eEF3 plays a role in frameshifting at CGA codon repeats. (A) Schematic of the selection for mutants that suppress frameshifting at CGA codon repeats when Mbf1 is defective. In the P15 selection strain, CGA codon repeats plus a single nucleotide were inserted upstream of the URA3 and HA epitope-GFP coding regions; the strain also contains a plasmid-borne copy of ASC1 to avoid mutations in ASC1. The mbf1-R89K mutant in the P15 strain results in an Ura+ GFP+ phenotype due to efficient frameshifting at CGA codon repeats (Wang et al. 2018). Mutants that suppress frameshifting in the mbf1-R89K mutant were selected as FOA resistant mutants that also exhibited reduced GFP expression. (B) The P15 suppressor (P15–30) exhibits an Ura− FOA-resistant phenotype, unlike its parent P15, but like its grandparent (YJYW290) (see Wang et al. 2018). Serial dilutions of the indicated strains were grown at 30°C on rich media (YPAD), complete minimal media (SDC), minimal media lacking uracil (SD-Ura), and minimal media containing FOA. (C) Expression of the GLN4(1–99)-(CGA)4+1-GFP frameshifted reporter is significantly reduced in the P15–30 suppressor relative to its parent (P15). (D) Expression of the GLN4(1–99)-(CGA)4 + 1-GFP frameshifted reporter is partially restored in the P15–30 suppressor by addition of a plasmid bearing the YEF3 gene (encoding eEF3). GFP/RFP was measured in the P15 parent strain and the P15–30 suppressor strain bearing 2µ plasmids with either no insert (V, vector) or genomic inserts from the yeast tiling collection (Jones et al. 2008). eEF3: plasmid with the YEF3 gene; C1 and C2: plasmids with flanking chromosomal sequences. (E) Expression of the GLN4(1–99)-(CGA)4 + 1-GFP frameshifted reporter is modulated by replacement of YEF3 alleles in the chromosome. In the P15 strain, integration of mutant yef3-fs1009 into the chromosome results in reduced expression of frameshifted GFP/RFP while in the suppressor P15–30 strain, integration of wild-type YEF3 into the chromosome results in increased expression of frameshifted GFP/RFP. (F) Amino acid sequence of the carboxy-terminal region of eEF3 from S. cerevisiae was aligned with six evolutionarily distant Ascomycete fungi and a verified eEF3 from the Chromista P. infestans (Mateyak et al. 2018) using MultAlin (http://multalin.toulouse.inra.fr/multalin/) (Corpet 1988). The yef3 G1007V K1009fs mutation is shown above. Numbering above the sequences is based on S. cerevisiae eEF3. The color text represents the level of consensus for each residue (blue: 50%–90%, red: >90%). In all panels, (***) indicates P-value of <0.001.
