N¹-Methylpseudouridine directly modulates translation dynamics.

Abstract

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 immunogenicity^2-5. 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.