Whole genome sequencing of Naumovozyma castellii RNA interference mutants

A genetic selection was designed to discover novel components of the RNA interference (RNAi) pathway in the budding-yeast species Naumovozyma castellii. This selection centered on an N. castellii strain that expressed RNA hairpins that were processed by Dicer to produce siRNAs targeting HIS3, URA3, and GFP mRNAs expressed from exogenous genes that had been integrated into the genome. The hairpin and GFP genes were under the control of the S. cerevisiae GAL1 and URA3 promoters, respectively, and the HIS3 and URA3 genes, together with their promoters, were obtained from S. cerevisiae and replaced their N. castellii counterparts in the genome of the selection strain. Because this parental strain was engineered to deploy the RNAi pathway to silence HIS3 and URA3, it did not grow on media lacking histidine and uracil. This strain was mutagenized with ethyl methane sulfonate (EMS), and the cells were plated on media lacking histidine and uracil with the goal of identifying mutants that had a His+ and Ura+ phenotype because they no longer efficiently silenced HIS3 and URA3. As a secondary screen, mutants were examined by flow cytometry for their ability to silence GFP, and those that had impaired GFP silencing were carried forward for complementation analysis. Mutations that lost silencing were selected in this parental selection strain (which contained duplicated AGO1 and DCR1 genes). As expected, isolated mutant strains but not the parental strain grew on selective media, as assessed by a serial-dilution growth assay. Each of the isolated strains carried recessive mutations, as assessed from analyses of diploid products of mating with a wild-type strain. 19 strains retained both Ago1p and Dcr1p activities, as indicated by fully restored GFP silencing in diploid products of both ago1 KO and dcr1 KO matings. These 19 strains were carried forward as potentially having mutations in genes encoding another protein needed for efficient RNAi. To identify mutated genes, we sequenced the genomes of the parental and mutant strains (130-fold and 65-248-fold coverage, respectively), which identified many protein-coding or tRNA changes in each of the mutant genomes. The data represented here are the whole-genome sequencing data of the parental selection strain and the 19 selected putative RNAi-mutant strains.