N1-Methylpseudouridine Directly Modulates Translation Dynamics

The remarkable effectiveness of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted synthetic mRNA as a promising technology. A critical feature of this approach is the incorporation of the modified ribonucleotide N1-methylpseudouridine (m¹Ψ), which enhances antigen expression while reducing immunogenicity. However, a comprehensive understanding of how this modification influences translation remains incomplete. Here, we analyze translation at sub-codon resolution using ribosome profiling and demonstrate that m¹Ψ increases ribosome density on synthetic mRNAs. This elevated ribosome load, together with the correlated increase in protein production, occurs independently of intrinsic cellular immunity or eIF2α phosphorylation. Our data reveal that m¹Ψ directly slows ribosome elongation in specific sequence contexts while concurrently enhancing translation initiation. Cryo-electron microscopy shows that m¹Ψ-modified mRNAs alter interactions within the ribosome decoding center, providing a mechanistic basis for slowed elongation dynamics. Furthermore, by introducing synonymous mutations that disrupt modification-mediated changes in elongation, we show that the m¹Ψ-dependent effect on protein output can be modulated, and that its impact is strongest in mRNAs containing non-optimal codons with uridines at the wobble position. Together, these findings demonstrate that m¹Ψ directly modulates translation elongation and initiation, thereby enhancing translational capacity and increasing protein output from synthetic mRNAs in defined sequence contexts.