Lim Boram, Lee Kangseok
Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
Department of Life Science, Chung-Ang University, Seoul, Republic of Korea
J Bacteriol. 2015 Apr;197(7):1297-305. doi: 10.1128/JB.02460-14. Epub 2015 Feb 2.
The enzymatic activity of Escherichia coli endo-RNase III determines the stability of a subgroup of mRNA species, including bdm, betT, and proU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability of proP mRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels of proP mRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell. In vitro and in vivo analyses of proP mRNA revealed RNase III cleavage sites in a stem-loop within the 5' untranslated region present only in proP mRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III on proP mRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance of proP mRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III via in vivo cross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress in E. coli.
Our results demonstrate that RNase III activity on proP mRNA degradation is downregulated in Escherichia coli cells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes.
大肠杆菌内切核糖核酸酶III的酶活性决定了一组mRNA种类的稳定性，包括bdm、betT和proU，其蛋白质产物与细胞对渗透胁迫的反应相关。在此，我们报告编码渗透保护剂转运蛋白的proP mRNA的稳定性受核糖核酸酶III调控以应对渗透胁迫。我们观察到proP mRNA和ProP蛋白的稳态水平与细胞核糖核酸酶III活性呈负相关，进而影响细胞的脯氨酸摄取能力。对proP mRNA的体外和体内分析揭示了仅在由渗透调节的P1启动子合成的proP mRNA种类的5'非翻译区内的一个茎环中有核糖核酸酶III切割位点。在切割位点引入核苷酸取代可抑制核糖核酸酶III对proP mRNA的核糖核酸水解活性，增加mRNA的稳态水平和半衰期。此外，在高渗胁迫条件下，核糖核酸酶III活性降低与proP mRNA的半衰期和丰度显著增加同时发生。通过体内交联和免疫沉淀分析与核糖核酸酶III结合的RNA表明，这种现象与核糖核酸酶III的RNA结合能力降低有关。我们的研究结果表明存在一个核糖核酸酶III介导的渗透调节网络，该网络可迅速平衡大肠杆菌中与细胞对渗透胁迫反应相关因子的表达水平。
我们的结果表明，在渗透胁迫下，大肠杆菌细胞中核糖核酸酶III对proP mRNA降解的活性下调。此外，我们表明核糖核酸酶III活性的下调与高渗条件下核糖核酸酶III的RNA结合能力降低有关。特别是，我们的研究结果证明了渗透胁迫与核糖核酸酶III活性之间的联系，强调了转录后调控在调节对环境变化的快速生理适应中的重要性日益增加。
