Fig. 3

CPEB4-repressor complex formation by interaction with the CCR4-NOT complex. A Co-IP of endogenous NOT1, NOT2, NOT7, NOT10, and TOB1 with ectopically expressed SBP-CPEB4 in HeLa cells. IP was carried out using streptavidin beads; precipitated proteins were detected by western blot analysis using specific antibodies as indicated. The asterisk denotes a non-specific band. B Co-IP of endogenous NOT1, NOT7, and TOB1 with ectopically expressed SBP-CPEB4 in HeLa cells following treatment with 20 nM RMD. After siRNA-mediated KD of TOB1 or overexpression of HA-TOB1, the IP was carried out as in A. C The amount of co-precipitated NOT1 was quantified after normalization to the SBP-CPEB4 signal; data are presented as mean ± SD (n = 3). p-values were calculated using a two-sided, one-sample t-test. D Schematic illustration of the tethering assay. HA-CPEB4 fused to the bacteriophage PP7 coat protein (PP7cp) is co-expressed with a β-globin reporter mRNA containing 6 repeats of the PP7binding site (PP7bs). E For the tethering assay, HeLa cells were transiently transfected with HA-PP7cp or HA-PP7cp-CPEB4 together with a β-globin reporter mRNA containing 6 repeats of the PP7bs. Transcription was shut-off with 5 μg/ml actinomycin D (actD), and total RNA was extracted at indicated time points. Decay of the β-globin reporter mRNA was visualized by northern blot analysis; 18S rRNA was visualized by ethidium bromide (EtBr) staining after blotting; nucleolin (NCL) mRNA serves as additional loading control (left side). Deadenylation was visualized by densitometric analysis of the β-globin mRNA signal (right side). RNA digested with RNase H in presence of oligo-dT serves as a reference for fully deadenylated (poly(A)-) RNA. The signal intensity was plotted as a function of the poly(A) tail length and the maximum of the 4-h timepoint is indicated by a red dashed line. F The decay of β-globin mRNA is depicted after normalization to 18S rRNA (mean values ± SD; n = 3). G Half-lives of the β-globin mRNA in the tethering assays were calculated following first-order decay kinetics (mean values ± SD; n = 3). p-values were calculated using a two-sided, paired t-test; * p < 0.05, ** p < 0.01