Proteolytic Control of an Auto-inhibitory Intrinsically Disordered Region Governs Small RNA Selectivity in Argonaute Proteins

SUMMARY Argonaute proteins are central to small RNA-mediated gene regulation, yet the mechanisms controlling their activity remain incompletely understood. We elucidate a novel regulatory mechanism governing small RNA loading into the C. elegans Argonaute proteins WAGO-1 and WAGO-3. We show that N-terminal intrinsically disordered regions (N-IDRs) of these proteins do not affect subcellular localization but play critical roles in small RNA loading. We demonstrate that the N-IDR-processing protease DPF-3 facilitates small RNA loading in a catalysis-independent manner. Catalysis by DPF-3 and a second protease APP-1 is required, however, for activity. Deletion of these N-IDRs results in loading of aberrant small RNA species that trigger erroneous gene silencing. Supported by atomistic molecular dynamics simulations, we propose a model in which N-IDRs can simultaneously act as tuneable gatekeepers that auto-inhibit small RNA loading and as regulators of Argonaute stability, representing a previously unrecognized layer of regulation in Argonaute activity in small RNA pathways.