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Comprehensive quantitation of RNA-protein interaction dynamics by orthogonal organic phase separation (OOPS)

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Author(s)

  • 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

Department/unit(s)

Publication details

JournalNature Biotechnology
DateAccepted/In press - 5 Oct 2018
DatePublished (current) - 3 Jan 2019
Number of pages18
Original languageEnglish

Abstract

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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