Journal
NATURE BIOTECHNOLOGY
Volume 37, Issue 2, Pages 169-+Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41587-018-0001-2
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Funding
- Wellcome Trust [110170/Z/15/Z, 110071/Z/15/Z]
- Medical Research Council [5TR00]
- FEBS Long-Term Fellowship
- IB Catalyst grant [BB/N01040X/1]
- BBSRC [BB/N01040X/1, BB/N010493/1] Funding Source: UKRI
- MRC [MC_UU_00025/7, MC_U105185859] Funding Source: UKRI
- Wellcome Trust [110071/Z/15/Z, 110170/Z/15/Z] Funding Source: Wellcome Trust
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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.
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