A synthetic‐lethal screen to identify non‐functional ribosomal RNA decay pathway factors in Saccharomyces cerevisiae

Abstract

Nonfunctional rRNA Decay (NRD) is a quality control pathway in eukaryotes which eliminates nonfunctional ribosomes that have been assembled with mutant rRNAs. To investigate the mechanistic details of NRD, a synthetic‐lethal screen was developed to identify trans‐acting factors associated with this pathway. Galactose‐inducible plasmids encoding rRNA genes with mutations in functionally important regions of 18S and 25S rRNA, as well as a control plasmid with wildtype 18S and 25S rRNA genes, were transformed into Saccharomyces cerevisiae. Transformants were exposed to ultra‐violet (UV) radiation in order to induce random genomic DNA mutations. Mutations in genes required for NRD may result in a synthetic‐lethal phenotype when mutant rRNAs are expressed in these cells. To determine which cells acquired a synthetic lethal phenotype, UV‐exposed colonies were replica plated onto galactose to allow expression of the plasmid‐encoded mutant rRNAs. Thus far, we have isolated eight different S. cerevisiae colonies with the 18S mutant plasmid and ten colonies with the 25S mutant plasmid that are unable to grow on galactose after UV exposure. Phenotypic analysis, rRNA expression analysis, and complementation studies are currently underway to characterize the mutant strains and to determine the identity of the NRD factor mutations. This work is supported by the Dreyfus Foundation and the Jeffress Memorial Trust.