N1-Methylpseudouridine Directly Modulates Translation Dynamics
The remarkable effectiveness of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted synthetic mRNA as a promising technology. A critical feature of this approach is the incorporation of the modified ribonucleotide N1-methylpseudouridine (m¹?), which enhances antigen expression while reducing immunogenicity. However, a comprehensive understanding of how this modification influences translation remains incomplete. Here, we analyze translation at sub-codon resolution using ribosome profiling and demonstrate that m¹? increases ribosome density on synthetic mRNAs. This elevated ribosome load, together with the correlated increase in protein production, occurs independently of intrinsic cellular immunity or eIF2a phosphorylation. Our data reveal that m¹? directly slows ribosome elongation in specific sequence contexts while concurrently enhancing translation initiation. Cryo-electron microscopy shows that m¹?-modified mRNAs alter interactions within the ribosome decoding center, providing a mechanistic basis for slowed elongation dynamics. Furthermore, by introducing synonymous mutations that disrupt modification-mediated changes in elongation, we show that the m¹?-dependent effect on protein output can be modulated, and that its impact is strongest in mRNAs containing non-optimal codons with uridines at the wobble position. Together, these findings demonstrate that m¹? directly modulates translation elongation and initiation, thereby enhancing translational capacity and increasing protein output from synthetic mRNAs in defined sequence contexts. Overall design: All experiments involved comparing in vitro transcripts (IVTs) synthesized with standard UTP to the same IVTs synthesized with either ? or m¹? substitutions at all uridine positions. Two specific IVTs were studied in detail: SARS-CoV-2 and luciferase (in two different nucleotide sequence variants). For these IVTs, we performed RNA-seq, Ribo-seq, and, in the case of luciferase, also a chase experiment.  In addition, we generated a library of luciferase-encoding sequences with varying uridine content. For this library, we performed polysome profiling, followed by sequencing of five gradient fractions as well as the total RNA.