Role of eEF2 diphthamide modification in translational fidelity

Diphthamide (DPH) is a conserved amino acid modification on eukaryotic translation elongation factor eEF2. We show that loss of DPH increases -1 ribosomal frameshifting at both programmed, including in SARS-CoV-2, and non-programmed sites. Ribosome profiling of yeast and mammalian cells lacking DPH reveals increased ribosomal drop-off during elongation. The drop-off defect on the long yeast MDN1 gene was suppressed by eliminating out-of-frame stop codons. Our data reveal that loss of DPH impairs the fidelity of translocation during translation elongation resulting in increased rates of ribosomal frameshifting and premature termination at out-of-frame stop codons.白喉酰胺（Diphthamide，DPH）是存在于真核翻译延伸因子2（eEF2）上的一类保守氨基酸修饰。本研究证实，DPH缺失会在程序性位点（涵盖新型冠状病毒SARS-CoV-2相关位点）与非程序性位点上，提升-1位核糖体移码事件的发生频率。对缺失DPH的酵母及哺乳动物细胞开展核糖体谱分析（ribosome profiling）后发现，细胞在翻译延伸过程中出现核糖体脱落的现象显著增多。针对酵母长度较长的MDN1基因所存在的核糖体脱落缺陷，可通过清除读码框外终止密码子的方式予以抑制。本研究数据表明，DPH缺失会损伤翻译延伸阶段的转位保真度，进而导致核糖体移码率升高，并在读码框外终止密码子处发生过早翻译终止。