Elucidating the role of the CCR4-NOT complex in miRNA-mediated gene silencing

Abstract

miRNAs are small non-coding RNAs involved in the regulation of almost all biological pathways in metazoans. miRNAs promote silencing of the mRNA targets by translational repression and mRNA degradation. They associate with Argonaute (AGO) proteins and bind to complementary mRNA targets. In animals, AGO interacts with GW182 family protein, which in turn recruits the main deadenylase complexes, the CCR4-NOT and PAN2-PAN3. These interactions trigger deadenylation which is followed by 5’ decapping and finally by mRNA degradation. GW182 proteins are essential for miRNA-mediated gene silencing. They are conserved in vertebrates and insects, and share a similar domain organization. By contrast, the Caenorhabditis elegans GW182 proteins, AIN-1 and AIN-2, are highly divergent and do not contain any identified domains. In the beginning of my doctoral studies, I investigated how C. elegans proteins mediate silencing and showed that only AIN-1, but not AIN-2, interacts with the deadenylases and poly(A)-binding protein (PABP). My studies demonstrated the evolutionary plasticity of GW182 proteins, which promote silencing via interaction with the deadenylase complexes and PABP across species. I used the information obtained from C. elegans proteins to design a functional GW182 protein, in order to understand the minimal requirements to mediate silencing. This study highlighted the importance of the CCR4-NOT complex and PABP for miRNA mediated translational repression and mRNA decay. In order to understand the molecular mechanism of the CCR4-NOT complex in miRNA mediated gene silencing, I worked in collaborative projects where I first showed that the NOT module containing the NOT1,2,3 proteins, is required for the mRNA degradation mediated by the complex. Next, I demonstrated that GW182 proteins interact directly with CNOT9 (CAF40) subunit of the complex through two tryptophan-binding pockets. The collaborative structural project also revealed an uncharacterized direct interaction between the scaffold subunit of the complex, NOT1 and a DEAD-box helicase, DDX6, which functions as translational repressor and decapping activator. This interaction provides a direct link between deadenylation, decapping and translational repression. Finally, I focused on the role of the CCR4-NOT complex in miRNA-mediated translational repression. Previous studies proposed that miRNAs inhibit translation initiation by blocking ribosomal scanning through either the direct interaction between NOT1 and eIF4A2 or the displacement of eIF4A1 and eIF4A2 from the mRNA targets. By contrast, my studies demonstrated that miRNAs can repress translation independently of deadenylation and scanning. Moreover, I showed that DDX6, which is directly interacting with NOT1, is required for the translational repression activity of the CCR4-NOT complex. Overall, my studies placed the CCR4-NOT complex as a major effector complex in miRNA mediated gene silencing.