Figure 5. The Fin_Major mimic y-gle1^{^PFQ} alleles have specific defects in mRNA export.

(A) y-gle1^{^PFQ} mutants exhibit no growth defect. Growth of the indicated strains in 5-fold serial dilution on YPD was monitored at the temperatures shown.(B) y-gle1^{^PFQ}mutants display genetic interactions with mRNA export mutants. Strains bearing the indicated mutation in combination with y-gle1Δ harboring a y-gle1^{^PFQ}-LEU plasmid and a yGLE1/URA3 plasmid were monitored for growth at 23°C. Failure to grow on synthetic complete media containing 5-FOA indicates synthetic lethality. (C) The yGle1 PFQ insertions that mimic Fin_Major perturb mRNA export, and y-gle1ΔCC expression does not rescue export defects. Nuclear accumulation of poly(A)⁺ RNA was detected by in situ oligo-dT hybridization following a shift to 37°C for 2 hours (ipk1Δy-gle1Δ) or 1 hour (y-gle1-4). Calculations were based on >100 cells/condition. See Figure S4D for representative FISH images. (D) y-gle1^{^PFQ} mutants exhibit no defect in translation termination. Ratios of luciferase and β-galactosidase activities were determined and read-through efficiency expressed as the percentage from the reporter with a stop codon inserted in-frame into the linker region between the tandem β-galactosidase and luciferase coding sequences (denoted the TMV reporter) compared to the reporter lacking a stop codon (the TQ control) (Stahl et al., 1995). Standard error of the mean was calculated from 3 independent experiments.(E) y-gle1ΔCC rescues the temperature sensitivity of y-gle1-2 nip1-1. Growth of serially diluted strains on -LEU media was monitored at the indicated temperatures.(F) Oligomerization of Gle1 is required for function in vivo. Mutant y-gle1-4 strains harboring plasmids expressing yGLE1, vector only, y-gle1ΔCC, y-gle1ΔCC+hGLE1-CC, y-gle1ΔCC+GCN4, or y-gle1ΔCC+Fin_Major-CC were monitored for growth in 5-fold serial dilution on -LEU media at the temperatures shown. (Left) Schematic representation of the coiled-coil chimeric proteins with yGle1 (red), GCN4 (yellow), hGLE1 (Green), and Fin_Major (Green with white bar indicating PFQ insertion).