 tmRNA 的抑制，而 AtmR 的高量表达解除了该抑制作用。.(6) 在不同的刺激条件下考察了 AtmR 表达水平的变化，发现热激和高盐条件 下 AtmR 的表达水平有一定程度的提升。. 综上所述，本项目首次发现了 tmRNA 的反义 RNA (AtmR)，并研究了 AtmR 对 tmRNA 的调控。这不仅拓宽了 tmRNA 及反式翻译的调控网络，也深刻阐明了反式翻译拯救系统的调控机制，为药物靶标的设计提供了新思路。