A Ψ-Ψ codon-anticodon pairing in nonsense suppression and translational recoding

Pseudouridine (Ψ) is known for decades but its flexibility in base pairing remains unclear. This study engineers artificial box H/ACA guide RNAs to direct pseudouridylation at the uridine of a premature termination codon (PTC; UAA, UAG or UGA) within an intronless mRNA and U36 of the anticodon of a matching tRNA in yeast and human cells. Targeted pseudouridylation leads to the formation of a Ψ-Ψ codon-anticodon pair, which, together with the other two Watson-Crick base pairs in the codon-anticodon duplex, greatly improves codon-anticodon recognition, robustly promoting PTC readthrough. The intronless mRNA level remains unchanged with or without guide RNAs. Additionally, pseudouridylation does not impact tRNA stability or charging. Our results show that nonsense suppression is promoted by the high affinity of the Ψ-Ψ pair, which is verified by melting curve analysis. This work identifies an unusual Ψ-Ψ base pair, which contributes greatly to codon-anticodon recognition and translational recoding.