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Abstract

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein (AGO) and a GW182 family protein. In turn, GW182 proteins interact with the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for silencing of miRNA targets. GW182 proteins are characterized by tryptophan (W)-containing motifs, which have been shown to mediate the interactions with the Argonaute pro- teins, and the PAN2-PAN3 and CCR4-NOT deadenylase com- plexes. In molecular terms, it has been speculated that the tryptophan residues are accommodated in hydrophobic pockets of the protein partners and that several such pockets and their spatial arrangement could confer increased affinity and specific- ity. We are combining cellular, biochemical and structural approaches to investigate how the GW182 proteins interact with their partners to mediate silencing. Our studies uncovered the presence of W-binding pockets in PAN3 and the CNOT9 subunit of the CCR4-NOT complex, revealing the structural basis for the recruitment of deadenylase complexes to miRNA targets. We fur- ther demonstrated that a central domain in the CNOT1 subunit of the CCR4-NOT complex interacts with the CAF1 and CCR4 deadenylases and with the RNA helicase DDX6. DDX6 is a translational repressor and decapping activator, which has been implicated in the miRNA pathway. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadeny- lation and decapping.