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N1-Methylpseudouridine directly modulates translation dynamics

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TL;DR

N1-methylpseudouridine (m1Ψ) in synthetic mRNA vaccines enhances protein production by slowing ribosome elongation in specific codon contexts while promoting translation initiation. Structural studies reveal m1Ψ alters ribosomal decoding center interactions, with strongest effects in mRNAs containing non-optimal codons with uridines at wobble positions.

Key Takeaways

  • •m1Ψ increases ribosome density and protein production independent of immune activation mechanisms
  • •m1Ψ directly slows ribosome movement in specific sequence contexts while enhancing translation initiation
  • •Cryo-EM structural studies show m1Ψ alters interactions within the ribosomal decoding center
  • •m1Ψ's enhancement effect is modulated by codon composition and strongest in mRNAs with non-optimal codons containing uridines at wobble positions
  • •The findings demonstrate m1Ψ directly modulates translation dynamics to increase protein yield from synthetic mRNAs

Tags

Nucleic-acid therapeuticsTranslationScienceHumanities and Social Sciencesmultidisciplinary

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 immunogenicity2,3,4,5. 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.

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Fig. 1: Modified transcripts exhibit higher ribosome occupancy.
Fig. 2: Modified nucleotides slow translation elongation in a specific sequence context.
Fig. 3: Atomic snapshot of codon–anticodon geometry in the m1Ψ–PRE ribosome complex.
Fig. 4: Enhanced translation initiation underlies increased expression from m1Ψ-modified mRNAs.
Fig. 5: m1Ψ impact is strongest in mRNAs with non-optimal codons.

Data availability

All next-generation sequencing data files have been deposited at the Gene Expression Omnibus under accession GSE309271. The cryo-EM density maps of the ribosome complexes have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-55091 and EMD-55083. Atomic coordinates and structure factors have been deposited in the PDB under accession codes 9SPI and 9SPF.

References

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