TY - UNPB
T1 - N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation
AU - Monroe, Jeremy G.
AU - Mitchell, Lili
AU - Deb, Indrajit
AU - Roy, Bijoyita
AU - Frank, Aaron T.
AU - Koutmou, Kristin
PY - 2022/6/14
Y1 - 2022/6/14
N2 - 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 (m1Y) 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 m1Y does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, insertion of m1Y can affect the selection of near-cognate tRNAs both in vitro and in human cells. Our observations demonstrate that m1Y, and the related naturally occurring pseudouridine (Y) 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 m1Y- or Y- 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 Y and m1Y 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.
AB - 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 (m1Y) 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 m1Y does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, insertion of m1Y can affect the selection of near-cognate tRNAs both in vitro and in human cells. Our observations demonstrate that m1Y, and the related naturally occurring pseudouridine (Y) 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 m1Y- or Y- 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 Y and m1Y 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.
U2 - 10.1101/2022.06.13.495988
DO - 10.1101/2022.06.13.495988
M3 - Preprint
BT - N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation
PB - BioRxiv
ER -
