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Nat Biotechnol. 2019 Jan 3. doi: 10.1038/s41587-018-0001-2. [Epub ahead of print]

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

Author information

1
Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK.
2
Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK. ev318@cam.ac.uk.
3
MRC Laboratory of Molecular Biology, Cambridge, UK.
4
MRC Toxicology Unit, University of Cambridge, Leicester, UK.
5
Department of Biology, University of York, York, UK.
6
Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK. k.s.lilley@bioc.cam.ac.uk.

Abstract

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.

PMID:
30607034
DOI:
10.1038/s41587-018-0001-2

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