Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing

EMBO J. 2010 Dec 15;29(24):4146-60. doi: 10.1038/emboj.2010.274. Epub 2010 Nov 9.

Abstract

miRNA-mediated gene silencing requires the GW182 proteins, which are characterized by an N-terminal domain that interacts with Argonaute proteins (AGOs), and a C-terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)-binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP-interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C-terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N-terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A-C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A-C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species-specific differences in the relative strength of the PABPC1-binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA-mediated silencing in animal cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution / genetics
  • Animals
  • Binding Sites
  • Cell Line
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster
  • Gene Silencing*
  • Genes, Reporter
  • Humans
  • Immunoprecipitation
  • Luciferases / genetics
  • Luciferases / metabolism
  • MicroRNAs / metabolism*
  • Mutagenesis, Site-Directed
  • Poly(A)-Binding Protein I / genetics
  • Poly(A)-Binding Protein I / metabolism*
  • Protein Interaction Mapping
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*

Substances

  • Drosophila Proteins
  • Gw protein, Drosophila
  • MicroRNAs
  • Poly(A)-Binding Protein I
  • RNA-Binding Proteins
  • TNRC6C protein, human
  • Luciferases