N1-methylpseudouridine and pseudouridine mRNA Modifications Modulate Translation Rate and Fidelity

Abstract

The discovery of modifications within the coding region of mRNA have highlighted the importance of uncovering how modifications alter the transformation of the information encoded in RNA transcripts into functional protein. Prominent among mRNA modifications are the N1-methylpseudouridine (m1Ψ) and pseudouridine (Ψ) modifications, which are both incorporated into RNA therapeutic platforms, with m1Ψ being a prominent component in the Pfizer and Moderna COVID-19 vaccines. Using a fully reconstituted prokaryotic in vitro system detailed molecular level insights can be gained about how mRNA modifications can modify the rate and fidelity of translation. Alterations to either the rate or fidelity of translation can result in varying levels of protein production or miscoded protein products. My studies have demonstrated that both Ψ and m1Ψ are able to modulate ribosome fidelity in a codon and anticodon dependent manner. This alteration of ribosome fidelity was confirmed by in cellular studies in HEK293 cells which displayed the same pattern of altered ribosomal accuracy. Using molecular modeling we deduced that isomerization and increased base stacking interactions are partially responsible for the changes in translational accuracy. Understanding if and how these modifications impact translation is important for understanding the epigenetic landscape and potential avenues of mRNA therapeutics development.