A Live-Cell NanoBRET Assay to Monitor RNA-Protein Interactions and Their Inhibition by Small Molecules

RNA-protein interactions are critical for cellular processes, including translation, pre-mRNA splicing, post-transcriptional modifications, and RNA stability. Their dysregulation is implicated in diseases such as myotonic dystrophy type 1 (DM1) and amyotrophic lateral sclerosis (ALS). To investigate RNA-protein interactions, here is described a live-cell NanoBioluminescence Resonance Energy Transfer (NanoBRET) assay to study the interaction between expanded r-(CUG) repeats [r-(CUG)^exp] and muscleblind-like 1 (MBNL1), central to DM1 pathogenesis. This r-(CUG)^exp sequesters MBNL1, a regulator of alternative pre-mRNA splicing, in nuclear foci causing splicing dysregulation. In the NanoBRET assay, r-(CUG)^exp acts as a scaffold to bring into proximity a BRET pair, MBNL1-NanoLuciferase (NanoLuc) and MBNL1-HaloTag, enabling a quantitative readout of RNA-protein interactions. Following assay optimization, an RNA-focused small molecule library was screened, identifying ten compounds with shared chemotypes that disrupt the r-(CUG)^exp-MBNL1 complex. Nuclear magnetic resonance (NMR) studies revealed these inhibitors bind to the 1 × 1 UU internal loops formed when r-(CUG)^exp folds. Five of these molecules rescued two cellular hallmarks of DM1 in patient-derived myotubes, alternative pre-mRNA splicing defects and formation of nuclear r-(CUG)/MBNL1-positive foci. These results demonstrate that the NanoBRET assay is a powerful tool to study RNA-protein interactions in live cells and to identify small molecules that alleviate RNA-mediated cellular pathology.