MicroRNAs (miRNAs) are derived from gene-encoded RNA hairpins and play critical roles in development, homeostasis, disease, and environmental responses. In C. elegans mature miRNAs are loaded in the Argonautes ALG-1 and ALG-2 and direct gene silencing from within the miRNA-Induced Silencing Complex (miRISC). In animals, miRNAs bind to 3’ untranslated regions (UTR) of messenger RNAs (mRNAs) through imperfect base pairing. Because of this imperfect base pairing, the identification of miRNA targets remains a challenge that can only be fully answered through direct functional validation. While several lines of evidence point to the importance of cooperative interactions between miRNA-binding sites on individual target mRNAs, they are still largely investigated as functionally independent regulatory units.
Using a unique C. elegans embryonic cell-free system, we had previously shown that miRNA-instigated deadenylation, an important mechanism of silencing, requires target site cooperation. Now exploiting an array of biochemical, computational, genome editing and genetic approaches, we decipher the mechanistic bases for cooperative miRNA-mediated silencing. Our findings reveal that miRNA-binding site cooperation is required to recruit miRISC to non-canonical sites on mRNAs, and that miRISC association is not sufficient for target deadenylation. We further demonstrate ALG-1/2 homo- or hetero-dimerization in vitro, thus providing an explanation for cooperation in target site recruitment. Using computational modeling of miRNA and 3’UTR-bound Argonaute dimers, we identified two putative miRISC-miRISC interaction interfaces. Turning to CRISPR/Cas-9 to engineer mutant strains, we systematically screened candidate dimerization determinants to identify Argonaute residues that are critical in vivo for miRNA function, for miRISC interactions, and for mRNA target deadenylation.
Our findings unveil molecular determinants for miRNA cooperation, and suggest that miRISC-miRISC interactions play a cornerstone role in the mechanisms of miRNA-mediated silencing.