1. Massively parallel dissection of RNA in RNA-protein interactions in vivo.
- Author
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Lee YH, Hass EP, Campodonico W, Lee YK, Lasda E, Shah JS, Rinn JL, and Hwang T
- Subjects
- Humans, Binding Sites, CCCTC-Binding Factor metabolism, CCCTC-Binding Factor genetics, Immunoprecipitation, Levivirus genetics, Levivirus metabolism, Mutation, Nucleic Acid Conformation, Protein Binding, RNA, Long Noncoding metabolism, RNA, Long Noncoding genetics, RNA, Long Noncoding chemistry, RNA, Viral metabolism, RNA, Viral chemistry, RNA, Viral genetics, Telomerase metabolism, Telomerase genetics, Models, Statistical, RNA metabolism, RNA chemistry, RNA genetics, RNA-Binding Proteins metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins chemistry
- Abstract
Many of the biological functions performed by RNA are mediated by RNA-binding proteins (RBPs), and understanding the molecular basis of these interactions is fundamental to biology. Here, we present massively parallel RNA assay combined with immunoprecipitation (MPRNA-IP) for in vivo high-throughput dissection of RNA-protein interactions and describe statistical models for identifying RNA domains and parsing the structural contributions of RNA. By using custom pools of tens of thousands of RNA sequences containing systematically designed truncations and mutations, MPRNA-IP is able to identify RNA domains, sequences, and secondary structures necessary and sufficient for protein binding in a single experiment. We show that this approach is successful for multiple RNAs of interest, including the long noncoding RNA NORAD, bacteriophage MS2 RNA, and human telomerase RNA, and we use it to interrogate the hitherto unknown sequence or structural RNA-binding preferences of the DNA-looping factor CTCF. By integrating systematic mutation analysis with crosslinking immunoprecipitation, MPRNA-IP provides a novel high-throughput way to elucidate RNA-based mechanisms behind RNA-protein interactions in vivo., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
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