Queiroz, Rayner M.L., Smith, Tom, Villanueva, Eneko et al. (10 more authors) (2019) Comprehensive identification of RNA–protein interactions in any organism using orthogonal organic phase separation (OOPS). Nature Biotechnology. pp. 169-178. ISSN 1087-0156
Abstract
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 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. |
Keywords: | 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 |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) |
Funding Information: | Funder Grant number UNSPECIFIED BB/N01040X/1 |
Depositing User: | Pure (York) |
Date Deposited: | 14 Dec 2018 11:20 |
Last Modified: | 16 Oct 2024 15:19 |
Published Version: | https://doi.org/10.1038/s41587-018-0001-2 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1038/s41587-018-0001-2 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:140009 |
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