Structural and functional characterization of eIF4E1 and eIF4E2 complexes involved in translational control

Abstract

Protein synthesis is one of the costliest processes in the cell. Therefore, the initiation of translation is a tightly regulated process. One major control mechanism targets the activity or formation of the so-called eIF4F (eukaryotic initiation factor 4F) complex bound to the 5’ cap structure of an mRNA. This heterotrimeric complex, consisting of the RNA helicase eIF4A, the cap-binding protein eIF4E and the scaffold subunit eIF4G, is ultimately required for the recruitment of the 43S PIC (pre-initiation complex) to the mRNA, leading to subsequent scanning and initiation. The formation of the eIF4F complex is under the control of a group of inhibitory proteins known as eIF4E-binding proteins (4E-BPs), which bind to eIF4E and prevent its interaction with eIF4G. 4E-BPs comprise a group of functionally distinct proteins and include global translational repressors such as the three human proteins 4E-BP1-3, or large, multidomain proteins that likely act on an mRNA-specific level. Alternatively, the assembly of the eIF4F complex can be prevented by the eIF4E-homologous protein (4EHP or eIF4E2), which competes with eIF4E in binding to the 5’cap structure of an mRNA. Compared to the global repression by 4E-BPs, the later mechanism only acts on a message specific level. Comprehensive molecular insight into eIF4E- and 4EHP-complexes involved in the regulation of translation initiation was lacking. My doctoral work provides a fundamental structural and mechanistic understanding of the formation of these regulatory complexes. In my initial studies, I characterized the binding of various 4E-BPs to eIF4E and provided the first structural insights into an extended eIF4E-binding mode of different 4E-BPs. The structures revealed a conserved mode of interaction with eIF4E, despite the lack of sequence conservation. Additionally, in a collaborative project, I observed that the eIF4E-binding mode characteristic of 4E-BP complexes is also present in eIF4E-eIF4G complexes, expanding the knowledge on the mechanism of translation initiation and its regulation. Another part of my doctoral studies focused on 4E-BPs very specific functions and architecture. Specifically, I investigated the binding mode of an invertebrate-specific 4E-BP called Mextli. My studies unveiled an unexpected variation and evolutionary plasticity in the eIF4E-binding mode of Mextli homologs across species, which confer distinct functional properties to the respective eIF4E-complexes. I also studied 4EHP, the second member of the eIF4E protein family, and its specific interaction partners, the Grb10-interacting GYF domain-containing (GIGYF) proteins 1 and 2, and obtained the first crystal structures of theses 4EHP-specific binding partners bound to 4EHP. The molecular details of the 4EHP-GIGYF translational repressor complex explain why GIGYF proteins bind to 4EHP and not to eIF4E. Overall, my doctoral studies revealed new insights on eIF4E-related complexes and their diverse roles in posttranscriptional gene regulation.