Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).
Queiroz, Rayner ML
Thomas, Gavin H
Willis, Anne E
Springer Science and Business Media LLC
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Queiroz, R. M., Smith, T., Villanueva, E., Marti-Solano, M., Monti, M., Pizzinga, M., Mirea, D., et al. (2019). Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).. Nat Biotechnol, 37 (2), 169-178. https://doi.org/10.1038/s41587-018-0001-2
Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.
Cell Line, Tumor, Humans, Escherichia coli, Nocodazole, Glycoproteins, RNA-Binding Proteins, Proteome, RNA, RNA, Bacterial, RNA, Messenger, Thymidine, Cross-Linking Reagents, Cluster Analysis, Sequence Analysis, RNA, Proteomics, Protein Binding, HEK293 Cells, Transcriptome, RNA, Long Noncoding
Wellcome Trust (110170/Z/15/Z)
Wellcome Trust (110071/Z/15/Z)
Biotechnology and Biological Sciences Research Council (BB/N010493/1)
External DOI: https://doi.org/10.1038/s41587-018-0001-2
This record's URL: https://www.repository.cam.ac.uk/handle/1810/287600