Targeted Inhibition of Translation Initiation via 2’O-Methylation of Start Codons

While protein synthesis typically initiates at an optimal AUG start codon, the 5’ untranslated region (5’UTR) of mRNAs harbors non-canonical start codons that result in the translation of upstream Open Reading Frames (uORFs). However, the mechanisms underlying the selection of non- canonical start codons remain poorly understood. Structural analysis of translation pre-initiation complexes showed that the 2’OH group of the first nucleotide within start codons is monitored by 18S rRNA, allowing optimal translation initiation. We identified a chemical modification of mRNA, 2’O-methylation (N _m ), that represses translation initiation by interfering with the interaction of mRNA and 18S rRNA, thereby preventing the pre-initiation complex from recognizing start codons. Significantly, we uncovered the presence of 2’O-methylation in the first nucleotide of non- canonical initiation sites within 5’UTRs, where it repressed upstream translation initiation. These findings revealed a potent regulatory function of 2’O-methylation in translation initiation and uORF expression, providing new insights into the mechanisms of non-canonical start codon selection in human cells.