Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS) (#205)

Eneko Villanueva 1 , Rayner ML Queiroz 1 , Tom Smith 1 , Maria Marti-Solano 2 , Mie Monti 1 , Mariavittoria Pizzinga 3 4 , Dan-Mircea Mirea 1 , Manasa Ramakrishna 4 , Robert F Harvey 3 , Veronica Dezi 4 , Gavin H Thomas 5 , Anne E Willis 4 , Kathryn S Lilley 1
  1. Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK
  2. MRC Laboratory of Molecular Biology, Cambridge, UK
  3. MRC Toxicology Unit, University of Cambridge, Leicester
  4. MRC Toxicology Unit, University of Cambridge, Leicester, UK
  5. Department of Biology, University of York, York, UK

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 poly-adenylated 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.