Kournoutou Georgia G, Giannopoulou Panagiota C, Sazakli Eleni, Leotsinidis Michel, Kalpaxis Dimitrios L
Department of Biochemistry, School of Medicine, University of Patras, 26504 Patras, Greece.
Laboratory of Public Health, School of Medicine, University of Patras, 26504 Patras, Greece.
Aquat Toxicol. 2017 Nov;192:136-147. doi: 10.1016/j.aquatox.2017.09.007. Epub 2017 Sep 6.
Numerous studies have shown the ability of trace metals to accumulate in marine organisms and cause oxidative stress that leads to perturbations in many important intracellular processes, including protein synthesis. This study is mainly focused on the exploration of structural changes, like base modifications, scissions, and conformational changes, caused in 18S and 5S ribosomal RNA (rRNA) isolated from the mussel Mytilus galloprovincialis exposed to 40μg/L Cu, 30μg/L Hg, or 100μg/L Cd, for 5 or 15days. 18S rRNA and 5S rRNA are components of the small and large ribosomal subunit, respectively, found in complex with ribosomal proteins, translation factors and other auxiliary components (metal ions, toxins etc). 18S rRNA plays crucial roles in all stages of protein synthesis, while 5S rRNA serves as a master signal transducer between several functional regions of 28S rRNA. Therefore, structural changes in these ribosomal constituents could affect the basic functions of ribosomes and hence the normal metabolism of cells. Especially, 18S rRNA along with ribosomal proteins forms the decoding centre that ensures the correct codon-anticodon pairing. As exemplified by ELISA, primer extension analysis and DMS footprinting analysis, each metal caused oxidative damage to rRNA, depending on the nature of metal ion and the duration of exposure. Interestingly, exposure of mussels to Cu or Hg caused structural alterations in 5S rRNA, localized in paired regions and within loops A, B, C, and E, leading to a continuous progressive loss of the 5S RNA structural integrity. In contrast, structural impairments of 5S rRNA in mussels exposed to Cd were accumulating for the initial 5days, and then progressively decreased to almost the normal level by day 15, probably due to the parallel elevation of metallothionein content that depletes the pools of free Cd. Regions of interest in 18S rRNA, such as the decoding centre, sites implicated in the binding of tRNAs (A- and P-sites) or translation factors, and areas related to translation fidelity, were found to undergo significant metal-induced conformational alterations, leading either to loosening of their structure or to more compact folding. These modifications were associated with parallel alterations in the translation process at multiple levels, a fact suggesting that structural perturbations in ribosomes, caused by metals, pose significant hurdles in translational efficiency and fidelity.
众多研究表明，痕量金属能够在海洋生物体内蓄积，并引发氧化应激，进而导致包括蛋白质合成在内的许多重要细胞内过程受到干扰。本研究主要聚焦于探索从暴露于40μg/L铜、30μg/L汞或100μg/L镉环境中5天或15天的地中海贻贝（Mytilus galloprovincialis）分离出的18S和5S核糖体RNA（rRNA）所发生的结构变化，如碱基修饰、断裂和构象变化。18S rRNA和5S rRNA分别是小核糖体亚基和大核糖体亚基的组成部分，它们与核糖体蛋白、翻译因子及其他辅助成分（金属离子、毒素等）结合存在。18S rRNA在蛋白质合成的各个阶段都发挥着关键作用，而5S rRNA则作为28S rRNA几个功能区域之间的主要信号转导分子。因此，这些核糖体成分的结构变化可能会影响核糖体的基本功能，进而影响细胞的正常代谢。特别是，18S rRNA与核糖体蛋白一起形成了解码中心，确保密码子 - 反密码子的正确配对。如酶联免疫吸附测定（ELISA）、引物延伸分析和二甲基亚砜（DMS）足迹分析所示，每种金属都会对rRNA造成氧化损伤，这取决于金属离子的性质和暴露时间。有趣的是，贻贝暴露于铜或汞会导致5S rRNA的结构改变，这些改变位于配对区域以及环A、B、C和E内，导致5S RNA结构完整性持续逐渐丧失。相比之下，暴露于镉的贻贝中5S rRNA的结构损伤在最初5天不断累积，然后到第15天逐渐降至几乎正常水平，这可能是由于金属硫蛋白含量的平行升高消耗了游离镉池。在18S rRNA中感兴趣的区域，如解码中心、与tRNA（A位和P位）或翻译因子结合相关的位点以及与翻译保真度相关的区域，都发现发生了明显的金属诱导构象改变，导致其结构松弛或折叠更紧密。这些修饰与翻译过程在多个层面的平行改变相关，这一事实表明金属引起的核糖体结构扰动对翻译效率和保真度构成了重大障碍。
