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Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS).
Villanueva, Eneko; Smith, Tom; Queiroz, Rayner M L; Monti, Mie; Pizzinga, Mariavittoria; Elzek, Mohamed; Dezi, Veronica; Harvey, Robert F; Ramakrishna, Manasa; Willis, Anne E; Lilley, Kathryn S.
Afiliación
  • Villanueva E; Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK. ev318@cam.ac.uk.
  • Smith T; Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK.
  • Queiroz RML; Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK.
  • Monti M; MRC Toxicology Unit, University of Cambridge, Leicester, UK.
  • Pizzinga M; MRC Toxicology Unit, University of Cambridge, Leicester, UK.
  • Elzek M; Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK.
  • Dezi V; MRC Toxicology Unit, University of Cambridge, Leicester, UK.
  • Harvey RF; MRC Toxicology Unit, University of Cambridge, Leicester, UK.
  • Ramakrishna M; MRC Toxicology Unit, University of Cambridge, Leicester, UK.
  • Willis AE; MRC Toxicology Unit, University of Cambridge, Leicester, UK.
  • Lilley KS; Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK. k.s.lilley@bioc.cam.ac.uk.
Nat Protoc ; 15(8): 2568-2588, 2020 08.
Article en En | MEDLINE | ID: mdl-32651564
RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Proteínas de Unión al ARN / Proteoma / Transcriptoma / Fraccionamiento Químico Límite: Humans Idioma: En Revista: Nat Protoc Año: 2020 Tipo del documento: Article Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Proteínas de Unión al ARN / Proteoma / Transcriptoma / Fraccionamiento Químico Límite: Humans Idioma: En Revista: Nat Protoc Año: 2020 Tipo del documento: Article Pais de publicación: Reino Unido