RNA–protein interaction networks govern many biological processes but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a hetero-bifunctional crosslinker to freeze interacting proteins in place on RNA and then maps multiple bound proteins on single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells.
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Raw and processed sequencing data sets analyzed in this report will be made available upon reasonable request and have been deposited in the Gene Expression Omnibus database (GSE152483).
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The work was supported by grants from the National Science Foundation (MCB-1121024) and the National Institutes of Health (R35 GM122532) to K.M.W. C.A.W. is a postdoctoral fellow at the American Cancer Society (ACS 130845-RSG-17-114-01-RMC). J.M.C. was supported by National Institutes of Health grant R01 GM121806. Xist and XIST antisense probes were provided by the M. Guttman laboratory (CalTech), and we thank M. Blanco (CalTech) for his initial support in their application. XIST eCLIP data from published works were provided by the G.W. Yeo laboratory (UCSD), and we thank G.W. Yeo (UCSD), M. Corley (UCSD) and D. Sprague (UNC) for support in formatting these data for integration into this work and for helpful comments on the project.
A.M.M. is an advisor to and K.M.W. is an advisor to and holds equity in Ribometrix, to which mutational profiling technologies have been licensed.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–10 and legends for Supplementary Data 1 and 2 and Supplementary Tables 1–3.
RNaseP-RMRP structural alignment
eCLIP sites used for analysis
MI linking eCLIP
Significantly excluded eCLIP pairs
Reporter and primer sequences
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Weidmann, C.A., Mustoe, A.M., Jariwala, P.B. et al. Analysis of RNA–protein networks with RNP-MaP defines functional hubs on RNA. Nat Biotechnol 39, 347–356 (2021). https://doi.org/10.1038/s41587-020-0709-7