Wang Xu, Qin Geng, Yang Jie, Zhao Chuanqi, Ren Jinsong, Qu Xiaogang
Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China.
University of Science and Technology of China, 96 JinZhai Road, Hefei, Anhui 230026, P. R. China.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1259.
G-quadruplexes (G4s), as an important type of non-canonical nucleic acid structure, have received much attention because of their regulations of various biological processes in cells. Identifying G4s-protein interactions is essential for understanding G4s-related biology. However, current strategies for exploring G4 binding proteins (G4BPs) include pull-down assays in cell lysates or photoaffinity labeling, which are lack of sufficient spatial specificity at the subcellular level. Herein, we develop a subcellular selective APEX2-based proximity labeling strategy to investigate the interactome of mitochondrial DNA (mtDNA) G4s in living cells. By this method, we have identified several mtDNA G4BPs. Among them, a previously unrecognized mtDNA G4BP, DHX30 has been selected as an example to explore its important biofunctions. DHX30 localizes both in cytoplasm and mitochondria and can resolve mtDNA G4s. Further studies have demonstrated that DHX30 unfolds mtDNA G4 in living cells, which results in a decrease in glycolysis activity of tumor cells. Besides, RHPS4, a known mtDNA G4 stabilizer, will reverse this inhibition effect. Benefiting from the high spatiotemporal resolution and the ability of genetically encoded systems to perform the labeling with exquisite specificity within living cells, our approach can realize the identification of subcellular localized G4BPs. Our work provides a novel strategy to map protein interactions of specific nucleic acid features in subcellular compartments of living cells.
G-四链体（G4s）作为一种重要的非经典核酸结构，因其对细胞内各种生物过程的调控作用而备受关注。识别G4s与蛋白质的相互作用对于理解G4s相关生物学至关重要。然而，目前探索G4结合蛋白（G4BPs）的策略包括细胞裂解物中的下拉分析或光亲和标记，这些方法在亚细胞水平上缺乏足够的空间特异性。在此，我们开发了一种基于亚细胞选择性APEX2的邻近标记策略，以研究活细胞中线粒体DNA（mtDNA）G4s的相互作用组。通过这种方法，我们鉴定了几种mtDNA G4BPs。其中，一个先前未被识别的mtDNA G4BP，DHX30被选为例子来探索其重要的生物功能。DHX30定位于细胞质和线粒体中，并且可以解开mtDNA G4s。进一步的研究表明，DHX30在活细胞中解开mtDNA G4，这导致肿瘤细胞糖酵解活性降低。此外，已知的mtDNA G4稳定剂RHPS4将逆转这种抑制作用。受益于高时空分辨率以及基因编码系统在活细胞内以精确特异性进行标记的能力，我们的方法可以实现亚细胞定位的G4BPs的鉴定。我们的工作为绘制活细胞亚细胞区室中特定核酸特征的蛋白质相互作用提供了一种新策略。
