Figure 7.—

Ccr4 modulates the half-life of WHI5 mRNAs. GAL-WHI5 constructs were transformed in whi5Δ and whi5Δccr4Δ cells. Subsequently, cultures were grown to mid-log phase in −URA medium containing 1% raffinose and 1% galactose. Cells were rapidly centrifuged and resuspended in −URA medium containing 2% glucose to repress the GAL-WHI5 construct. Subsequently, cells were harvested over a time course. Total RNA was isolated from each time point. Blots with RNA from cells with (A) or without CCR4 (B) were hybridized with a WHI5 probe. ACT1 probes were used as loading controls. WHI5 mRNA expression levels were quantitated, and the relative WHI5/ACT1 ratios were plotted as a function of time to determine the effects of CCR4 on WHI5 mRNA half-life (C). Analyses revealed that the half-life of WHI5 mRNA was ∼1 min in the presence of CCR4 compared to ∼10 min in ccr4Δ cells. To assess how overexpression of CCR4 affects proliferation, wild type, ccr4Δ, and cln3Δ cells were transformed with either GAL-WHI5 or a control vector. Subsequently, cells were serially diluted 10-fold onto either −URA glucose (D) or −URA RG (E) plates and grown at 30° for 4 days. Analyses revealed that overexpression of WHI5 inhibited proliferation in ccr4Δ cells almost as much as in cln3Δ cells as previously reported (Costanzo et al. 2004).