Although strong evidence supports the importance of their cooperative interactions, microRNA (miRNA)- binding sites are still largely investigated as functionally independent regulatory units. Here, a survey of alternative 3-UTR isoforms implicates a non-canonical seedless site in cooperative miRNAmediated silencing. While required for target mRNA deadenylation and silencing, this site is not sufficient on its own to physically recruitmiRISC. Instead, it relies on facilitating interactions with a nearby canonical seed-pairing site to recruit the Argonaute complexes. We further show that cooperation between miRNA target sites is necessary for silencing in vivo in the C. elegans embryo, and for the recruitment of the Ccr4-Not effector complex. Using a structural model of cooperating miRISCs, we identified allosteric determinants of cooperative miRNAmediated silencing that are required for both embryonic and larval miRNA functions. Our results delineate multiple cooperative mechanisms in miRNAmediated silencing and further support the consideration of target site cooperation as a fundamental characteristic of miRNA function.
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