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.

Keywords: cysteine tRNA; near-cognate tRNA; readthrough-inducing tRNA; stop codon readthrough; translation.

FIGURE 1.

tRNA^Cys isodecoders with the highest tRNA mature score promote SC-RT on UGA when overexpressed. Cys01 and Cys02 (A); Cys03, Cys04, and Cys05 (B); and Cys06–09 (C) were overexpressed in HEK293T cells and SC-RT at UGA was measured using the p2luci system. Changes in the readthrough levels relative to the reporter with no tRNA (eV) were analyzed by the Student's t-test (mean + SD; n = 2); statistically significant increase with P < 0.01 is marked with asterisk (*).

FIGURE 2.

Cys01 displays the highest readthrough levels at UGA also with the pSGDluc system. The changes in the readthrough levels relative to the reporter with no tRNA (eV) were analyzed by Student's t-test (mean + SD; n = 2); statistically significant increase with P < 0.01 is marked with asterisk (*).

FIGURE 3.

Comparison of primary sequences and secondary structures of tRNA^Cys isodecoders. Cys01 (A) was taken as a reference sequence/structure—all individual differences between Cys01 and other isodecoders (B–I) are highlighted in red. The secondary structure predictions were done with the help of http://gtrnadb.ucsc.edu.