The Silencing Domain of GW182 Interacts with PABPC1 To Promote Translational Repression and Degradation of MicroRNA Targets and Is Required for Target Release

Abstract

GW182 family proteins are essential in animal cells for microRNA (miRNA)-mediated gene silencing, yet the molecular mechanism that allows GW182 to promote translational repression and mRNA decay remains largely unknown. Previous studies showed that while the GW182 N-terminal domain interacts with Argonaute proteins, translational repression and degradation of miRNA targets are promoted by a bipartite silencing domain comprising the GW182 middle and C-terminal regions. Here we show that the GW182 C-terminal region is required for GW182 to release silenced mRNPs; moreover, GW182 dissociates from miRNA targets at a step of silencing downstream of deadenylation, indicating that GW182 is required to initiate but not to maintain silencing. In addition, we show that the GW182 bipartite silencing domain competes with eukaryotic initiation factor 4G for binding to PABPC1. The GW182-PABPC1 interaction is also required for miRNA target degradation; accordingly, we observed that PABPC1 associates with components of the CCR4-NOT deadenylase complex. Finally, we show that PABPC1 overexpression suppresses the silencing of miRNA targets. We propose a model in which the GW182 silencing domain promotes translational repression, at least in part, by interfering with mRNA circularization and also recruits the deadenylase complex through the interaction with PABPC1.

Structural features of Argonaute–GW182 protein interactions

“…The silencing domain contains two interaction sites for the cytoplasmic poly(A)-binding proteins (PABPC). One interaction is mediated by a PABPC-interaction motif 2 (PAM2), whereas the second interaction platform is less well defined and located at the Cterminal end of the protein (12,(21)(22)(23). In the current model, the silencing domain interacts with PABPC, leading to inhibition of translation by preventing mRNA circularization mediated by PABPC interaction with the cap-binding complex (12).…”

The p90 ribosomal S6 kinase–UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor–associated factor 3

“…In addition to accelerating mRNA degradation, GW182 is also involved in translational repression through recruiting CCR4-NOT complex and DEAD-box ATPase DDX6 (4,5). GW182 also interact with poly(A)-binding protein (PABP) (6). PABP circularizes mRNA by interacting with both eIF4G (scaffold protein of cap recruit complex, eIF4F) and poly (A) tail, and promotes translational initiation (6,7).…”

“…PABP circularizes mRNA by interacting with both eIF4G (scaffold protein of cap recruit complex, eIF4F) and poly (A) tail, and promotes translational initiation (6,7). The GW182-PABP interaction represses translation by interfering with mRNA circularization (6).…”

To polyadenylate or to deadenylate

“…It has been shown that a conserved motif in GW182 interacts with the C-terminal domain of PABPC1 and that this interaction and the activity of PABPC1 contribute to miRNA-mediated poly(A) removal. [133][134][135] One of the best studied deadenylases involved in miRISCs is the CAF1-CCR4-NOT1 complex, which contains two protein factors with deadenylase activity, CCR4 and CAF1. It has been shown that the CAF1-CCR4-NOT complex associates with PABPC1, and the deadenylase activity of the CAF1-CCR4-NOT complex is necessary for the miRNA-mediated deadenylation.…”

“…[129][130][131] Interestingly, GW182 dissociates from miRISCs at a step of silencing downstream of deadenylation, indicating that GW182 is essential to initiate silencing by activating deadenylation but is not required to maintain silencing. 132,133 The cytoplasmic polyadenylate binding protein 1 (PABPC1) is an additional protein component that is critical for the miRNAmediated deadenylation. It has been shown that a conserved motif in GW182 interacts with the C-terminal domain of PABPC1 and that this interaction and the activity of PABPC1 contribute to miRNA-mediated poly(A) removal.…”