Cysteine tRNA acts as a stop codon readthrough-inducing tRNA in the human HEK293T cell line

Abstract

Under certain circumstances, any of the three termination codons can be read through by a near-cognate tRNA; i.e., a tRNA whose two out of three anticodon nucleotides base pair with those of the stop codon. Unless programed to synthetize C-terminally extended protein variants with expanded physiological roles, readthrough represents an undesirable translational error. On the other side of a coin, a significant number of human genetic diseases is associated with the introduction of nonsense mutations (premature termination codons [PTCs]) into coding sequences, where stopping is not desirable. Here, the tRNA's ability to induce readthrough opens up the intriguing possibility of mitigating the deleterious effects of PTCs on human health. In yeast, the UGA and UAR stop codons were described to be read through by four readthrough-inducing rti-tRNAs—tRNA^Trp and tRNA^Cys, and tRNA^Tyr and tRNA^Gln, respectively. The readthrough-inducing potential of tRNA^Trp and tRNA^Tyr was also observed in human cell lines. Here, we investigated the readthrough-inducing potential of human tRNA^Cys in the HEK293T cell line. The tRNA^Cys family consists of two isoacceptors, one with ACA and the other with GCA anticodons. We selected nine representative tRNA^Cys isodecoders (differing in primary sequence and expression level) and tested them using dual luciferase reporter assays. We found that at least two tRNA^Cys can significantly elevate UGA readthrough when overexpressed. This indicates a mechanistically conserved nature of rti-tRNAs between yeast and human, supporting the idea that they could be used in the PTC-associated RNA therapies.