Figure 9.

Overexpression of the CNOT1-M domain inhibits silencing in a dominant negative manner. (A) Domain organization of human CNOT1. CNOT1-N, N-terminal domain; CNOT1-M, middle domain containing the MIF4G domain (31); CNOT1-C, C-terminal domain containing the NOT homology domain. Amino acid positions at domain boundaries are indicated below the protein outlines and correspond to the NCBI protein sequence NP_057368.3. (B) Interaction of GFP-tagged human CNOT1 (wild-type or fragments) and human HA-TNRC6A. Inputs (1%) and immunoprecipitates (5% for CNOT1 or 10% for TNRC6A) were analyzed by western blotting. F-Luc-GFP served as a negative control. (C and D) Human cells were transfected with the indicated Let-7 reporters or the corresponding controls (Mut) as described in Figure 8. The transfection mixtures also contained plasmids encoding GFP-CNOT1-M and GFP-CNOT7 (catalytically inactive mutant, D40A+E42A), as indicated. A plasmid encoding GFP-MBP served as a negative control. For each condition, R-Luc activities were normalized to that of a F-Luc transfection control and set at 100% for the Mut reporters (gray bars, shown only for control cells). Error bars represent standard deviations from three independent experiments.