Stop codon readthrough-the decoding of a stop codon by a near-cognate tRNA-is employed by viruses to balance levels of enzymatic and structural proteins and by eukaryotic cells to enable isoform-specific protein synthesis in response to external stimuli. Owing to the prevalence of premature termination codons in human disease, readthrough has emerged as an attractive therapeutic target. A growing list of various features, for example the +4 nucleotide immediately following the stop codon, modulate readthrough levels, underscoring the need for systematic investigation of readthrough. Here, we identified and described a complete group of yeast tRNAs that induce readthrough in the stop-codon tetranucleotide manner when overexpressed, designated readthrough-inducing tRNAs (rti-tRNAs). These rti-tRNAs are the keystones of YARIS (yeast applied readthrough inducing system), a reporter-based assay enabling simultaneous detection of readthrough levels at all twelve stop-codon tetranucleotides and as a function of the complete set of rti-tRNAs. We demonstrate the utility of YARIS for systematic study of translation readthrough by employing it to interrogate the effects of natural rti-tRNA modifications, as well as various readthrough-inducing drugs (RTIDs). This analysis identified a variety of genetic interactions demonstrating the power of YARIS to characterize existing and identify novel RTIDs.

Biology Department and Biochemistry Program Vassar College 124 Raymond Avenue Poughkeepsie 12601 NY USA

Laboratory of Regulation of Gene Expression Institute of Microbiology ASCR Videnska 1083 142 20 Prague the Czech Republic

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Guidelines for minimal reporting requirements, design and interpretation of experiments involving the use of eukaryotic dual gene expression reporters (MINDR)

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

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uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3