Mutational analysis of CAF1 proteins reveals requirement for NOT1 interaction. (A) Residues mutated are depicted on the human CAF1a structure (PDB 2D5R) and colored by the interaction disrupted. (B) Outline of the tethered function assay. (FF) firefly luciferase, (Ren) Renilla luciferase. (C) Results of tethered function assay, normalized to “no MS2 protein” control (none). Three independent experiments were performed, with four oocyte replicates each time. Error bars represent 1 SD. Student's two-tailed t-test was used to determine significant changes between CAF1 WT and mutants, and only NOT1-interaction-defective mutants were significantly (P < 0.005) different. (D) Outline of coimmunoprecipitation assay from Xenopus oocytes. (E) NOT1 interacts with wild-type CAF1 but not with the M141R mutant. A CAF1-interaction-defective mutant of NOT1 served as a negative control. Western blots depict a single representative experiment, and three biological replicates were conducted.
