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 intron-less 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, significantly improves codon-anticodon recognition, robustly promoting PTC read-through. The intron-less 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 has identified an unusual Ψ-Ψ base pair, which contributes significantly to codon-anticodon recognition and translational recoding. BY4741 strain was transformed with mCherry(K14UAG) reporter gene and guide RNA (gRNA) expression cassette for targeted pseudouridylation. The cassette contains targeted gRNAs, including PTC(K14ΨAG)-gRNA and tRNA_Lys(CUΨ36)-gRNA, for treated samples, or control gRNAs for control samples. Cells were cultured to mid-log phase before harvesting. Samples were processed by TB-SEQ, Inc. for RNA-seq and ribosome profiling (RIBO-seq).