RNA accessibility sequencing reveals transcriptome-wide RBP binding and RNA structure

We introduce RAD‑seq (RNA Accessibility via Deamination sequencing), a simple and scalable method that quantifies RNA accessibility by protein binding in vivo. RAD‑seq uses TadA8.20-mediated adenosine deamination to report the local exposure of each adenosine to its molecular environment, integrating the combined effects of base pairing, RNA folding, and RNA‑binding protein (RBP) occupancy in a single experiment. RAD‑seq profiles recapitulate known structural signatures, reveal high‑resolution RBP footprints, report on the influence of RNA modifications, and capture dynamic remodeling of RNA accessibility during oxidative and heat‑shock stress. By integrating RAD-seq with RNA Bind-n-Seq motifs, we detected local enrichment of the RNA N6-methyladenosine (m6A) methyltransferase recruiter TRA2A following knockdown of the exon junction anchor protein EIF4A3, revealing a potential mechanism that links RNA accessibility to enhanced TRA2A recruitment and m6A deposition. Extending this framework, we introduce an unsupervised statistical model that combines RAD-seq with motif priors to infer binding landscapes for multiple RBPs, reproducing the information from numerous CLIP-seq datasets from a single RAD-seq experiment. These results indicate RAD-seq as a generalizable approach for joint profiling of RNA structure and protein occupancy, defining enzymatic RNA accessibility as a new dimension to investigate regulatory elements on RNA.