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. 2018 Nov 22;7:e39637. doi: 10.7554/eLife.39637

Figure 1. MBF1 (Multi-protein Bridging Factor 1) prevents frameshifting at CGA codon repeats.

(A) Schematic of selection for mutants that frameshift at CGA codon repeats. The indicated CGA codon repeats plus one extra nucleotide were inserted upstream of the URA3 and GFP coding region (with an upstream HA tag shown in purple), resulting in an Ura- GFP- parent strain. Additional copies of the ASC1 gene were introduced on a LEU2 plasmid to avoid recessive mutations in the native ASC1 gene. To obtain mutants with increased frameshifting efficiency, Ura+ mutants were selected and screened for increased GFP/RFP. (B) Expression of GLN4(1-99)-(CGA)4+1-GFP is increased in the MATa Ura+ mutants from this selection. Flow cytometry scatter plot showing GFP fluorescence versus RFP fluorescence for three mutants from this selection (P15: mbf1-R89K, light blue; P25: mbf1Δ125–151, black; P38: mbf1-K64E, dark blue), for the asc1Δ mutant (red) and for the wild-type parent strain (gray). (C) Expression of the non-native tRNAArg(UCG)* suppressed the Ura+ phenotype of mutant P25 at 30°C. Serial dilutions of the indicated strains with empty vector or expressing the mutant tRNAArg(UCG)* were grown on the indicated media. (D) Mutations in the MBF1 mutants map in conserved amino acids in both the MBF1-specific domain and the Helix-Turn-Helix (HTH) domain of Mbf1 protein. Alignment of yeast Mbf1 amino acids 60–100 with other eukaryotic species is shown (full alignment see Figure 1—figure supplement 3A). GFP/RFP of frameshifted (CGA)4+1 reporter is shown for mutants obtained from MATa (circles) and MATα (triangles) strains, with the color of markers corresponding to the consensus level of this residue (Blue: 50–90%, Red: >90%), however the conserved residue for R61 is N, and for S86 is Q, with all others identical to yeast.

Figure 1.

Figure 1—figure supplement 1. Classification of dominant and recessive mutations and complementation of a recessive mutation.

Figure 1—figure supplement 1.

(A) Analysis of complementation and dominant/recessive nature of mutations. Twelve MATa mutants were crossed with 20 MATα mutants, as well as with the wild type strains. An Ura+ phenotype of resulting diploids with the wild type strain indicated that three mutants were dominant while the Ura+ phenotype of mutants crossed with each other indicated one major complementation group among recessive mutants. (B) Introduction of the Prelich library pool 15 DNA resulted in FOA-resistant cells (Ura-) which indicated suppression of the frameshifting phenotype.
Figure 1—figure supplement 2. Confirmation that mutations in MBF1 are responsible for frameshifting.

Figure 1—figure supplement 2.

(A) Plasmid-borne MBF1 gene suppressed the Ura+ phenotype of mutants P25 and P38 at 30°C. (B) Deletion of the MBF1 coding sequence in the parent GFP- strain resulted in GFP+ phenotype.
Figure 1—figure supplement 3. Mbf1 is conserved and frameshifting mutations do not exhibit sensitivity to 3-AT.

Figure 1—figure supplement 3.

(A) Amino acid sequence alignment of Mbf1 protein from 11 eukaryotic species using MultAlin (http://multalin.toulouse.inra.fr/multalin/) (Corpet, 1988). The color of text corresponds to the consensus level of this residue (Blue: 50–90%, Red: >90%) (B) Frameshifting mbf1-K64E and I85T mutants grow like wild type on plates with 3-aminotriazole at 30°C and do not display a gcn4Δ phenotype. The mbf1Δ strains are more resistant to 3-AT than gcn4Δ strains.