Beharry Aruun, Ward Cian, Moore Henry, Hoffman Kyle S, Chan Patricia P, Lowe Todd M, O'Donoghue Patrick
Department of Biochemistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Department of Biomolecular Engineering, Baskin School of Engineering & UCSC Genomics Institute, University of California Santa Cruz, Santa Cruz, CA 95064, United States.
Nucleic Acids Res. 2025 Jul 19;53(14). doi: 10.1093/nar/gkaf730.
Nonsense mutations generate premature termination codons (PTCs) that are responsible for 11% of genetic disease alleles. The arginine (Arg, CGA) to stop (UGA) mutation is the most common PTC. Humans encode >600 transfer RNA (tRNA) genes with many identical and similar copies. We developed a dual fluorescent reporter to quantify PTC readthrough in live cells and found single nucleotide mutations of human tRNAArg gene variants enabled differential nonsense suppression that depended on the tRNA sequence and the cell type. We investigated G36A variants of all six human tRNAArgUCG isodecoders, and only the TCG-6-1 tRNA, where G36A occurs in 0.01% of human genomes, was unable to translate nonsense codons. With tRNA sequencing, we showed that a suppressor tRNA derived from the TCG-3-1 gene was expressed 2.1-fold higher and generated 1.8-fold more nonsense suppression than a tRNA derived from the TCG-4-1 gene. In a neuroblastoma model of frontotemporal dementia, we observed >70% readthrough of progranulin R493X with a suppressor tRNA that represented 5%-18% of the total tRNAArg pool. The tRNAs outperformed aminoglycoside-induced nonsense suppression in efficacy, tolerability to the cells, and translation fidelity according to mass spectrometry. Our studies show that human nonsense suppressor tRNAs can correct genetic defects that cause disease.
无义突变会产生过早终止密码子（PTC），这些密码子导致了11%的遗传疾病等位基因。精氨酸（Arg，CGA）突变为终止密码子（UGA）是最常见的PTC。人类编码超过600个转移RNA（tRNA）基因，其中有许多相同和相似的拷贝。我们开发了一种双荧光报告基因来定量活细胞中的PTC通读情况，发现人类tRNAArg基因变体的单核苷酸突变能够实现差异无义抑制，这取决于tRNA序列和细胞类型。我们研究了所有六种人类tRNAArgUCG同功受体的G36A变体，只有TCG-6-1 tRNA（在0.01%的人类基因组中发生G36A突变）无法翻译无义密码子。通过tRNA测序，我们发现源自TCG-3-1基因的抑制性tRNA表达量高2.1倍，产生的无义抑制比源自TCG-4-1基因的tRNA多1.8倍。在额颞叶痴呆的神经母细胞瘤模型中，我们观察到携带一种抑制性tRNA时，前颗粒蛋白R493X的通读率超过70%，该抑制性tRNA占总tRNAArg池的5%-18%。根据质谱分析，这些tRNA在疗效、对细胞的耐受性和翻译保真度方面优于氨基糖苷诱导的无义抑制。我们的研究表明，人类无义抑制性tRNA可以纠正导致疾病的遗传缺陷。
