Exploring the effects of N1-methylpseudouridine in mRNA translation

  • Dec 7, 2023
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Exploring the effects of N1-methylpseudouridine in mRNA translation

In a recent study published in Nature, a group of researchers explored how the incorporation of N1-methylpseudouridine (1-methylΨ) into messenger ribonucleic acids (mRNAs) affects ribosomal frameshifting and the overall fidelity of mRNA translation.

Study: N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Image Credit: MattL_Images/


Therapeutic in vitro-transcribed (IVT) mRNAs, like those in COVID-19 vaccines, often contain modified ribonucleotides such as 1-methylΨ to reduce immunogenicity and increase stability, enhancing their therapeutic effectiveness. While some modifications like 5-methylcytidine (5-methylC) are naturally occurring in eukaryotic mRNA, others like 1-methylΨ are not. These modifications, including 5-methoxyuridine (5-methoxyU) and 5-methylC, aim to boost recombinant protein synthesis for mRNA therapies.

Further research is needed to comprehensively understand how modified ribonucleotides like 1-methylΨ, 5-methoxyU, and 5-methylC affect the fidelity of mRNA translation, particularly in therapeutic IVT mRNAs, given their widespread use in therapies and vaccines and the current limited knowledge about their impact on protein synthesis.

About the study 

In the present study, the researchers employed a variety of methods to synthesize and analyze modified mRNA. For plasmids and mRNA synthesis, they used Phusion High-Fidelity deoxyribonucleic acid (DNA) polymerase reagents and created template DNA for wild-type (WT) and frameshifted firefly luciferase (Fluc). Custom genes were transcribed for different mRNA modifications using the TranscriptAid T7 High Yield Transcription Kit. The transcripts underwent modifications with nucleotides like 5-methoxyUTP, N1-methylpseudoUTP, or 5-methylCTP and were purified and quantified for further experiments.

For RNA gel electrophoresis, samples were prepared with formamide and dyes, then resolved on agarose gels, stained with ethidium bromide, and visualized under UV light. The study involved culturing and transfecting HeLa cells with modified mRNAs and assessing luciferase activity post-transfection. They also performed in vitro translation using the Flexi Rabbit Reticulocyte Lysate System, incorporating [35S]methionine for labeling.

The study utilized peptide Liquid Chromatography–Tandem Mass Spectrometry LC–MS/MS) analysis for identifying translation products, and mass spectrometry to analyze in-gel digests. Data from mass spectrometry were processed using Proteome Discoverer software. Additionally, they conducted RNA-sequencing analysis of IVT mRNAs using the NextFlex Rapid Directional RNA-seq kit and Illumina MiSeq sequencing.

For further insights, they used Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) autoradiography and quantified incorporated [35S]methionine in translated mRNAs. The immunological aspect was explored through mouse immunization studies, Interferon Gamma Enzyme-Linked ImmunoSpot (IFNγ ELISpot) assays to measure immune responses, and Human Leukocyte Antigen (HLA) genotyping of human donors to understand genetic composition related to vaccine responses.

Statistical analyses of the collected data were performed using R software. This comprehensive approach allowed the researchers to investigate how modified ribonucleotides affect mRNA translation thoroughly.

Study results 

In their exploration of how ribonucleotide modifications influence reading frame maintenance during mRNA translation, the researchers developed in vitro transcribed (IVT) mRNAs (Fluc+1FS) as reporters for out-of-frame protein synthesis. These mRNAs were designed to encode an amino-terminal segment of NFluc and a complementary carboxy-terminal segment of Fluc (CFluc) in the +1 reading frame. Normally translated, these mRNAs would produce an inactive NFluc, but ribosomal frameshifting could yield active polypeptides containing residues from both segments.

The team synthesized various modified Fluc+1FS mRNAs, incorporating 5-methoxyU, 5-methylC, and 1-methylΨ, and compared their translation with unmodified mRNAs. They discovered that 1-methylΨ significantly increased ribosomal +1 frameshifting in Fluc+1FS mRNA translation, a phenomenon not observed with other ribonucleotides. This finding was also replicated in HeLa cells transfected with 1-methylΨ Fluc+1FS mRNA.

Further investigation was conducted to understand these +1 frameshift translation products better. Western blotting revealed that 1-methylΨ mRNAs, unlike other modified mRNAs, generated additional higher molecular weight bands, indicative of frameshift polypeptides. Given 1-methylΨ’s use in SARS-CoV-2 mRNA vaccines, they extended their study in vivo, using BNT162b2, a vaccine containing 1-methylΨ. They found that vaccinated mice showed significant T cell responses to +1 frameshift spike peptides. This response was also observed in humans vaccinated with BNT162b2, indicating that 1-methylΨ mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting.

To further dissect the mechanism behind this frameshifting, they conducted LC-MS/MS analysis on the translation products of 1-methylΨ Fluc+1FS mRNA, identifying several peptides derived from the +1 frame. This supported the notion that these elongated polypeptides were indeed chimeric, combining in-frame and frameshift residues. Additionally, they investigated whether frameshifted products of 1-methylΨ mRNA translation were due to transcriptional errors. RNA sequencing of unmodified and 1-methylΨ Fluc+1FS mRNA suggested that the frameshifted products were a result of bona fide ribosomal frameshifting rather than transcriptional inaccuracies.

The study also examined how 1-methylΨ in IVT mRNA affects translation elongation, discovering that translation of 1-methylΨ mRNA was slower compared to unmodified mRNA, indicating ribosome stalling. They hypothesized that this stalling could be due to altered aminoacyl-tRNA binding and found that the addition of paromomycin improved polypeptide elongation in 1-methylΨ mRNA translation.


Given these findings, the researchers emphasized the importance of careful mRNA sequence design in future mRNA technology applications to minimize ribosomal frameshifting events. They demonstrated that synonymous mutations in slippery sequences of 1-methylΨ Fluc+1FS mRNA could significantly reduce +1 frameshifting. This suggests that with targeted mRNA design, it is possible to mitigate the effects of ribosomal frameshifting induced by N1-methylpseudouridylation.

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