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.

RNA 结合蛋白 (RBP) 在真核生物的基因表达调控中起着核心作用。阐明复杂的 RNA-蛋白质相互作用模式和特异性结合位点一直是近年来深入研究的主题。然而，由于缺乏广泛且公正的 RBP 富集方法，大规模 RNA-蛋白结合位点 (RBS) 鉴定仍然是一个紧迫的问题。在这项研究中，通过使用补骨脂素探针 (PP) 进行大规模 RBP 富集，开发了一种新的 RBS 识别策略。通过质谱法鉴定了总共 1957 个具有良好重现性的高置信度 RBP。共有 1783 个蛋白质组 (91.1%) 之前被注释为 RBP。为了识别 RBS，富集的 RBP 用两种不同的方法处理：通过核糖核酸酶 (RNases) 或氢氟酸 (HF) 消化。使用前一种策略共识别出448个高置信度RBS，使用后一种方法识别出1301个高置信度RBS。通过比较两种方法中交联位点的鉴定结果，观察到RNA与RBP的结合不太可能发生在酸性氨基酸上。此外，HF 方法可以比 RNase 方法更公正地识别 RBS。上述结果表明这两种方法都具有用于大规模 RBS 识别的潜力。HF 方法可以比 RNase 方法更公正地识别 RBS。上述结果表明这两种方法都具有用于大规模 RBS 识别的潜力。HF 方法可以比 RNase 方法更公正地识别 RBS。上述结果表明这两种方法都具有用于大规模 RBS 识别的潜力。