Chemical modifications of RNAs have long been appreciated as key modulators of non-coding RNA structure and function in cells. However, it has recently become apparent that nucleoside modifications are also sometimes present in the sequences of mRNAs, which direct protein synthesis. It is widely hypothesized that mRNA modification may serve to modulate gene expression because the enzymatic incorporation of mRNA modifications has the potential to impact mRNA stability, protein-recruitment, and translation in a programmed manner. One major obstacle to defining how RNA modifications contribute to gene expression is the difficulty of de-coupling the effects on protein synthesis from protein and mRNA stability in cells. We overcame this challenge by using a fully-purified translation system to directly assess how protein synthesis is impacted by several modifications present in mRNA coding sequences. Our work reveals that replacing a single nucleotide with a modified base in an mRNA codon can reduce the rate of amino acid incorporation, and prevent peptide hydrolysis by class I release factors during translation termination. Additionally, we find that incorporation of pseudouridine (Ψ) promotes the synthesis of multiple peptide products from a single mRNA sequence in both a reconstituted translation system and human embryonic kidney cells. We have a crystal structure that further supports these findings, and suggests that tRNA accommodation may be impacted by the presence of Ψ. These studies provide support for the hypothesis that chemical modifications in mRNA could potentially provide a distinct way for cells to quickly and directly regulate and alter protein production.

mRNA modifications alter translation elongation and fidelity

2019 Fall National Meeting

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