The success of mRNA vaccines against COVID-19 highlights the promise of synthetic mRNA in medicine. A key ingredient in these vaccines is a modified nucleoside called N1-methylpseudouridine, which helps produce more proteins while reducing unwanted immune responses. This study examines how this modification affects the process of translating mRNA into proteins. We found that N1-methylpseudouridine increases the presence of ribosomes on mRNAs, leading to higher protein levels. It slows down the movement of ribosomes while also helping to start the translation process. Our structural studies show that this modification changes how ribosomes interact with mRNA, providing insights into how it enhances protein production, especially in mRNAs with specific sequences. Overall, our findings reveal that N1-methylpseudouridine plays a crucial role in improving protein yield from synthetic mRNAs.
Simplified
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.
