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

Abstract

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.

mi RISC and the CCR 4– NOT complex silence mRNA targets independently of 43S ribosomal scanning

“…We and others have shown that DDX6 acts downstream of the CCR4–NOT complex to mediate translational repression and stimulate decapping in human cells (Chen et al , 2014; Mathys et al , 2014; Rouya et al , 2014). To further confirm that DDX6 function lies downstream of CCR4–NOT, we used tethering assays to analyze the repressive activity of a NOT1 mutant that it is impaired in binding to DDX6 but binds to CNOT2 and CNOT3 (Fig 6G, NOT1 Mut; Appendix Table S1).…”

“…One possible model is that the recruitment of the CCR4–NOT complex is sufficient to mediate silencing (Jonas & Izaurralde, 2015). This model is based on the following observations: First, the interaction of GW182 proteins with the CCR4–NOT complex is not only required for degradation but also required for translational repression of miRNA reporters (Braun et al , 2011; Chekulaeva et al , 2011; Fabian et al , 2011; Huntzinger et al , 2013; Zekri et al , 2013; Chen et al , 2014; Mathys et al , 2014). Second, like miRISC, the CCR4–NOT complex represses translation in the absence of deadenylation (Cooke et al , 2010; Braun et al , 2011; Chekulaeva et al , 2011; Bawankar et al , 2013; Zekri et al , 2013).…”

“…Translational repression by the CCR4–NOT complex can, at least in part, be explained by a direct interaction between the NOT1 subunit and the DEAD‐box ATPase DDX6 (also known as RCK). Human DDX6 and its Drosophila melanogaster ( Dm ) ortholog Me31B repress translation, activate decapping, and play a role in silencing (Chu & Rana, 2006; Eulalio et al , 2007; Nishihara et al , 2013; Chen et al , 2014; Mathys et al , 2014; Rouya et al , 2014). …”

From guide to target: molecular insights into eukaryotic RNA-interference machinery

“…2b). In addition, CCR4-NOT interacts via its CNOT1 subunit with the DEAD-box helicase DDX6 which functions as a translational repressor and activator of decapping [102,103] (Fig 2c) Piwis: After primary biogenesis, piRNA loaded RNA-induced silencing complexes (piRISCs) are loaded with 26-31 nt guides that are not only typically longer than si-or miRNAs, but are also 2′-O-methylated at their 3′ end. Our current understanding of the molecular mechanisms of Piwi proteins is primarily based on numerous genetic screens, biochemical assays, and comparisons to their Ago counterparts.…”

“…2b). In addition, CCR4-NOT interacts via its CNOT1 subunit with the DEAD-box helicase DDX6 which functions as a translational repressor and activator of decapping [102,103] (Fig 2c) (Box 2). Finally, post-translational modification of Argonautes by numerous factors can also affect silencing activity by influencing small RNA binding or protein stability (reviewed in [104]), thus adding another layer of regulation, particularly to miRNA silencing.…”