GIGYF recruits mRNA decay factors to repress target mRNA expression

Abstract

Gene expression is fundamental for all living organisms and involves different pathways that act to regulate the amount and quality of gene products. Translation initiation is one of the most heavily regulated steps. It is negatively modulated by a protein called 4E-homologous protein (4EHP), which is recruited to different mRNAs through the binding to specific RNA binding proteins (RBPs), forming transcriptspecific repressor complexes. One such complex, contains the Grb10-interacting GYF (GIGYF) protein that in turn associates with the zinc finger RBPs ZNF598 and tristetraprolin (TTP). These RBPs are involved in mouse embryonic development and AU-rich element-containing mRNAs regulation, respectively. Moreover, the 4EHP/GIGYF complex interacts with the CCR4-NOT deadenylase complex altering mRNA abundance. This preliminary information suggests an important role of the 4EHP/GIGYF complex in translational repression and possibly in mRNA degradation, although the interacting partners, the mechanism and the significance remain substantially elusive. In my doctoral studies, I demonstrated that the 4EHPGIGYF interaction is conserved between human and fly and is characterised by a tripartite binding mode. 4EHP repressor function is strongly dependent on the interaction with GIGYF, which post-transcriptionally regulates mRNA abundance by inducing deadenylation-dependent mRNA decay. Subsequently, I identified novel Drosophila melanogaster (Dm) GIGYF-interacting mRNA decay factors such as NOT1, Me31B, and HPat and I mapped their interaction to previously uncharacterized motifs/domains. By generating several GIGYF mutants that disrupt single or multiple protein interactions, I analysed the relevance of these interactions in GIGYF-mediated mRNA repression. I observed that both Me31B and HPat contribute to the repressor function of GIGYF. Moreover, since 4EHP requires GIGYF to repress translation of target mRNA, I tested the relevance of the interactions of GIGYF with Me31B/HPat in the control of mRNA expression by 4EHP. I observed that 4EHP can only reduce mRNA expression if GIGYF is able to interact with the mRNA decay partners. Taken together, these results indicate that 4EHP and GIGYF do not only elicit translational repression but also induce mRNA degradation via the recruitment of several mRNA decay factors.