Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation

Mol Cell. 2009 Sep 24;35(6):868-80. doi: 10.1016/j.molcel.2009.08.004. Epub 2009 Aug 27.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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 / genetics
  • Proteins / metabolism*
  • RNA Processing, Post-Transcriptional*
  • RNA Stability
  • RNA, Messenger / metabolism*
  • RNA-Induced Silencing Complex / genetics
  • RNA-Induced Silencing Complex / metabolism*
  • Receptors, CCR4 / metabolism
  • Ribonucleases
  • Transfection

Substances

  • Ago2 protein, mouse
  • Argonaute Proteins
  • Autoantigens
  • Ccr4 protein, mouse
  • Eukaryotic Initiation Factor-2
  • Eukaryotic Initiation Factor-4G
  • MicroRNAs
  • Poly(A)-Binding Proteins
  • Proteins
  • RNA, Messenger
  • RNA-Induced Silencing Complex
  • Receptors, CCR4
  • mirnlet7 microRNA, mouse
  • Cnot7 protein, mouse
  • Ribonucleases