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

  • Queiroz, Rayner ML
  • Smith, Tom
  • Villanueva, Eneko
  • Marti-Solano, Maria
  • Monti, Mie
  • Pizzinga, Mariavittoria
  • Mirea, Dan-Mircea
  • Ramakrishna, Manasa
  • Harvey, Robert F
  • Dezi, Veronica
  • Thomas, Gavin H
  • Willis, Anne
  • Lilley, Kathryn
Publication Date
Jan 08, 2019
Apollo - University of Cambridge Repository
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Existing high-throughput methods to identify RNA-binding proteins (RBPs) involving capture of polyadenylated RNAs can not recover proteins that interact with nonadenylated RNAs, including lncRNA, pre-mRNA and bacterial RNAs. We present orthogonal organic phase separation (OOPS) which does not require molecular tagging or capture of polyadenylated RNA. We verify OOPS in HEK293, U2OS and MCF10A human cell lines, finding 96% of proteins recovered are bound to RNA. We demonstrate that all long RNAs can be crosslinked to proteins and recover 1838 RBPs, including 926 putative novel RBPs. Importantly, OOPS is approximately 100-fold more efficient than current techniques, enabling analysis of dynamic RNA-protein interactions. We identified 749 proteins with altered RNA binding following release from nocodazole arrest. Finally, OOPS allowed the characterisation of the first RNA-interactome for a bacterium, Escherichia coli. OOPS is an easy to use and flexible technique, compatible with downstream proteomics and RNA sequencing and applicable to any organism.

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