Ccr4-Not Regulates RNA Polymerase I Transcription and Couples Nutrient Signaling to the Control of Ribosomal RNA Biogenesis (original) (raw)
Fig 4
Loss of Ccr4-Not function disrupts regulation of Rrn3-Pol I complex formation.
(A) ChIP for Rrn3 at the rDNA promoter. Three independent experiments were performed and the average and SD are reported. (B) Rrn3-Pol complex formation and basal Rrn3 protein levels are both increased from ccr4Δ cells cultured in nutrient rich media. α-HA immunoprecipitations were performed from no tag control, wild-type and ccr4Δ extracts. Samples were resolved by 8% SDS-PAGE and Rrn3 co-association determined by α-Myc immunoblot. Inputs represent 30 μg of cell extract. (C) Expression of the indicated genes was evaluated using the indicated gene-specific primers and then normalized to the expression of the SPT15 housekeeping gene. Wild-type was set to a value of 1 for each gene and the expression in ccr4Δ was then expressed relative to wild-type. Data are the average and SD of four independent experiments. (D) Spotting assay for WT cells expressing control vector or ccr4Δ cells reconstituted either with control, CCR4, or ccr4-1 expression vectors. The indicated strains were grown in SC-ura media overnight before equal numbers of cells were serially diluted 5-fold and spotted to the indicated plates. Plates were incubated for four days at 30°C. (E) Culturing WT and ccr4Δ cells in nutrient defined media (SC-ura) prevents increased Rrn3-Pol I complex formation. Cell extracts from strains cultured in nutrient defined (SC-ura) media were used in α-HA immunoprecipitations to measure Rrn3-Pol I complex formation. α-IgG is the negative control co-IP. Inputs represent 30 μg of cell extracts while the asterisk represents a residual signal that was not completely removed after stripping the previous immunoblot. Arrow indicates the Ccr4-FLAG specific bands. (F) WT and ccr4Δ cells without plasmids were cultured in nutrient defined (SC) media before harvesting total RNA and quantifying ETS1 and ITS1 expression. Average and SD of four independent experiments are presented. (G) A not4Δ results in accumulation of polyubiquitylated Rrn3. The Rrn3 pull-down was performed as described in the Methods. Duplicate sets of samples were resolved by 8% SDS-PAGE and one set was probed with α-ubiquitin and the other with 0α-Myc. Student’s t-test determined statistical significance. **-p< 0.01.