title: The DDX6-4E-T interaction mediates translational repression and P-body assembly.
abstract: 4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB21QW, UK.
authors.forename: Anastasiia
authors.initials: A
authors.lastname: Kamenska
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB21QW, UK.
authors.forename: Clare
authors.initials: C
authors.lastname: Simpson
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB21QW, UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Vindry
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB21QW, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Broomhead
authors.affiliation: Sorbonne Universités, UPMC, CNRS, IBPS, Developmental Biology Laboratory, 75005 Paris, France.
authors.forename: Marianne
authors.initials: M
authors.lastname: Bénard
authors.affiliation: Sorbonne Universités, UPMC, CNRS, IBPS, Developmental Biology Laboratory, 75005 Paris, France.
authors.forename: Michèle
authors.initials: M
authors.lastname: Ernoult-Lange
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Barrack Road, Exeter EX2 5DW.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Lee
authors.affiliation: Institute of Biomedical and Clinical Sciences, University of Exeter Medical School, Barrack Road, Exeter EX2 5DW.
authors.forename: Lorna W
authors.initials: LW
authors.lastname: Harries
authors.affiliation: Sorbonne Universités, UPMC, CNRS, IBPS, Developmental Biology Laboratory, 75005 Paris, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Weil
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB21QW, UK nms23@cam.ac.uk.
authors.forename: Nancy
authors.initials: N
authors.lastname: Standart
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001665:Binding Sites; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D060066:Protein Interaction Maps / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C528690:CNOT1 protein, human; C079950:CSDE1 protein, human; D004268:DNA-Binding Proteins; C411571:EIF4ENIF1 protein, human; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D029543:Nucleocytoplasmic Transport Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C094683:DDX6 protein, human; D053487:DEAD-box RNA Helicases