Large-scale enrichment of RNA-binding proteins and binding site identification using the psoralen cycloaddition reaction

RNA-binding proteins (RBPs) play central roles in the regulation of gene expression in eukaryotes. The elucidation of complex RNA‒protein interaction patterns and specific binding sites has been the subject of intensive research in recent years. However, large-scale RNA‒protein binding site (RBS) identification remains an urgent issue due to the lack of broad and unbiased RBP enrichment methods. In this study, a novel strategy for RBS identification was developed by using a psoralen probe (PP) for large-scale RBP enrichment. A total of 1957 high-confidence RBPs with good reproducibility were identified via mass spectrometry. A total of 1783 protein groups (91.1%) were previously annotated as RBPs. To identify RBSs, the enriched RBPs were treated with two different methods: digestion via ribonucleases (RNases) or hydrofluoric acid (HF). A total of 448 high-confidence RBSs were identified by using the former strategy, and 1301 high-confidence RBSs were identified by using the latter method. By comparing the identification results of cross-linking sites in both methods, it was observed that the binding of RNA to RBP was less likely to occur on acidic amino acids. Furthermore, the HF method can identify RBS in a more unbiased manner than the RNase method. The above mentioned results suggest the potential of both methods for large-scale RBS identification.