Probing direct interactions between nuclear proteins in cells with nxReLo.

Abstract

Nearly all biological processes depend on protein-protein interactions (PPIs). While various methods exist to study these interactions, investigating those that involve nuclear proteins, including structurally complex proteins containing long disordered regions, remains a significant challenge. Here, we developed nxReLo, a simple and fast cell culture-based colocalization assay, designed to identify and characterize interactions between nuclear proteins. PIWI-interacting RNAs (piRNAs) safeguard germline genome integrity, and the PPIs that facilitate piRNA production are therefore essential for animal reproduction, yet remain incompletely understood. We used nxReLo to investigate interactions between members of the <i><i>Drosophila melanogaster</i></i> Rhino-Deadlock-Cutoff (RDC) complex and two associated components, Bootlegger and Moonshiner, a nuclear protein network required for piRNA expression. We demonstrate the utility of the nxReLo assay by systematically screening pairwise interactions within the RDC network and by assembling a multiprotein complex from four components. By combining nxReLo assays with AlphaFold structural prediction, we characterized the Cutoff-Deadlock and the Bootlegger-Deadlock complexes in detail, providing molecular and structural insights. Specifically, we refined the domains involved in the interaction and identified interface point mutations that interfered with complex formation, validating the predicted structures. In conclusion, nxReLo facilitates rapid and simple testing of direct interactions between nuclear proteins in a cellular context, which is particularly important when working with structurally challenging proteins or when established interaction assays prove unsuccessful.