Abstract
tRNAs are highly mobile molecules that are trafficked back and forth between the nucleus and cytoplasm by several proteins. However, characterization of the movement of tRNAs and the proteins mediating these movements can be difficult. Here, we describe an easy and cost-effective assay to discover genes that are involved in two specific tRNA trafficking events, retrograde nuclear import and nuclear re-export for yeast, Saccharomyces cerevisiae. This assay, referred to as the hydrochloric acid (HCl)/aniline assay, identifies the presence or absence of a unique modification on tRNA(Phe)(GAA) called wybutosine (yW) that requires mature, spliced tRNA(Phe)(GAA) to undergo retrograde nuclear import and subsequent nuclear re-export for its addition. Therefore, the presence/absence of yW-modified tRNA(Phe)(GAA) serves as a readout of retrograde nuclear import and nuclear re-export. This simple assay can be used to determine the role of any gene product in these previously elusive tRNA trafficking events.