The DDX6-4E-T interaction mediates translational repression and P-body assembly.
Lee, Benjamin P
Harries, Lorna W
Nucleic Acids Res
MetadataShow full item record
Kamenska, A., Simpson, C., Vindry, C., Broomhead, H., Bénard, M., Ernoult-Lange, M., Lee, B. P., et al. (2016). The DDX6-4E-T interaction mediates translational repression and P-body assembly.. Nucleic Acids Res, 44 (13), 6318-6334. https://doi.org/10.1093/nar/gkw565
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.
Amino Acid Sequence, Binding Sites, DEAD-box RNA Helicases, DNA-Binding Proteins, Eukaryotic Initiation Factor-4E, Eukaryotic Initiation Factor-4G, Gene Expression Regulation, HEK293 Cells, HeLa Cells, Humans, Nucleocytoplasmic Transport Proteins, Protein Binding, Protein Biosynthesis, Protein Interaction Maps, Proto-Oncogene Proteins, RNA, Small Interfering, RNA-Binding Proteins, Transcription Factors
BBSRC [BB/J00779X/1 to N.S.]; CNRS PICS (to D.W.); Agence Nationale pour la Recherche [ANR-14-CE09-0013-01ANR to D.W.]; Gates Cambridge Foundation (to A.K.); Fondation Wiener – Anspach of the Université Libre de Bruxelles and the Cambridge Newton Trust (C.V.). Funding for open access charge: BBSRC.
External DOI: https://doi.org/10.1093/nar/gkw565
This record's URL: https://www.repository.cam.ac.uk/handle/1810/256711
Attribution 4.0 International
Licence URL: http://creativecommons.org/licenses/by/4.0/
Recommended or similar items
The following licence files are associated with this item: