G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4’s biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins. Implementing this innovative strategy in live cells, we unveiled a cohort of 149 potential G4-interacting proteins, which exhibiting multifaceted functionalities. We then substantiate the directly binding affinity of 7 candidate G4-interacting-proteins (SF3B4, FBL, PP1G, BCL7C, NDUV1, ILF3, GAR1) in vitro. Remarkably, we verified that splicing factor 3B subunit 4 (SF3B4) binds preferentially to the G4-rich 3′ splice site and the corresponding splicing sites are modulated by the G4 stabilizer PDS, indicating the regulating role of G4s in mRNA splicing procedure. The PLGPB strategy could biotinylate multiple proteins simultaneously, which providing an opportunity to map G4-interacting proteins network in living cells.

中文翻译：

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使用人工 G4 靶向生物素连接酶鉴定活细胞中的 G-四链体相互作用蛋白

G-四链体 (G4) 是对细胞过程和疾病途径至关重要的非典型核酸结构。破译 G4 相互作用蛋白对于阐明 G4 的生物学意义至关重要。在这项研究中，我们开发了一种名为 G4PID 的 G4 靶向生物素连接酶，并在体外和体内仔细评估了其结合亲和力和特异性。利用 G4PID，我们设计了一种称为 G 四链体相互作用蛋白特异性生物素连接程序 (PLGPB) 的定制方法，以精确分析 G4 相互作用蛋白。在活细胞中实施这一创新策略，我们揭示了一组 149 种潜在的 G4 相互作用蛋白，它们表现出多方面的功能。然后，我们在体外证实了 7 种候选 G4 相互作用蛋白（SF3B4、FBL、PP1G、BCL7C、NDUV1、ILF3、GAR1）的直接结合亲和力。值得注意的是，我们验证了剪接因子 3B 亚基 4 (SF3B4) 优先结合富含 G4 的 3' 剪接位点，并且相应的剪接位点受到 G4 稳定剂 PDS 的调节，表明 G4 在 mRNA 剪接过程中的调节作用。PLGPB 策略可以同时对多种蛋白质进行生物素化，这为绘制活细胞中 G4 相互作用蛋白质网络提供了机会。