Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany.
Biomolecules. 2020 May 7;10(5):729. doi: 10.3390/biom10050729.
Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of deletion on the essential decoding of CAG (Gln) codons by tRNACUG in yeast. Synthetic lethality was observed upon combining the modification defect with destabilized variants of tRNACUG, pointing to a severe CAG-decoding defect of the hypomodified tRNA. In addition, we demonstrated that misreading of UAG stop codons by a tRNACUG variant is positively affected by Pus1. Genetic approaches further indicated that mildly elevated temperature decreases the decoding efficiency of CAG and UAG via destabilized tRNACAG variants. We also determined the misreading of CGC (Arg) codons by tRNAGUG, where the CGC decoder tRNAICG contains Pus1-dependent pseudouridine, but not the mistranslating tRNA. We found that the absence of Pus1 increased CGC misreading by tRNA, demonstrating a positive role of the modification in the competition against non-synonymous near-cognate tRNA. Part of the in vivo decoding defects and phenotypes in mutants and strains carrying destabilized tRNACAG were suppressible by additional deletion of the rapid tRNA decay (RTD)-relevant , suggesting the involvement of RTD-mediated tRNA destabilization.
Pus1 依赖性假尿嘧啶化发生在许多 tRNA 中，并在多个位置发生，但这种修饰的功能影响尚未完全了解。我们分析了缺失对酵母中 tRNACUG 对 CAG（Gln）密码子的基本解码的影响。当与 tRNACUG 的不稳定变体结合时，观察到合成致死性，这表明修饰不足的 tRNA 存在严重的 CAG 解码缺陷。此外，我们证明了 tRNACUG 变体对 UAG 终止密码子的误读受到 Pus1 的正向影响。遗传方法进一步表明，通过不稳定的 tRNACAG 变体，轻度升高的温度会降低 CAG 和 UAG 的解码效率。我们还确定了 tRNAGUG 对 CGC（Arg）密码子的误读，其中 CGC 解码器 tRNAICG 包含 Pus1 依赖性假尿嘧啶，但不包含翻译错误的 tRNA。我们发现 Pus1 的缺失增加了 tRNA 对 CGC 的误读，这表明该修饰在与非同义近同功 tRNA 的竞争中具有积极作用。部分体内解码缺陷和突变体和携带不稳定 tRNACAG 的菌株的表型可以通过额外缺失快速 tRNA 衰变（RTD）相关的 来抑制，这表明涉及 RTD 介导的 tRNA 不稳定。
