N¹-Methylpseudouridine directly modulates translation dynamics.

Area of Science:

• Biomedical technology
• Molecular biology
• Synthetic biology

Background:

• Synthetic messenger RNA (mRNA) technology has shown significant success, particularly in vaccines.
• N¹-methylpseudouridine (m¹Ψ) is a key modified nucleoside used in synthetic mRNA to improve protein expression and reduce unwanted immune responses.
• The precise mechanisms by which m¹Ψ influences mRNA translation are not fully understood.

Purpose of the Study:

• To elucidate the detailed effects of m¹Ψ on mRNA translation dynamics at a subcodon level.
• To investigate how m¹Ψ impacts ribosome movement, initiation, and overall protein output.
• To determine the sequence-specific contexts where m¹Ψ exerts its strongest effects on translation.

Main Methods:

• Ribosome profiling at subcodon resolution to analyze ribosome density and movement.
• Cryo-electron microscopy to visualize structural changes induced by m¹Ψ in the ribosome.
• Synonymous recoding strategies to probe the role of codon composition in m¹Ψ-mediated effects.

Main Results:

• m¹Ψ increases ribosome density on synthetic mRNAs, leading to higher protein production.
• m¹Ψ directly slows ribosome elongation in specific sequence contexts while promoting translation initiation.
• Structural analysis reveals m¹Ψ alters ribosomal decoding center interactions, explaining the slowed elongation.
• The enhancing effect of m¹Ψ on protein output is dependent on mRNA codon composition, particularly with non-optimal codons containing uridines.

Conclusions:

• m¹Ψ directly modulates mRNA translation dynamics, enhancing protein yield.
• The impact of m¹Ψ is context-dependent, influenced by specific mRNA sequences and codon usage.
• These findings provide a mechanistic understanding of m¹Ψ's role in optimizing synthetic mRNA performance.