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

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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.

Source: ACS Central Science

DOI: 10.1021/acscentsci.5c00705