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Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS)
Eneko Villanueva1, Tom Smith2, Rayner M L Queiroz2
1Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK. ev318@cam.ac.uk.
Nature Protocols
|July 12, 2020
Summary
Orthogonal Organic Phase Separation (OOPS) offers a rapid, unbiased method to study RNA-protein interactions. This technique efficiently purifies cross-linked RNA-protein adducts from various organisms, simplifying research into cell homeostasis and disease.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- RNA-protein interactions are crucial for cellular functions and implicated in diseases.
- Existing methods for studying these interactions are often material-intensive, biased, or time-consuming.
Purpose of the Study:
- To present a detailed protocol for the Orthogonal Organic Phase Separation (OOPS) method.
- To demonstrate the versatility and efficiency of OOPS across different biological systems.
- To enable unbiased purification of RNA-protein complexes and their components.
Main Methods:
- Orthogonal Organic Phase Separation (OOPS) utilizes the interface of TRIzol extraction to enrich RNA-binding proteins (RBPs) and bound RNA.
- Enzymatic digestion (RNases or proteases) of the enriched interface allows for specific purification of RNA-protein adducts, free protein, or free RNA.
- The protocol is applicable to diverse organisms, including human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe, and Escherichia coli.
Main Results:
- OOPS provides a quick, efficient, and reproducible method for purifying cross-linked RNA-protein adducts.
- The method is unbiased, avoiding molecular tagging or selection of polyadenylated RNA.
- OOPS successfully isolates RNA-protein adducts, free protein, and free RNA from a single sample.
Conclusions:
- OOPS is a versatile and robust technique for studying RNA-protein interactions and RBP dynamics.
- The method simplifies the analysis of RNA-protein complexes, overcoming limitations of previous approaches.
- OOPS facilitates research in cell homeostasis, disease mechanisms, and RBP function across various species.
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