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Analysis of RNA–protein networks with RNP-MaP defines functional hubs on RNA

Abstract

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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Fig. 1: RNP-MaP strategy for probing RNA–protein interaction networks in cells.
Fig. 2: RNP-MaP defines protein interaction networks in the U1 snRNP.
Fig. 3: RNP-MaP reveals conserved protein interaction networks in RNase P and RMRP RNAs.
Fig. 4: RNP-MaP identifies conserved protein interaction networks in the XIST lncRNA.
Fig. 5: Communities of XIST-binding proteins.
Fig. 6: PTBP1 and MATR3 interactions with E region and functional control of XIST particle formation.

Data availability

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).

Code availability

ShapeMapper2, ΔSHAPE, SuperFold and RingMapper software used for analysis are available at http://weeks.chem.unc.edu/software.html and https://github.com/Weeks-UNC. MEME, VARNA, PyMol and Gephi are all third-party, open-source software.

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Acknowledgements

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.

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C.A.W. and P.B.J. conducted experiments. C.A.W., A.M.M. and K.M.W. analyzed data. C.A.W., J.M.C. and K.M.W. designed and interpreted experiments. The manuscript was written by C.A.W. and K.M.W. with input from all authors.

Corresponding author

Correspondence to Kevin M. Weeks.

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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.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and legends for Supplementary Data 1 and 2 and Supplementary Tables 1–3.

Reporting Summary

Supplementary Data 1

RNaseP-RMRP structural alignment

Supplementary Data 2

eCLIP sites used for analysis

Supplementary Table 1

MI linking eCLIP

Supplementary Table 2

Significantly excluded eCLIP pairs

Supplementary Table 3

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

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