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Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

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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, Cluster Analysis, Cross-Linking Reagents, Escherichia coli, Glycoproteins, HEK293 Cells, Humans, Nocodazole, Protein Binding, Proteome, Proteomics, RNA, RNA, Bacterial, RNA, Long Noncoding, RNA, Messenger, RNA-Binding Proteins, Sequence Analysis, RNA, Thymidine, Transcriptome

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Nat Biotechnol

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Springer Science and Business Media LLC
Wellcome Trust (110170/Z/15/Z)
Wellcome Trust (110071/Z/15/Z)
Biotechnology and Biological Sciences Research Council (BB/N010493/1)