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

Chemical damage to ribosomal RNA (rRNA) during oxidative or inflammatory stress can impact protein synthesis. Human cells were exposed to an H₂O₂ titration to induce oxidative stress, or tumor necrosis factor-α (TNF-α) over a time course to induce inflammation, followed by direct nanopore RNA sequencing of cytosolic and mitochondrial rRNAs to reveal damage sites. Guanosine (G) oxidation and deamination of adenosine to inosine (A-to-I) and cytidine to uridine (C-to-U) were identified by changes in the base-called data. Both stressors induced G oxidation in cytosolic rRNA, whereas mitochondrial rRNA was less oxidatively modified. Nitrosative stress during inflammation caused deamination lesions in rRNAs in both compartments. Inspection of highly modified sites found that GC-rich tentacles in the 28S rRNA expansion sequences were hotspots for G oxidation and C deamination in the cytosolic ribosome. Outside of tentacles, lesions generally occurred on surface nucleotides. The minimalist mitochondrial ribosome structure, compared to the cytosolic ribosome, alters reaction patterns such that nucleotides on the surface or in functionally relevant regions are damaged. These patterns suggest that tentacles on cytosolic rRNA direct reactive oxygen and nitrogen species away from the catalytic core to maintain activity during stress, while the mitochondrial ribosome is damaged in regions that can deactivate protein synthesis. These results provide molecular insight into metabolic dysfunction and suggest a new function for the GC-rich tentacles evolved in mammalian cells.