MicroRNAs (miRNAs), small RNA molecules derived from gene-encoded hairpins, play roles in various biological phenomena ranging from development to environmental responses. From within the miRNA-Induced Silencing Complex (miRISC), miRNAs base-pair with 3’ un-translated regions (3’UTRs) of mRNAs, and direct a series of gene silencing mechanisms. At the core of the miRISC are specialized Argonautes; ALG-1 and ALG-2 bind to miRNAs in C. elegans. While several lines of evidence point to the importance of cooperating interactions between multiple miRNA-binding sites on individual target mRNAs, they are still largely investigated as functionally independent regulatory units. We have previously shown that mRNA deadenylation, a prominent mechanism of miRNA-mediated silencing, strictly requires target site cooperation.
Investigation of the mechanistic basis for cooperative silencing by miRNAs in C. elegans unveiled its multi-faceted nature. Firstly, miRNA-binding sites cooperate in miRISC recruitment; binding to a canonical site enables the subsequent recruitment of miRISC to weaker, and even non-canonical sites on mRNAs, a process we coined trans-seeding. Secondly, we find that interactions between miRISC units are required to trigger miRNA-mediated silencing. At least part of such interactions may occur through homo- or hetero-dimerization of the ALG-1/2 Argonautes. Structural and thermodynamic modeling of ALG-1 proteins associated to cooperating miRNA-binding sites identified residues that may be required for cooperation. We are systematically validating these candidates in vivo and using embryonic cell-free assays by exploiting CRISPR/Cas-9 mediated genome-editing strategies. Furthermore, we found that two interactors of cooperative miRISC, the poly(A) binding proteins PAB-1 and PAB-2, are required for the full functions of miRNAs in vivo. Surprisingly, while they are dispensable for miRNA-mediated deadenylation, they are required for all miRNA-mediated translation repression.
Insight in the mechanistic nature of cooperative miRNA-mediated silencing will enable more reliable prediction of the identity of targets, the extent of their silencing, and their biological impact.
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