title: Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression.
abstract: In mammalian cells, microRNAs (miRNAs) are incorporated into miRNA-induced silencing complexes (miRISCs), which regulate protein expression post-transcriptionally through binding to 3'-untranslated regions of target mRNAs. Argonaute2 (Ago2), a key component of the miRISC, recruits GW182, a component of the processing body (GW/P-body), to the target mRNAs. To elucidate the function of GW182 in an miRNA-mediated translational repression, we analyzed Argonaute-binding sites in GW182. We found that human GW182 contains three binding sites for Ago2, within the amino-terminal glycine tryptophan (GW/WG)-repeated region that is characteristic of the GW182 family proteins. We also found that the first and second Ago2-binding site is conserved within the amino-terminal half of TNRC6B, which is a paralog of GW182. Each of the Ago-binding sites is alone sufficient to bind Ago2. Furthermore, we demonstrated that multiple Argonaute proteins were connected via the GW182 protein. A GW182 fragment containing the Ago2-binding region partially relieved let-7-mediated repression of protein synthesis in a mammalian cell-free system. Coincidentally, let-7-directed target mRNA deadenylation was delayed. Together, these results strongly suggested that the interactions of GW182 with Argonautes may induce the formation of large complexes containing miRNA target mRNAs, and may be critical for miRNA-mediated translational repression.
authors.affiliation: Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Takimoto
authors.forename: Motoaki
authors.initials: M
authors.lastname: Wakiyama
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins; D001324:Autoantigens / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D017124:Conserved Sequence; D004330:Drosophila / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D001324:Autoantigens; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; C465663:TNRC6A protein, human; D014364:Tryptophan