Translational regulation by oxidative desulfuration of tRNA modifications

Modifications in the anticodon region of transfer RNA (tRNA) are essential for accurate and efficient protein synthesis. 5-Methyl-2-thiouridine derivatives (xm⁵s²U) are major modifications at the wobble position of tRNA anticodons decoding purine-ending two-codon sets. Although the thiocarbonyl group of xm⁵s²U enhances decoding efficiency, it is chemically susceptible to oxidative desulfuration, yielding 4-pyrimidinone derivatives (xm⁵h²U). Here, we identify xm⁵h²U derivatives in human cells and mouse tissues and confirm their cellular formation by spike-in experiments. Desulfurized tRNAs carrying 5-methoxycarbonylmethyl-4-pyrimidinone (mcm⁵h²U) show impaired codon recognition in a human reconstituted in vitro translation system. The mcm⁵h²U modification reduces aminoacylation of tRNAs for lysine, glutamate, and glutamine, but not arginine. Cryogenic electron microscopy reveals the structural basis of altered AAA/AAG decoding by mcm⁵h²U at the ribosomal A-site. These findings reveal a mechanism by which oxidative desulfuration of tRNA modifications dynamically regulates codon recognition and protein synthesis under oxidative stress conditions in human and mammalian cells.