Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.
MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.
Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland; University of Basel, 4003 Basel, Switzerland.
Hansruedi
H
Mathys
Max Planck Institute of Biochemistry, Department of Structural Cell Biology, 82152 Martinsried/Munich, Germany.
Jérôme
J
Basquin
Max Planck Institute of Biochemistry, Department of Structural Cell Biology, 82152 Martinsried/Munich, Germany.
Sevim
S
Ozgur
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-109 Warsaw, Poland; Faculty of Biology, University of Warsaw, 02-109 Warsaw, Poland; International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.
Mariusz
M
Czarnocki-Cieciura
Max Planck Institute of Biochemistry, Department of Structural Cell Biology, 82152 Martinsried/Munich, Germany.
Fabien
F
Bonneau
Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland.
Aafke
A
Aartse
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-109 Warsaw, Poland; Faculty of Biology, University of Warsaw, 02-109 Warsaw, Poland.
Andrzej
A
Dziembowski
International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.
Marcin
M
Nowotny
Max Planck Institute of Biochemistry, Department of Structural Cell Biology, 82152 Martinsried/Munich, Germany. Electronic address: conti@biochem.mpg.de.
Elena
E
Conti
Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland; University of Basel, 4003 Basel, Switzerland. Electronic address: witold.filipowicz@fmi.ch.
Witold
W
Filipowicz
D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C528690:CNOT1 protein, human; C468527:CNOT9 protein, human; D035683:MicroRNAs; D046912:Multiprotein Complexes; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases