Genome-wide mapping of RNA-protein associations through sequencing

RNA-protein interactions critically regulate gene expression and cellular processes, yet their comprehensive mapping remains challenging due to their structural diversity. We introduce PRIM-seq (protein-RNA interaction mapping by sequencing), a method for concurrent de novo identification of RNA-binding proteins and their associated RNAs. PRIM-seq generates unique chimeric DNA sequences by proximity ligation of RNAs with protein-linked DNA barcodes, which are subsequently decoded through sequencing. We apply PRIM-seq to two human cell lines and construct a human RNA-protein association network (HuRPA), encompassing >350,000 associations involving ~7,000 RNAs and ~11,000 proteins, including 2,610 proteins that each interact with at least 10 distinct RNAs. We experimentally validate the tumorigenesis-associated lincRNA LINC00339, the RNA with the highest number of protein associations in HuRPA, as a protein-associated RNA. We further validate the RNA-associating abilities of chromatin-conformation regulators SMC1A, SMC3 and RAD21, as well as the metabolic enzyme PHGDH. PRIM-seq enables systematic discovery and prioritization of RNA-binding proteins and their targets without gene- or protein-specific reagents.