Adaptation of fluorescence rescue reporters of mistranslation for use in Saccharomyces cerevisiae

Abstract

Specific mutations to tRNA genes can cause mistranslation - the incorporation of amino acids mismatched to the mRNA code during translation. The effects of mistranslation are hugely varied, depending on the form of mistranslation and various contextual factors. A key aspect of mutant tRNA mistranslation investigation is the reliable detection of mistranslation events. While mistranslation confirmation and quantification are commonly accomplished via mass spectrometry, others have devised a means of detecting mistranslation via fluorescent signal. Here, we successfully adapt this method for use in Saccharomyces cerevisiae, a model in which a great deal of mistranslation research is currently conducted. We demonstrate that mistranslation fluorescence rescue is detectable using both microscopy as well as fluorescent plate reader measurement, and that fluorescence intensity varies with the form of mistranslation assessed. Interestingly, the quantity of mistranslation measured by fluorescence rescue differs from measurements by standard mass spectrometry methods, potentially due to capturing a different timepoint of proteins that contain mistranslation errors. As such, this tool has high utility in detecting mutant tRNA mistranslation presence in yeast models, while its quantification of mistranslation frequency requires further investigation.