A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.
CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.
Ying
Y
Chen
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.
Andreas
A
Boland
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.
Duygu
D
Kuzuoğlu-Öztürk
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.
Praveen
P
Bawankar
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.
Belinda
B
Loh
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.
Chung-Te
CT
Chang
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany. Electronic address: oliver.weichenrieder@tuebingen.mpg.de.
Oliver
O
Weichenrieder
Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany. Electronic address: elisa.izaurralde@tuebingen.mpg.de.
Elisa
E
Izaurralde
D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster; D057809:HEK293 Cells; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D035683:MicroRNAs / Q000235:genetics*; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D034622:RNA Interference; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
C528690:CNOT1 protein, human; C468527:CNOT9 protein, human; D035683:MicroRNAs; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases