Abstract
In contrast to prokaryotes wherein GUG and UUG are permissive start codons, initiation frequencies from non-AUG codons are generally low in eukaryotes, with CUG being considered as strongest. Here, we report that combined 5-cytosine methylation (5mC) and pseudouridylation (Ψ) of near-cognate non-AUG start codons convert GUG and UUG initiation strongly favored over CUG initiation in eukaryotic translation under a certain context. This prokaryotic-like preference is attributed to enhanced NUG initiation by Ψ in the second base and reduced CUG initiation by 5mC in the first base. Molecular dynamics simulation analysis of tRNA(i)(Met) anticodon base pairing to the modified codons demonstrates that Ψ universally raises the affinity of codon:anticodon pairing within the ribosomal preinitiation complex through partially mitigating discrimination against non-AUG codons imposed by eukaryotic initiation factor 1. We propose that translational control by chemical modifications of start codon bases can offer a new layer of proteome diversity regulation and therapeutic mRNA technology.