Structural insight into the molecular pathway that links miRNA target recognition to silencing

摘要: 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 PABPC and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the translational repression, deadenylation and decay of miRNA targets. GW182 proteins are characterized by their richness in tryptophan (W) residues, which are usually located in a sequence environment of predicted structural disorder. These W-containing motifs are either flanked by glycine (GW or WG, termed GW motifs) or by serine/threonine (S/TW or WS/T, termed S/TW motifs). The tryptophan residues in these sequence contexts have been shown to mediate the interactions of GW182 proteins with the Argonaute proteins, and the PAN2-PAN3 and CCR4-NOT deadenylase complexes via cumulative avidity effects. 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 specificity. However, the molecular details of these interactions have remained unclear. We have combined cellular, biochemical and structural approaches to investigate how the GW182 proteins specifically 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 …