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

Luca Ducoli, Brian J. Zarnegar, Douglas F. Porter, Robin M. Meyers, Weili Miao, Nicholas M. Riley, Suhas Srinivasan, Leandra V. Jackrazi, Yen-Yu Yang, Zhouxian Li, Yinsheng Wang, Carolyn R. Bertozzi, Ryan A. Flynn and Paul A. Khavari ()
Additional contact information
Luca Ducoli: Stanford University
Brian J. Zarnegar: Stanford University
Douglas F. Porter: Stanford University
Robin M. Meyers: Stanford University
Weili Miao: Stanford University
Nicholas M. Riley: Stanford University
Suhas Srinivasan: Stanford University
Leandra V. Jackrazi: Stanford University
Yen-Yu Yang: University of California
Zhouxian Li: University of California
Yinsheng Wang: University of California
Carolyn R. Bertozzi: Stanford University
Ryan A. Flynn: Boston Children’s Hospital
Paul A. Khavari: Stanford University

Nature, 2025, vol. 641, issue 8063, 769-778

Abstract: Abstract RNA-binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport and translation1–3. 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.

Date: 2025
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DOI: 10.1038/s41586-025-08787-5

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