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N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.

Published version
Peer-reviewed

Repository DOI


Type

Article

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Authors

Mulroney, Thomas E 
Pöyry, Tuija 
Yam-Puc, Juan Carlos  ORCID logo  https://orcid.org/0000-0003-4395-8844
Rust, Maria 
Harvey, Robert F 

Abstract

In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.

Description

Keywords

31 Biological Sciences, 3102 Bioinformatics and Computational Biology, 32 Biomedical and Clinical Sciences, Lung, Biotechnology, Prevention, Biodefense, Immunization, Emerging Infectious Diseases, Genetics, Pneumonia & Influenza, Vaccine Related, Infection

Journal Title

Nature

Conference Name

Journal ISSN

0028-0836
1476-4687

Volume Title

Publisher

Springer Science and Business Media LLC
Sponsorship
Wellcome Leap (R3-2021-0890067726)
MRC (Unknown)