Pseudouridinylation of mRNA coding sequences alters translation

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Psi), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with psi in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNA(Phe) bound to a Psi UU codon in the A site supports these findings. We also find that the presence of Psi can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNA(Val) reacting on a Psi UU codon. The vast majority of Psi moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Psi can be to modestly alter both translation speed and mRNA decoding.