N1-Methylpseudouridine directly modulates translation dynamics.

The considerable success of mRNA vaccines against SARS-CoV-2 has underscored the potential of synthetic mRNA as a transformative biomedical technology1. A critical feature of this approach is the incorporation of the modified nucleoside N1-methylpseudouridine (m1Ψ), which enhances antigen expression while reducing immunogenicity2-5. However, a comprehensive understanding of how m1Ψ influences translation remains incomplete. Here we use ribosome profiling at the subcodon resolution to show that m1Ψ increases ribosome density on synthetic mRNAs, leading to higher protein production independent of innate immune activation or eIF2α phosphorylation. We find that m1Ψ directly slows ribosome movement in defined sequence contexts while simultaneously promoting translation initiation. Structural studies using cryo-electron microscopy reveal that m1Ψ alters interactions within the ribosomal decoding centre, providing a mechanistic basis for slowed elongation. Furthermore, by introducing synonymous recoding that disrupts the modification-mediated changes in elongation, we show that the m1Ψ-dependent enhancement of protein output is modulated by codon composition, and that m1Ψ impact is strongest in mRNAs containing non-optimal codons with uridines at the wobble position. Together, these findings demonstrate that m1Ψ directly modulates translation dynamics, thereby increasing protein yield from synthetic mRNAs in specific sequence contexts.