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
- Exoribonucleases
- 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
- Repressor Proteins
- 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
- Repressor Proteins
- mirnlet7 microRNA, mouse
- Cnot7 protein, mouse
- Exoribonucleases
- Ribonucleases