Site-specific inhibition of translation initiation via 2'-O-methylation

Translation initiation involves a concerted set of intermolecular interactions that efficiently recognize optimal AUG start codons. However, translation initiation complexes often start at upstream non-optimal AUGs or near-cognate codons, leading to the expression of upstream open reading frames (uORFs). Using retrospective analyses of translation preinitiation cryo-EM structures, we identified putative hydrogen bonds between the 2'-OH groups of messenger RNA (mRNA) start codons with 18S ribosomal RNA. Disruption of these interactions using a chemical modification of mRNA, 2'-O-methylation (Nm), repressed translation initiation by preventing the preinitiation complex from recognizing start codons. Notably, 2'-O-methylation in upstream AUG and near-cognate codons inhibits upstream translation initiation while enhancing the expression of canonical ORFs. These findings revealed a transcript- and site-specific inhibitory role for 2'-O-methylation in translation initiation, providing novel insights into the mechanisms of start codon selection in human transcriptomes.