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N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

ABSTRACT: Mass spectrometry on affinity-purified luciferase produced from constitutively modified or unmodified mRNA in HEK293 cells.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Early Embryonic Cell

SUBMITTER: Daniel Eyler

LAB HEAD: Kristin Koutmou

PROVIDER: PXD053919 | Pride | 2025-05-07

REPOSITORIES: Pride

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			Action	DRS
	20180521_SwissProt_Human_Luc2.fasta	Fasta
	20181023_NEB_BR5.raw	Raw
	20181023_NEB_BR6.raw	Raw
	20181101_NEB_BR10.raw	Raw
	20181101_NEB_BR11.raw	Raw

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Publications

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation.

Monroe Jeremy J Eyler Daniel E DE Mitchell Lili L Deb Indrajit I Bojanowski Abigail A Srinivas Pooja P Dunham Christine M CM Roy Bijoyita B Frank Aaron T AT Koutmou Kristin S KS

Nature communications 20240916 1

The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid and accurate protein production. Chemical modification of mRNA nucleobases can adjust the strength and pattern of this hydrogen bonding to alter protein synthesis. We investigate how the N1-methylpseudouridine (m<sup>1</sup>Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the speed and fidelity of translation. We find that m<sup>1</sup>Ψ does not substantia ...[more]

PMID: 39284850

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2025-05-07 | PXD057811 | Pride

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation.

Project description:The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid and accurate protein production. Chemical modification of mRNA nucleobases can adjust the strength and pattern of this hydrogen bonding to alter protein synthesis. We investigate how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the speed and fidelity of translation. We find that m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, we also find that m1Ψ can subtly modulate the fidelity of amino acid incorporation in a codon-position and tRNA dependent manner in vitro and in human cells. Our computational modeling shows that altered energetics of mRNA:tRNA interactions largely account for the context dependence of the low levels of miscoding we observe on Ψ and m1Ψ containing codons. The outcome of translation on modified mRNA bases is thus governed by the sequence context in which they occur.

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Impact of Pus1 Pseudouridine Synthase on Specific Decoding Events in Saccharomyces cerevisiae.

Project description:Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of PUS1 deletion on the essential decoding of CAG (Gln) codons by tRNAGlnCUG in yeast. Synthetic lethality was observed upon combining the modification defect with destabilized variants of tRNAGlnCUG, pointing to a severe CAG-decoding defect of the hypomodified tRNA. In addition, we demonstrated that misreading of UAG stop codons by a tRNAGlnCUG variant is positively affected by Pus1. Genetic approaches further indicated that mildly elevated temperature decreases the decoding efficiency of CAG and UAG via destabilized tRNAGlnCAG variants. We also determined the misreading of CGC (Arg) codons by tRNAHisGUG, where the CGC decoder tRNAArgICG contains Pus1-dependent pseudouridine, but not the mistranslating tRNAHis. We found that the absence of Pus1 increased CGC misreading by tRNAHis, demonstrating a positive role of the modification in the competition against non-synonymous near-cognate tRNA. Part of the in vivo decoding defects and phenotypes in pus1 mutants and strains carrying destabilized tRNAGlnCAG were suppressible by additional deletion of the rapid tRNA decay (RTD)-relevant MET22, suggesting the involvement of RTD-mediated tRNA destabilization.

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Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons.

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