Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.
MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.
Animals, Ascites, Autoantigens, Binding Sites, Carcinoma, Krebs 2, Cell-Free System, Eukaryotic Initiation Factor-2, Eukaryotic Initiation Factor-4G, Gene Silencing, Hela Cells, Humans, Kinetics, Mice, MicroRNAs, Poly(A)-Binding Proteins, Protein Biosynthesis, Protein Structure, Tertiary, Proteins, RNA Processing, Post-Transcriptional, RNA Stability, RNA, Messenger, RNA-Induced Silencing Complex, Receptors, CCR4, Transfection