Does an N1-methyl-pseudouridine substitution in the first codon position (in mRNA) result in a different amino acid?

Question

Because of the possibility of ‘wobble’ in the base-pair made between the third (5’) position of the anticodon and the third (3’) base of the the mRNA codon , single tRNAs with appropriate bases in this position are often employed by the cell to decode more than one codon specifying the same amino acid (synonymous codons). This is not possible and does not occur in the first or second positions of the codon.

N1-methyl-pseudouridine has been used to replace uracil in the preparation of mRNA for vaccines because it renders the mRNA less immunogenic. It has the same potential for base–pairing as uracil, so that even if its orientation in space is not absolutely identical, one would expect it to be able to form a base-pair with the wobble position of the appropriate anticodon.

But what is the situation with N1-methyl-pseudouridine at the first or second codon positions, where base-pairing is more stringent? Is there a potential for miscoding there, producing a proteins with incorrect amino acids?

Answer 1

Summary

The answer would seem to be NO, from the very fact that the vaccine works. My answer discusses why this should be so. It takes a somewhat different tack to well-documented, but different, answer from @endolith.

Answer

The term ‘wobble’ was originally introduced by Crick to describe and predict instances of non-Watson–Crick base-pairing between the 3′- base of a mRNA codon and the 5′-base of a tRNA anticodon. This was provoked by the finding of the base, inosine, at the 5′-anticodon position of some of the first sequenced tRNAs, and other examples (such as a 5′-anticodon G being used to decode both 3′- codon C or U) were found in situations where the pair of codons involved specified the same amino acid. (See also this SE answer)

The term ‘wobble’ was specifically applied the base pairing at this position as it did not occur at the other positions, where it would generally cause the incorporation of the wrong amino acid. Moreover, when the three-dimensional structure of tRNA was elucidated an explanation for this was found in the strong base-stacking at the 3′- and preceding anticodon position, but not at the 5′-anticodon position (see G34 below — explanation in answer cited above).

Nevertheless, the term ‘wobble’ is sometimes used to mean any sort of non-Watson-Crick base-pairing, which can be the cause of ambiguity in cases where the context does not clarify the situation. I shall try to be clear in this answer.

The question is concerned with the possibility of non-Watson–Crick base-pairing — and consequent mistranslation — between tRNA and m1Ψ (N1-Methylpseudouridine — above) at codon positions 1 or 2 in the artificial Covid spike protein mRNA used in the Pfizer and Moderna vaccines. I would suggest that this does not happen to an appreciable extent otherwise very little spike protein would be produced and the vaccine would be ineffective, which is certainly not the case. The developers of these vaccines were clearly aware of the possibility that m1Ψ in the mRNA might have a greater mispairing potential than the unmodified U it replaced, as they changed a number of codons in consequence. However these changes were specifically U→C at the 3′-codon position (red shading, below), as the tRNA that decodes cognate pairs of codons ending in U or C always has a 5′-anticodon G. Of course, it would have been very difficult to do anything if there were miscoding of m1Ψ at the first two codon positions, but one can assume that the developers either checked that this did not occur or guessed correctly that it would not occur there to an appreciable extent.

A possible objection to the above argument is that readthrough of termination codons (UAG, UAA, UGA) occurs to an increased extent in mRNA in which m1Ψ has replaced U at the 5′-codon position. (This may be one reason for the use of multiple termination codons in the artificial mRNA — see this answer to the SE question on that topic.) However the situation here is different from the miscoding of, say, tyrosine codon UAC. In that case miscoding would involve competition between the tRNA for tyr and those for his, asn and asp (see green shading, below). However termination involves competition for the codon between tyr-tRNA and the termination factors. The modification of the codon U may conceivable decrease its affinity for the termination factor, which depends on RNA–protein interactions, not base-pairing.

Answer 2

Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines says that this can happen:

all U nucleotides were replaced by N1-methylpseudouridine (Ψ) [14,15]. However, Ψ wobbles more in base-pairing than U and can pair not only with A and G, but also, to a lesser extent, with C and U [16]. This is likely to increase misreading of a codon by a near-cognate tRNA. When nucleotide U in stop codons was replaced by Ψ, the rate of misreading of a stop codon by a near-cognate tRNAs increased [17]. Such readthrough events would not only decrease the number of immunogenic proteins, but also produce a longer protein of unknown fate with potentially deleterious effects.
…
There is another reason for recoding U-ending codons to C-ending codons. All U nucleotides in the two mRNA vaccines were replaced by N1-methylpseudouridines (Ψ) which can wobble with all [four] nucleotides and, therefore, should not be used in 2-fold codon families.

However, citation 16 is Kierzek 2014, which is about Ψ, not m1Ψ, and two others seem to contradict this:

The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines says:

However, Ψ contains an extra hydrogen bond donor group (N1H) that contributes to a universal base character, i.e., it can not only pair A but also wobble base-pair with G, U, or C in the context of a duplex (Kierzek et al., 2014). On the other hand, N1-methyl-Ψ has a methyl group instead in the N1-position (Figure 1), thus eliminating the extra hydrogen bond donor. Consequently, N1-methyl-Ψ can only use its Watson-Crick face to base-pair with another nucleoside, thus preventing it from wobble-pairing with other nucleotides (G, U, and C).

It cites the same Kierzek source for wobble-pairing of Ψ, but doesn't cite anything for lack of wobble-pairing in m1Ψ.

N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells seems to say the same thing?

Interestingly, Ψ oligonucleotides showed two Tm values, suggesting the existence of an additional unstable binding pattern. This observation, together with the fact that Ψ has an extra N1 imino proton (26,27) for hydrogen bonding (Supplementary Figure S5), agrees with previous research, which concluded that Ψ can act as a universal base to form wobble base-pairing with multiple bases (28) and may alter structural stability (29). Thus, together with the results above, we conclude that m1Ψ-containing miRNA-sensing switches outperform other switches because m1Ψ allows higher basal protein expression and enhanced miRNA sensitivity.

Since m1Ψ only has one Tm value, this implies that it doesn't act as a universal base?