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

Abstract

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- 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. Significance StatementInfection and injury trigger a cellular inflammatory response resulting in the release of free radical species capable of DNA and RNA damage. We used RNA direct nanopore sequencing to map oxidized guanosine sites in human ribosomal RNA via base-calling error analysis. Reactive nitrogen species derived from peroxynitrite result in deamination reactions, principally cytidine to uridine and adenosine to inosine, which can be directly read by nanopore sequencing. Importantly, cytosolic ribosomes behave very differently than mitochondrial ribosomes; the latter rRNA is somewhat protected from G oxidation by high levels of bicarbonate as well as a protein-coated ribosome structure. In contrast, cytosolic rRNA tentacles are hotspots for both G oxidation and C and A deamination, which might explain their evolved function.