Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine

mRNA function is influenced by modifications that modulate canonical nucleobase behavior. We show that a single modification mediates distinct impacts on mRNA translation in a position-dependent manner. Although cytidine acetylation (ac4C) within protein-coding sequences stimulates translation, ac4C within 5′ UTRs impacts protein synthesis at the level of initiation. 5′ UTR acetylation promotes initiation at upstream sequences, competitively inhibiting annotated start codons. Acetylation further directly impedes initiation at optimal AUG contexts: ac4C within AUG-flanking Kozak sequences reduced initiation in base-resolved transcriptome-wide HeLa results and in vitro utilizing substrates with site-specific ac4C incorporation. Cryo-EM of mammalian 80S initiation complexes revealed that ac4C in the −1 position adjacent to an AUG start codon disrupts an interaction between C and hypermodified t6A at nucleotide 37 of the initiator tRNA. These findings demonstrate the impact of RNA modifications on nucleobase function at a molecular level and introduce mRNA acetylation as a factor regulating translation in a location-specific manner. [Display omitted] •mRNA acetylation impacts translation in a position-dependent manner•5′ UTR ac4C promotes upstream initiation and inhibits canonical start codons•ac4C within Kozak sequences structurally alters the interaction with tRNAiMet•ac4C within mRNA CDSs enhances translation mRNA modifications alter canonical nucleobase behavior. Arango et al. show that a single modification, ac4C, influences mRNA translation in a position-dependent manner. ac4C within coding sequences promotes elongation, whereas 5′ UTR ac4C inhibits initiation through the generation of repressive structures and through the direct modulation of tRNAiMet interactions.