A dominant‐lethal screen to identify non‐functional rRNA decay pathway factors in yeast

Abstract

Nonfunctional rRNA Decay (NRD), which eliminates nonfunctional ribosomes that have assembled with mutant rRNAs, is a recently discovered quality control mechanism in eukaryotes. To investigate the mechanistic details of NRD, a dominant‐lethal screen was developed to identify trans‐acting factors associated with this pathway. Galactose‐induceable plasmids that encode rRNA genes with mutations in functionally important regions of 18S and 25S rRNA were transformed into Saccharomyces cerevisiae. Transformants were exposed to ultra‐violet (UV) radiation in order to induce genomic DNA mutations. Mutations in genes required for NRD may result in a dominant‐lethal phenotype when mutant rRNAs are expressed in these cells. To determine which cells acquired a dominant lethal phenotype, UV exposed colonies were replica plated onto galactose to allow expression of the plasmid‐encoded mutant rRNAs. To date, we have isolated ten different S. cerevisiae colonies that cannot grow on galactose after UV exposure. Complementation experiments are now underway to determine the identity of the resulting mutant trans‐acting factors and to establish their roles in NRD. Supported by the Dreyfus Foundation and the Jeffress Memorial Trust.