Structural and Biochemical Insights to the Role of the CCR4-NOT Complex and DDX6 ATPase in MicroRNA Repression

MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general. [Display omitted] •Central CNOT1 regions, CN9BD and MIF4G, function as mediators in miRNA repression•CNOT9 acts as adaptor in the Trp motif-dependent interaction of TNRC6 with CCR4-NOT•The CNOT1 MIF4G recruits the DEAD-box ATPase DDX6 and stimulates its activity•X-ray structures of CN9BD-CNOT9 and MIF4G-DDX6 complexes support the interactions The CCR4-NOT complex acts downstream of GW182/TNRC6 in the miRNA silencing pathway. Using functional and structural approaches, Mathys et al. show that the CNOT1 subunit of CCR4-NOT interacts, via CNOT9, with TNRC6. CNOT1 also recruits and activates the DEAD-box ATPase DDX6, a translational inhibitor.