Investigating How eIF4E Interacting Factors Modulate Protein Synthesis in Yeast

Abstract

Protein synthesis is a core biological process with stringent regulatory mechanisms at the initiation phase in eukaryotic cells. Translation initiation is promoted through an interaction between the translation initiation factors eIF4E and eIF4G at the 5' cap of a selected mRNA, thus mediating assembly of a complex of initiation factors and ribosomes to commence translation in a cap-dependent pathway. In eukaryotes, this process is regulated via a family of 4E-BPs controlling eIF4E-eIF4G binding to repress translation. In the yeast Saccharomyces cerevisiae these include Caf20 and Eap1, with Caf20 being more abundant in cells. A competition model between Caf20 and eIF4G for eIF4E binding was proposed due to the presence of a conserved eIF4E-binding sequence. In mammals, eIF4E association with 4E-BPs is regulated through mTOR-mediated phosphorylation of the 4E-BPs. Since this regulatory effect has not been identified in yeast, it is unclear how Caf20 regulates eIF4E-eIF4G association. This study aimed to further understanding of the mechanisms of this regulation and the roles of Caf20 and eIF4G in modulating translation initiation. In vitro pull-down investigations of FLAG-tagged Caf20 variants were implemented to demonstrate that eIF4E-Caf20 interaction is tight and entirely dependent on the Caf20 N-terminal canonical eIF4E interacting region. Through in vitro competition studies it was shown that the conserved eIF4E-binding motif is insufficient for the suggested competition mechanism between Caf20 and eIF4G and potentially other elements are involved in regulating eIF4E binding with either factor. Throughout the studies, a cell-free translation system in yeast was developed to assess contributions of Caf20 to translation in vitro. We found that Caf20 has a more intricate role in governing yeast translation by either up- or down-regulating the initiation cascade. We next employed an inhibitor of eIF4E-eIF4G interaction originally used in mammalian cells, 4EGI-1. By use of cap affinity chromatography and cell-free translation system studies we showed isoform-specific functions of eIF4G in yeast. Yeast has two eIF4G isoforms: eIF4G1 and eIF4G2. Intriguingly, 4EGI-1 compound exhibited more specificity towards inhibiting eIF4G2 binding to eIF4E and the eIF4G2-mediated stimulatory effect on translation over eIF4G1. 4EGI-1 also showed an unexpected broad inhibitory impact on yeast translation beyond targeting eIF4E-eIF4G interaction at the 5' cap. These findings suggest that eIF4G isoforms have different affinities for eIF4E near the cap and for poly A tails and introduce 4EGI-1 as a novel compound that can be exploited in the yeast studies directed to examining distinct roles of eIF4G isoforms in translation. In summary, Caf20 and eIF4G apparently contribute to the yeast regulatory mechanism of translation through more sophisticated mechanisms than current models of their actions imply and that require further research to unravel.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Graham Pavitt (Supervisor) & Martin Pool (Supervisor)