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Unbiased dynamic characterization of RNA-protein interactions by OOPS

Rayner M. L. Queiroz, Tom Smith, Eneko Villanueva, Mie Monti, Mariavittoria Pizzinga, Maria Marti-Solano, Dan-Mircea Mirea, Manasa Ramakrishna, Robert F. Harvey, Veronica Dezi, Sven Degroeve, Lennart Martens, Gavin H. Thomas, Anne E. Willis, Kathryn S. Lilley
doi: https://doi.org/10.1101/333336
Rayner M. L. Queiroz
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK
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Tom Smith
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK
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Eneko Villanueva
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK
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  • For correspondence: ev318@cam.ac.uk ksl23@cam.ac.uk
Mie Monti
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK
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Mariavittoria Pizzinga
2MRC Toxicology Unit, University of Cambridge, Lancaster Rd, Leicester LE1 9NH, UK
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Maria Marti-Solano
3MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK
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Dan-Mircea Mirea
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK
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Manasa Ramakrishna
2MRC Toxicology Unit, University of Cambridge, Lancaster Rd, Leicester LE1 9NH, UK
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Robert F. Harvey
2MRC Toxicology Unit, University of Cambridge, Lancaster Rd, Leicester LE1 9NH, UK
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Veronica Dezi
2MRC Toxicology Unit, University of Cambridge, Lancaster Rd, Leicester LE1 9NH, UK
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Sven Degroeve
4VIB, Ghent University Ghent, Belgium
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Lennart Martens
4VIB, Ghent University Ghent, Belgium
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Gavin H. Thomas
5Department of Biology, University of York, Wentworth Way, York YO10 5DD, UK
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Anne E. Willis
2MRC Toxicology Unit, University of Cambridge, Lancaster Rd, Leicester LE1 9NH, UK
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Kathryn S. Lilley
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK
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  • For correspondence: ev318@cam.ac.uk ksl23@cam.ac.uk
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Abstract

Current methods for the identification of RNA–protein interactions require a quantity and quality of sample that hinders their application, especially for dynamic biological systems or when sample material is limiting. Here, we present a new approach to enrich RNA-Binding Proteins (RBPs): Orthogonal Organic Phase Separation (OOPS), which is compatible with downstream proteomics and RNA sequencing. OOPS enables recovery of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in an unbiased manner. By applying OOPS to human cell lines, we extract the majority of known RBPs, and importantly identify additional novel RBPs, including those from previously under-represented cellular compartments. The high yield and unbiased nature of OOPS facilitates its application in both dynamic and inaccessible systems. Thus, we have identified changes in RNA-protein interactions in mammalian cells following nocodazole cell-cycle arrest, and defined the first bacterial RNA-interactome. Overall, OOPS provides an easy-to-use and flexible technique that opens new opportunities to characterize RNA-protein interactions and explore their dynamic behaviour.

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Posted May 29, 2018.
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Unbiased dynamic characterization of RNA-protein interactions by OOPS
Rayner M. L. Queiroz, Tom Smith, Eneko Villanueva, Mie Monti, Mariavittoria Pizzinga, Maria Marti-Solano, Dan-Mircea Mirea, Manasa Ramakrishna, Robert F. Harvey, Veronica Dezi, Sven Degroeve, Lennart Martens, Gavin H. Thomas, Anne E. Willis, Kathryn S. Lilley
bioRxiv 333336; doi: https://doi.org/10.1101/333336
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Unbiased dynamic characterization of RNA-protein interactions by OOPS
Rayner M. L. Queiroz, Tom Smith, Eneko Villanueva, Mie Monti, Mariavittoria Pizzinga, Maria Marti-Solano, Dan-Mircea Mirea, Manasa Ramakrishna, Robert F. Harvey, Veronica Dezi, Sven Degroeve, Lennart Martens, Gavin H. Thomas, Anne E. Willis, Kathryn S. Lilley
bioRxiv 333336; doi: https://doi.org/10.1101/333336

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