Pan Yi, Kierzek Elzbieta, Kierzek Ryszard, Mathews David H, Yu Yi-Tao
Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY, USA.
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Nat Chem Biol. 2025 Sep 12. doi: 10.1038/s41589-025-02025-9.
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.
假尿苷（Ψ）已被知晓数十年，但其碱基配对的灵活性仍不清楚。本研究设计了人工盒式H/ACA引导RNA，以指导在酵母和人类细胞中无内含子mRNA内的提前终止密码子（PTC；UAA、UAG或UGA）的尿苷以及匹配tRNA反密码子的U36处进行假尿苷化。靶向假尿苷化导致形成Ψ-Ψ密码子-反密码子对，该对与密码子-反密码子双链体中的其他两个沃森-克里克碱基对一起，极大地改善了密码子-反密码子识别，有力地促进了PTC通读。有无引导RNA时，无内含子mRNA水平保持不变。此外，假尿苷化不影响tRNA稳定性或电荷。我们的结果表明，Ψ-Ψ对的高亲和力促进了无义抑制，这通过熔解曲线分析得到验证。这项工作鉴定出一种不寻常的Ψ-Ψ碱基对，其对密码子-反密码子识别和翻译重编码有很大贡献。
