Figure 2. Disrupting sumoylation rescues Pol III mutations.

(A) Tetrad analysis of a cross between rpc160-M809I and siz1Δ. Tetrads were dissected on YPD, then incubated at 30°C for 4 days. The offspring of one representative tetrad was shown with genotypes labeled. (B) Similar tetrad analysis for rpc128-A704T and siz1Δ. (C) Similar tetrad analysis for rpc160-M809I and siz2Δ. (D) 2 μg of RNA extracted from the indicated strains was run on a 2.8% agarose gel containing ethidium bromide, then visualized with UV. (E) RNA from (C) was reverse transcribed into cDNA, followed by real-time PCR analysis. GAPDH transcripts were used as loading control. Data are mean ± standard deviation calculated from six data points (two biological replicates and three technical replicates), presented as relative amount compared to wild type. The intron-containing pre-mature tRNA (pre-tL(CAA)A) is short-lived, so its abundance reflects the Pol III transcriptional activity.

Figure 2—figure supplement 1. Position of the mutated residues in Pol III structure.

Figure 2—figure supplement 2. siz1Δ rescued a wide spectrum of Pol III mutations.

(A) Plasmid shuffle experiments to test the growth phenotype of known rpc160 and rpc31 mutants in wild-type SIZ1 or siz1Δ background. The RPC160 or RPC31 alleles were on LEU2 vectors. The parental rpc160Δ and rpc31Δ strains contain a URA3 plasmid carrying wild-type RPC160 or RPC31, respectively. (B) Wild-type RPC160 was placed under control of the GAL1 promoter on a LEU2 vector then transformed into an rpc160 strain carrying a URA3 RPC160 plasmid, which was lost in the presence of 5FOA. The 5FOA plate contained glucose as the only carbon source, which strongly repressed RPC160 expression, making the cells grow extremely slowly. siz1Δ partially alleviated this growth defect. (C–E) Similar plasmid shuffle experiments showing the rescue effect of siz1Δ on disease causing mutations. rpc160-DN: D384N, N789I.

Figure 2—figure supplement 3. Growth phenotype of Pol III disease mutations in yeast.

CEN LEU2 plasmids containing the indicated mutant alleles (e.g. rpc160 mutants) were transformed into a corresponding null strain in wild-type SIZ1 or siz1Δ background (e.g. rpc160Δand rpc160Δ siz1Δ strains) covering by a wild-type URA3 plasmid (e.g. a URA3 RPC160 plasmid). Transformants were then patched on a SC-Leu plate, replica plated to 5FOA plates, and incubated at 30°C or 37°C. Growth was scored after 2 days of incubation. The human gene names were shown in parenthesis underneath the yeast gene names.