N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

Abstract

The ribosome relies on hydrogen bonding interactions between mRNA codons and incoming aminoacyl-tRNAs to ensure rapid and accurate protein production. The inclusion of chemically modified bases into mRNAs has the potential to alter the strength and pattern of hydrogen bonding between mRNAs and aminoacyl-tRNAs to alter protein synthesis. We investigated how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the ability of codons to react with cognate and near-cognate tRNAs and release factors. We find that the presence of a single m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, insertion of m1Ψ can affect the selection of near-cognate tRNAs both in vitro and in human cells. Our observations demonstrate that m1Ψ, and the related naturally occurring pseudouridine (Ψ) modification, exhibit the ability to both increase and decrease the extent of amino acid misincorporation in a codon-position and tRNA dependent manner. To ascertain the chemical logic for our biochemical and cellular observations, we computationally modeled tRNAIle(GAU) bound to unmodified and m1Ψ- or Ψ-modified phenylalanine codons (UUU). Our modeling suggests that changes in the energetics of mRNA:tRNA interactions largely correlate with the context specificity of Ile-miscoding events we observe on Ψ and m1Ψ containing Phe codons. These studies reveal that the sequence context of a given modification within an mRNA plays a large role in determining how (and if) the modification impacts the number and distribution of proteoforms synthesized by the ribosome.

Structural and thermodynamic consequences of base pairs containing pseudouridine and N1-methylpseudouridine in RNA duplexes

“…Particularly, N1-methylpseudouridine has been used in the development of mRNA vaccines against COVID-19 disease32 and there is an immensely growing interest for this modification in the field of mRNA therapeutics in the current years. Such mRNA vaccines containing m 1 Ψ have been reported to show high stability and efficacy over those containing the canonical counterpart 32,A recent study by Monroe et al71 revealed the alteration of mRNA decoding as a result of Ψ and m 1 Ψ modifications during translation process and their study also suggested the context-dependent modulation of codon:anticodon base pairing interactions by Ψ and m 1 Ψ. Our study suggests the ability of m 1 Ψ to form more stable base pairs with the canonical residues compared to U or Ψ.…”

Translation of in vitro-transcribed RNA therapeutics

“…Consistent with this expectation, translation elongation rates on fully Ψ-modified transcripts in reconstituted bacterial translation systems were reduced 3-fold and thus greater than expected if the reduced GTPase activation had been the sole contributing factor. Moreover, increased amino acid misincorporation was observed specifically on Ψ-containing codons in bacterial and cultured human cells ( Eyler et al, 2019 ), Ψ incorporation into stop codons was observed to cause increased non-sense suppression ( Karijolich and Yu, 2011 ), and a recent study provided direct evidence for changes in near-cognate tRNA interactions on m1Ψ-modified codons ( Monroe et al, 2022 ). Together these results suggest that modification of transcripts with Ψ or m1Ψ alters competition from near-cognate tRNAs, thereby leading to general ribosome slow-down as well as reduced translational accuracy.…”