Ribosomal RNA tentacles are targets of free radical damage in mammalian cells during oxidative and inflammatory stress

Chemical damage to ribosomal RNA (rRNA) during oxidative or inflammatory stress can impact protein synthesis. Human cells were exposed to a H2O2 titration series to induce oxidative stress or to tumor necrosis factor-α to induce inflammation over a time course followed by RNA direct nanopore sequencing of cytosolic and mitochondrial rRNAs. The guanosine (G) oxidation sites and deamination of adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) lesion sites were revealed by changes in the base-called data. Both stressors induced G oxidation in cytosolic rRNA, whereas mitochondrial rRNA was less oxidatively modified. Nitrosative stress generated during inflammation resulted in deamination lesions in rRNAs in both compartments. Inspection of highly modified sites showed the GC-rich tentacles in the 28S rRNA sequence were hotspots for G oxidation and C deamination in the cytosolic ribosome. Outside of tentacles, lesions were generally found on nucleotides on the ribosome surface exposed to solvent, where diffusible reactive species exist. The minimalist structure of the mitochondrial ribosome compared to the cytosolic ribosome alters the reaction patterns observed to target nucleotides on the surface or in functionally relevant regions. These patterns support the hypothesis that tentacles in cytosolic ribosomes direct reactive oxygen and nitrogen species away from the catalytic core to maintain ribosome activity during stress, while the mitochondrial ribosome is damaged in regions that can deactivate protein synthesis. The results provide molecular insight into metabolic dysfunction during oxidative and inflammatory stress and suggest a new function for the GC-rich tentacles that have evolved in mammalian cells.氧化应激或炎症应激下的核糖体RNA（ribosomal RNA, rRNA）化学损伤会影响蛋白质合成。本研究将人类细胞暴露于过氧化氢（hydrogen peroxide, H₂O₂）梯度浓度体系以诱导氧化应激，或暴露于肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）以诱导炎症应激，并设置时间进程，随后对胞质与线粒体核糖体RNA开展RNA直接纳米孔测序。通过碱基识别数据的变化，可揭示鸟苷（G）氧化位点，以及腺苷脱氨为肌苷（A-to-I）、胞苷脱氨为尿苷（C-to-U）的损伤位点。两种应激原均可诱导胞质核糖体RNA发生G氧化，但线粒体核糖体RNA的氧化修饰程度更低。炎症应激过程中产生的亚硝化应激，会导致两个分区的核糖体RNA均出现脱氨损伤。对高修饰位点的分析显示，胞质核糖体28S rRNA序列中的GC富集触手结构是G氧化与C脱氨的热点区域。在触手结构之外，损伤位点通常位于核糖体表面暴露于溶剂的核苷酸位置，这类区域存在扩散性活性物种。相较于胞质核糖体，线粒体核糖体的极简结构改变了靶向表面或功能相关区域核苷酸的反应模式。这些反应模式支持以下假说：胞质核糖体的触手结构可引导活性氧与活性氮远离催化核心，从而在应激状态下维持核糖体活性；而线粒体核糖体则会在可使蛋白质合成失活的区域受到损伤。本研究结果为氧化与炎症应激下的代谢功能障碍提供了分子层面的见解，并提示哺乳动物细胞演化出的GC富集触手结构具有全新的功能。