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

TY - JOUR

T1 - Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS)

AU - Queiroz, Rayner M.L.

AU - Smith, Tom

AU - Villanueva, Eneko

AU - Marti-Solano, Maria

AU - Monti, Mie

AU - Pizzinga, Mariavittoria

AU - Mirea, Dan-Mircea

AU - Ramakrishna, Manasa

AU - Harvey, Robert F.

AU - Dezi, Veronica

AU - Thomas, Gavin Hugh

AU - Willis, Anne E.

AU - Lilley, Kathryn S.

N1 - © 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.

PY - 2019/2

Y1 - 2019/2

N2 - 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.

AB - 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.

KW - Cell Line, Tumor

KW - Cluster Analysis

KW - Cross-Linking Reagents/chemistry

KW - Escherichia coli

KW - Glycoproteins/chemistry

KW - HEK293 Cells

KW - Humans

KW - Nocodazole/chemistry

KW - Protein Binding

KW - Proteome

KW - Proteomics

KW - RNA, Bacterial/chemistry

KW - RNA, Long Noncoding/chemistry

KW - RNA, Messenger/chemistry

KW - RNA-Binding Proteins/chemistry

KW - RNA/chemistry

KW - Sequence Analysis, RNA

KW - Thymidine/chemistry

KW - Transcriptome

UR - http://www.scopus.com/inward/record.url?scp=85059560229&partnerID=8YFLogxK

U2 - 10.1038/s41587-018-0001-2

DO - 10.1038/s41587-018-0001-2

M3 - Article

C2 - 30607034

SN - 1087-0156

VL - 37

SP - 169

EP - 178

JO - Nature Biotechnology

JF - Nature Biotechnology

IS - 2

ER -