irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein assemblies on RNA

• 成果类型: Article
• 署名作者: 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.
• 署名单位: Stanford University; Stanford University; Howard Hughes Medical Institute; Stanford University; University of California System; University of California Riverside; Harvard University; Harvard University Medical Affiliates; Boston Children's Hospital; Harvard University; Harvard University; Stanford University; US Department of Veterans Affairs; Veterans Health Administration (VHA); VA Palo Alto Health Care System
• 刊物名称: Nature
• ISSN/ISSBN: 0028-0888
• DOI: 10.1038/s41586-025-08787-5
• 发表日期: 2025-05-15
• 关键词: epidermal-growth-factor; messenger-rna; gene-expression; binding; hnrnp; quantification; transcription; purification; platform; cells

摘要

RNA-binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport and translation1, 2-3. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease1,4,5; 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 immunoprecipitation6 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.