Ducoli Luca, Zarnegar Brian J, Porter Douglas F, Meyers Robin M, Miao Weili, Riley Nicholas M, Srinivasan Suhas, Jackrazi Leandra V, Yang Yen-Yu, Li Zhouxian, Wang Yinsheng, Bertozzi Carolyn R, Flynn Ryan A, Khavari Paul A
Program in Epithelial Biology, Stanford University, Stanford, CA, USA.
Department of Chemistry and Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
Nature. 2025 May;641(8063):769-778. doi: 10.1038/s41586-025-08787-5. Epub 2025 Mar 26.
RNA-binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport and translation. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease; however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. Here, to address this, non-isotopic ligation-based ultraviolet-light-induced cross-linking and immunoprecipitation was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, revealing patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodelling in response to epidermal growth factor (EGF), revealing that EGF-induced recruitment of UPF1 adjacent to HNRNPC promotes splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships observed in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
RNA结合蛋白（RBPs）控制着多种过程，包括RNA剪接、稳定性、运输和翻译。功能失调的RNA-RBP相互作用会导致人类疾病的发病机制；然而，描述RNA上多蛋白组装体的性质和动态变化一直具有挑战性。在此，为了解决这个问题，基于非同位素连接的紫外线诱导交联和免疫沉淀与质谱分析相结合（irCLIP-RNP），以鉴定与任何感兴趣的RBP共同结合在RNA上的RNA依赖性相关蛋白（RDAPs）。irCLIP-RNP确定了RNA上多聚体蛋白组装体的图谱，揭示了RBP-RNA关联模式，包括RDAPs与主要RBPs之间的细胞类型选择性组合关系。irCLIP-RNP还确定了响应表皮生长因子（EGF）的动态RDAP重塑，揭示了EGF诱导的UPF1在HNRNPC附近的募集促进了细胞增殖mRNA的剪接监测。为了鉴定多个研究的RBPs同时共同结合的RNA，还开发了一种顺序免疫沉淀irCLIP（Re-CLIP）方法。Re-CLIP证实了在irCLIP-RNP中观察到的结合关系，并确定了HNRNPC和UPF1 RBP在RND3和DDX3X mRNA上的共同结合。irCLIP-RNP和Re-CLIP提供了一个框架，用于鉴定和表征活细胞中动态的RNA-蛋白质组装体。
