Human transfer RNA anticodon variants suppress pathogenic nonsense mutations

Aruun Beharry, Western University

Abstract

Nonsense mutations disrupt protein synthesis by introducing a premature termination codon (PTC) into messenger RNA (mRNA), leading to truncated protein products and severe diseases. Transfer RNAs (tRNAs) can be mutated at their anticodon, allowing them to suppress these nonsense mutations, restoring full-length proteins to the cell. I demonstrate that three human tRNA^Arg_UCG isodecoders, bearing the same engineered anticodons (G36A) and amino acids but with differences in body sequence, can mediate significant and differential suppression in a live-cell fluorescent reporter and with a clinically relevant PTC. Experiments in multiple mammalian cell lines demonstrate that suppressor tRNA^Arg_UCA (sup-tRNAs ) promotes PTC readthrough levels that depend on the sequence of the tRNA and the cell type. Suppressor tRNA^Arg variants produced wildtype progranulin (PGRN/GRN) from a frontotemporal dementia (FTD)-causative allele (R493X). The suppressor tRNAs significantly outperform the aminoglycoside nonsense suppressor (G418) in efficacy, tolerability to the cells, and in translation fidelity as validated by mass spectrometry. The data indicates that suppressor tRNAs are an important area for further therapeutic development with applications in FTD and other diseases caused by PTCs.