RAPseq enables large-scale identification of RBP-RNA interactions and reveals essentials of post-transcriptional gene regulation.

Over the past decade, thousands of putative human RNA-binding proteins (RBPs) have been identified, increasing the need for methods that define their RNA-binding capacities across diverse biological settings. Existing methods rely either on antibody-based in vivo capture (e.g. CLIP-seq), which depends on cross-linking efficiency and antibody availability, or on synthetic oligonucleotide-based assays (e.g. RNAcompete), which use artificial RNA substrates and cannot assess binding across the native transcriptome. To bridge this gap, we developed RNA affinity purification followed by sequencing (RAPseq), an in vitro method that profiles RBP-binding to native cellular RNA, enabling large-scale transcriptome-wide characterization of RNA-protein interactions without antibodies or synthetic probes. Using RAPseq, we characterized the RNA interactomes of 11 canonical RBPs and 26 non-canonical RBPs, and uncovered novel and specialized moonlighting RNA-binding activities. Applying RAPseq to vertebrate HUR proteins revealed recognition of a conserved RNA-binding motif but showed species-specific binding preferences. Profiling of five pathological IGF2BP family variants exhibited distinct gain- and loss-of-function binding patterns, with implications for cancer biology. Our combinatorial RBP-binding assay (co-RAPseq) uncovered cooperative RNA-binding by HUR and PTBP1, including de novo estimation of the optimal binding distance. Lastly, we introduce a modification-sensitive assay (mod-RAPseq) to distinguish between modification-dependent and -independent RNA-binding sites of YTHDF1 and YBX1. Overall, our simple, scalable, and versatile method enables exploration of complex RNA-protein interactions and the regulatory layers that shape post-transcriptional gene regulation.