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

Rayner M.L. Queiroz, Tom Smith, Eneko Villanueva, Maria Marti-Solano, Mie Monti, Mariavittoria Pizzinga, Dan-Mircea Mirea, Manasa Ramakrishna, Robert F. Harvey, Veronica Dezi, Gavin Hugh Thomas, Anne E. Willis, Kathryn S. Lilley

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)169-178
Number of pages10
JournalNature Biotechnology
Issue number2
Early online date3 Jan 2019
Publication statusPublished - Feb 2019

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© 2019 Springer Nature Publishing AG. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details.


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

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