Investigation of the mechanism of 4E-BP Caf20 eIF4E-Independent Repression of mRNA Translation

  • Ebelechukwu Nwokoye

Student thesis: Phd


The translation factor, eIF4F is a complex made up of the eIF4E, eIF4G and eIF4A. The eIF4F is a major determinant in mRNA selection for recruitment of ribosomes for protein synthesis initiation. eIF4E recognises and binds the 5’ cap. On actively translating mRNAs, eIF4G associates with eIF4E leading to increased synthesis of the encoded proteins. However, eIF4E-eIF4G interactions are regulated across all eukaryotes by a group of inhibitory binding proteins called the 4E-BPs that can displace eIF4G from eIF4E and repress translation. In yeast, Caf20 and Eap1 are the two 4E-BPs identified that associate with eIF4E and inhibit translation. Prior studies in Graham Pavitt’s lab identified a new role for Caf20, whereby it was shown to interact with ribosomes and specific mRNAs independently of its binding to eIF4E. The research presented in this thesis describes an investigation of the elements of Caf20 important of its association with eIF4E, the ribosome and itself; and mechanisms of Caf20 interaction with other proteins. By a combination of systematic mutagenesis and immunoprecipitation experiments it was shown that Caf20 requires a short motif within its amino terminal region (NTD) to interact with eIF4E. In contrast Caf20 requires multiple elements driven largely by an extended region of the N-terminus to interact with the ribosome. By using a double tagging and immunoprecipitation approach, it was demonstrated that Caf20 interacts with its binding partners as a monomer rather than a homodimer or other higher order complex. In vivo crosslinking of whole cell extracts and polysome enriched fractions combined with western blotting identified some specific proteins including a novel approximately 10 KDa Caf20-interacting protein. Mass-spectrometry provided some candidates of which ribosomal proteins, Rps5, Rps24, Rps27, Rpl10, Rpl27 and Rpl30 of the small and large subunits were identified. The crosslinked ribosomal proteins are located around the interface of the 40S and 60S subunits. Results from different phenotypic characterisations for Caf20 indicated that it affects cell growth when there is a switch from glucose medium to respiratory medium, especially at low temperatures (16°C). Caf20 was found not to be a target of the TOR pathway, but Caf20 presence did increase strain sensitivity to clioquinol drug and excess CuSO4 treatments. CAF20 deletion in three RP-TAP strains was found to have synthetic growth defects when two strains (Rpl27-TAP and Rps27B-TAP) were grown at different temperatures. Mild phenotypes identified in this study all appear to be largely explained by the eIF4E-Caf20 interaction rather than the novel Caf20-ribosome interaction. In summary, this study broadens our understanding of how Caf20 binds to the ribosome.
Date of Award1 Aug 2020
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorGraham Pavitt (Supervisor) & Anil Day (Supervisor)


  • Regulation
  • Translation initiation
  • ribosome
  • Caf20
  • eIF4E
  • 4E-BP
  • mRNA

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