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

Abstract

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.