An approach for the systematic profiling of drug-induced remodeling of RNA-RBP (RNA-binding protein) interactions.

Nucleic acid-based therapeutics targeting RNA-related regulatory networks have received substantial attention in drug development, among which the interventions of RNA-RBP (RNA-binding protein) interactions or RNA-protein complex assemblies represent a promising strategy for treating various diseases. RBPs play essential roles in post-transcriptional regulation, and their interactions with RNA are closely associated with physiological homeostasis and pathological progression. Although RNA-RBP interactions are increasingly recognized as therapeutic targets, the lack of systematic methods for screening the drugs that intervene in these interactions and evaluating their off-target effects remains a major bottleneck in the field. Here, we established an integrated approach combining biotinylated furocoumarin probe (BFP)-mediated RNA-tagging and RPC enrichment with quantitative proteomics to systematically profile the drug-induced remodeling of RNA-RBP interactions, thereby facilitating therapeutic drug discovery and off-target effect assessment. Applying this approach to MS-444-treated cells, we confirmed therapeutic target engagement by detecting inhibited HuR-RNA binding (the primary target of MS-444) and simultaneously revealed widespread binding suppression of RNA-processing RBPs, particularly those harboring RRM domains, as potential off-target effects. When applied to risdiplam-treated cells (an FDA-approved splicing modulator for spinal muscular atrophy), we verified the expected dissociation of hnRNP G from RNA transcripts and revealed extensive remodeling of SR and hnRNP splicing factors involved in SMN2 exon 7 regulation, providing insights into both its therapeutic mechanism and potential off-target perturbations. We anticipate that this approach will serve as a powerful platform for screening the drugs targeting RNA-RBP interactions and evaluating their off-target effects, thereby advancing the development of RNA-targeted therapeutics.