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 tRNAPheGAA called wybutosine (yW) that requires mature, spliced tRNAPheGAA to undergo retrograde nuclear import and subsequent nuclear re-export for its addition. Therefore, the presence/absence of yW-modified tRNAPheGAA 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.
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Acknowledgments
This work was supported by funding from the National Institutes of Health [grant number GM122884 to A.K.H.].
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Nostramo, R.T., Hopper, A.K. (2023). A Simple Method for the Detection of Wybutosine-Modified tRNAPheGAA as a Readout of Retrograde tRNA Nuclear Import and Re-export: HCl/Aniline Cleavage and Nonradioactive Northern Hybridization. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 2666. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3191-1_1
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DOI: https://doi.org/10.1007/978-1-0716-3191-1_1
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