Mammalian miRNA RISC Recruits CAF1 and PABP to Affect PABP-Dependent Deadenylation

• Published in: Molecular cell Vol. 35; no. 6; pp. 868 - 880
• Main Authors: Fabian, Marc R., Mathonnet, Géraldine, Sundermeier, Thomas, Mathys, Hansruedi, Zipprich, Jakob T., Svitkin, Yuri V., Rivas, Fabiola, Jinek, Martin, Wohlschlegel, James, Doudna, Jennifer A., Chen, Chyi-Ying A., Shyu, Ann-Bin, Yates, John R., Hannon, Gregory J., Filipowicz, Witold, Duchaine, Thomas F., Sonenberg, Nahum
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.09.2009
• Subjects: Animals; Argonaute Proteins; Ascites - genetics; Ascites - metabolism; Autoantigens - metabolism; Binding Sites; Carcinoma, Krebs 2 - genetics; Carcinoma, Krebs 2 - metabolism; Cell-Free System; Eukaryotic Initiation Factor-2 - metabolism; Eukaryotic Initiation Factor-4G - metabolism; Gene Silencing; HeLa Cells; Humans; Kinetics; Mice; MicroRNAs - metabolism; Poly(A)-Binding Proteins - genetics; Poly(A)-Binding Proteins - metabolism; Protein Biosynthesis; Protein Structure, Tertiary; PROTEINS; Proteins - genetics; Proteins - metabolism; Receptors, CCR4 - metabolism; RNA; RNA Processing, Post-Transcriptional; RNA Stability; RNA, Messenger - metabolism; RNA-Induced Silencing Complex - genetics; RNA-Induced Silencing Complex - metabolism; Transfection; RNA; PROTEINS
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2009.08.004

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.