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dc.contributor.authorQueiroz, Rayner ML
dc.contributor.authorSmith, Tom
dc.contributor.authorVillanueva, Eneko
dc.contributor.authorMarti-Solano, Maria
dc.contributor.authorMonti, Mie
dc.contributor.authorPizzinga, Mariavittoria
dc.contributor.authorMirea, Dan-Mircea
dc.contributor.authorRamakrishna, Manasa
dc.contributor.authorHarvey, Robert F
dc.contributor.authorDezi, Veronica
dc.contributor.authorThomas, Gavin H
dc.contributor.authorWillis, Anne
dc.contributor.authorLilley, Kathryn
dc.date.accessioned2019-01-08T00:30:49Z
dc.date.available2019-01-08T00:30:49Z
dc.date.issued2019-02
dc.identifier.issn1087-0156
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/287600
dc.description.abstractExisting 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.
dc.format.mediumPrint-Electronic
dc.languageeng
dc.publisherSpringer Science and Business Media LLC
dc.subjectCell Line, Tumor
dc.subjectHumans
dc.subjectEscherichia coli
dc.subjectNocodazole
dc.subjectGlycoproteins
dc.subjectRNA-Binding Proteins
dc.subjectProteome
dc.subjectRNA
dc.subjectRNA, Bacterial
dc.subjectRNA, Messenger
dc.subjectThymidine
dc.subjectCross-Linking Reagents
dc.subjectCluster Analysis
dc.subjectSequence Analysis, RNA
dc.subjectProteomics
dc.subjectProtein Binding
dc.subjectHEK293 Cells
dc.subjectTranscriptome
dc.subjectRNA, Long Noncoding
dc.titleComprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).
dc.typeArticle
prism.endingPage178
prism.issueIdentifier2
prism.publicationDate2019
prism.publicationNameNat Biotechnol
prism.startingPage169
prism.volume37
dc.identifier.doi10.17863/CAM.32300
dcterms.dateAccepted2018-11-19
rioxxterms.versionofrecord10.1038/s41587-018-0001-2
rioxxterms.versionAM
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserved
rioxxterms.licenseref.startdate2019-02
dc.contributor.orcidSmith, Tom [0000-0002-0697-8777]
dc.contributor.orcidVillanueva, Eneko [0000-0002-3585-8846]
dc.contributor.orcidMarti-Solano, Maria [0000-0003-0373-8927]
dc.contributor.orcidMonti, Mie [0000-0002-0617-9457]
dc.contributor.orcidRamakrishna, Manasa [0000-0002-5736-8311]
dc.contributor.orcidHarvey, Robert F [0000-0002-0023-1146]
dc.contributor.orcidWillis, Anne [0000-0002-1470-8531]
dc.contributor.orcidLilley, Kathryn [0000-0003-0594-6543]
dc.identifier.eissn1546-1696
rioxxterms.typeJournal Article/Review
pubs.funder-project-idWellcome Trust (110170/Z/15/Z)
pubs.funder-project-idWellcome Trust (110071/Z/15/Z)
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (BB/N010493/1)
cam.issuedOnline2019-01-03
rioxxterms.freetoread.startdate2019-07-03


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