dirCLIP profiles variant-specific RNA-protein interactions via nanopore long-read sequencing

RNA binding proteins (RBPs) control gene expression through their activities in essentially all processing steps of coding and noncoding RNAs and are dysregulated in disease. With >95% of human multi-exon genes undergoing alternative splicing, the inability to assign RBP sites to distinct RNA variants fundamentally limits our understanding of RNA regulation. We present dirCLIP, a method combining UV crosslinking and immunoprecipitation (CLIP) with amplification-free direct nanopore long-read sequencing preserving full-length transcripts. dirCLIP employs two complementary strategies: direct RNA sequencing detecting amino acid adduct-induced perturbations in current signals and direct cDNA sequencing capturing binding sites as mutations. Benchmarking dirCLIP with SRSF3 demonstrated >75% concordance with short-read-based data, revealed isoform-selective RNA binding and enabled detection of co-occurring binding sites in single RNA molecules. Application to the noncanonical RBP HMGA1 uncovered new AT-hook mediated RNA variant-specific interactions. dirCLIP fundamentally transforms our ability to interrogate RNA regulation of distinct isoforms and transcript variants with direct implications to disease mechanisms and development of RNA therapeutics.