Regulation of gene expression relies on various molecular mechanisms, which affect different stages of mRNA biogenesis. In animals, microRNAs (miRNAs) suppress gene expression by base-pairing with target mRNAs through partial sequence complementarity, which results in mRNA deadenylation and decay and in translation inhibition. These processes are mediated by Argonaute proteins and associated factors such as trinucleotide repeat-containing gene 6B protein (TNRC6B), which together with miRNAs constitute the miRNA-induced silencing complex (miRISC). How the structure and organization of miRISC modulates its function is unknown. Sheu-Gruttadauria and MacRae now show that binding of Ago2 to TNRC6B promotes phase separation and the formation of miRISC-containing liquid droplets both in vitro and in cells, which can increase the functionality of miRISC.

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Structural data of purified human Ago2 and TNRC6B identified three tryptophan (Trp)-binding pockets in the PIWI domain of Ago2, of which two were previously reported, and revealed they are arranged at equal distances from each other on the surface of Ago2. Biochemical binding studies showed that all three pockets can recognize multiple distinct Trp residues in the glycine–tryptophan (GW)-rich N-terminal domain of TNRC6B. This suggested that the interactions between Ago2 and TNRC6B are complex and that the underlying structure of miRISC is heterogeneous at the atomic level.

In vitro, a solution of TNRC6B became turbid with the introduction of purified Ago 2; microscopic, phase-separated liquid droplets were formed in dependence on Ago2 concentration. Moreover, fluorescence recovery after photobleaching experiments using GFP–TNRC6B and mCherry–Ago2 indicated that, also in cells, phase separation drives the formation of viscoelastic TNRC6B–Ago2 droplets.

Importantly, in the separated phase, catalytically active Ago2 bound to miRNAs that specifically sequestered target mRNAs from the surrounding solution, and cleaved them. Furthermore, in the presence of Ago2, TNRC6B was able to recruit and concentrate components of the miRISC complex, including components of the CCR4–NOT complex, from soluble lysates.

To assess whether miRISC-associated gene silencing processes remain active in the identified droplets, the authors examined mRNA deadenylation. Using 5ʹ cap-radiolabeled target mRNA, they found that the rate of poly(A)-dependent 3ʹ end shortening increased tenfold after adding higher amounts of TNRC6B to induce miRISC condensation. The increase in deadenylation was 100-fold when the reaction was performed with the molecular crowding agent polyethylene glycol to enhance miRISC phase separation.

In summary, human miRISC undergoes phase separation through the formation of multivalent interactions between Ago2 and the GW-rich sequence of TNRC6B, which helps sequester target mRNAs and promotes their deadenylation. Thus, the capacity to form multivalent interactions could have a role in the regulation of miRNA-mediated gene silencing.