Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing.
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.
Department of Biochemistry, Max Planck Institute for Developmental Biology, Tübingen, Germany.
Eric
E
Huntzinger
Joerg E
JE
Braun
Susanne
S
Heimstädt
Latifa
L
Zekri
Elisa
E
Izaurralde
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D039122:Poly(A)-Binding Protein I / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D029721:Drosophila Proteins; C513405:Gw protein, Drosophila; D035683:MicroRNAs; D039122:Poly(A)-Binding Protein I; D016601:RNA-Binding Proteins; C540399:TNRC6C protein, human; D008156:Luciferases