The DDX6-4E-T interaction mediates translational repression and P-body assembly

Nucleic Acids Res. 2016 Jul 27;44(13):6318-34. doi: 10.1093/nar/gkw565. Epub 2016 Jun 24.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence / genetics
  • Binding Sites
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Eukaryotic Initiation Factor-4E / genetics
  • Eukaryotic Initiation Factor-4E / metabolism*
  • Eukaryotic Initiation Factor-4G / genetics
  • Eukaryotic Initiation Factor-4G / metabolism
  • Gene Expression Regulation / genetics
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Nucleocytoplasmic Transport Proteins / genetics
  • Nucleocytoplasmic Transport Proteins / metabolism*
  • Protein Binding
  • Protein Biosynthesis*
  • Protein Interaction Maps / genetics
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • RNA, Small Interfering / genetics
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*


  • CNOT1 protein, human
  • CSDE1 protein, human
  • DNA-Binding Proteins
  • EIF4ENIF1 protein, human
  • Eukaryotic Initiation Factor-4E
  • Eukaryotic Initiation Factor-4G
  • Nucleocytoplasmic Transport Proteins
  • Proto-Oncogene Proteins
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Transcription Factors
  • DDX6 protein, human
  • DEAD-box RNA Helicases