Compositional CNOT requirements for PUM-mediated repression. Plotted data, listed in Supplemental Table S1, are graphed as mean log2 fold change values ±SEM. (A) Luciferase reporter constructs with Nluc followed by a 3′UTR containing three copies of WT or mutant (mt) Pumilio response element, PRE. Fluc served as control. (B) Architecture and module organization of the human CNOT deadenylase complex. (C) Western blot of PUM1&2 and CNOT1 confirming RNAi depletion in HCT116 cells. GAPDH served as loading control. (NTC) Nontargeting control siRNA. (D) Effect of depletion of PUM1&2 or CNOT1 on repression of the Nluc 3xPRE, relative to the Nluc 3xPRE mt reporter. NTC RNAi serves as a control. (*) P-value <0.05 relative to the PRE mt reporter (above x-axis) or between the indicated RNAi conditions (below the x-axis). n = 9. (E) Western blot of RNAi depletion of CNOT7 and PUM1&2. (F) RT-qPCR confirmed RNAi depletion of CNOT7&8 mRNAs, relative to NTC. (*) P-value of <0.05. n = 9. (G) Effect of RNAi depletion of CNOT7&8 and PUM1&2 on PRE-mediated repression. n = 9. (H) Western blot of RNAi depletion of CNOT6 and PUM1&2. (I) RT-qPCR measurement of RNAi-mediated depletion of CNOT6&6L mRNAs. n = 9. (J) Effect of RNAi-mediated depletion of CNOT6&6L and PUM1&2 on PRE-mediated repression.
