Yeast applied readthrough inducing system (YARIS): an invivo assay for the comprehensive study of translational readthrough
Abstrakt
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 isoformspecific 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 stopcodon tetranucleotide manner when overexpressed, designated readthrough-inducing tRNAs (rti-tRNAs).
These rti-tRNAs are the keystones of YARIS (yeast applied readthrough inducing system), a reporterbased 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 rtitRNA modifications, as well as various readthroughinducing drugs (RTIDs).
This analysis identified a variety of genetic interactions demonstrating the power of YARIS to characterize existing and identify novel RTIDs.