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Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS)

Nature Protocols volume 15, pages 2568–2588 (2020)Cite this article

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Abstract

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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Fig. 1: Schematic representation of OOPS workflow.
Fig. 2: Graphical index of OOPS workflow.
Fig. 3: Representation of different SILAC controls compatible with OOPS workflow.
Fig. 4: Schematic of TMT experimental design.
Fig. 5: OOPS RNA recovery.
Fig. 6: Silver staining illustrating different stages of the OOPS protocol.
Fig. 7: OOPS MS results.

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Data availability

All data presented herein are taken from ref. 5. The specific data sets presented in Fig. 7 are additionally made available alongside the plotting code in Supplementary Data Set 1.

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Acknowledgements

E.V., T.S., R.Q., R.F.H., M.P., M.R., V.D., M.M. and M.E. are supported by the Medical Research Council, grant/award no. 5TR00, and by the Wellcome Trust, grant/award nos. 110170/Z/15/Z and 110071/Z/15/Z.

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Author notes

  1. These authors contributed equally: Eneko Villanueva, Tom Smith, Rayner M. L. Queiroz.

Authors and Affiliations

  1. Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, UK

    Eneko Villanueva, Tom Smith, Rayner M. L. Queiroz, Mohamed Elzek & Kathryn S. Lilley

  2. MRC Toxicology Unit, University of Cambridge, Leicester, UK

    Mie Monti, Mariavittoria Pizzinga, Veronica Dezi, Robert F. Harvey, Manasa Ramakrishna & Anne E. Willis

Authors
  1. Eneko Villanueva
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Contributions

All author contributions are based on CRediT standards. E.V.: conceptualization, methodology, writing—original draft, visualization, project administration and writing—review and editing. T.S.: conceptualization, methodology, writing—original draft, visualization, data curation, formal analysis and writing—review and editing. R.M.L.Q.: conceptualization, methodology, writing—original draft and writing—review and editing. M.M.: conceptualization, methodology, investigation, visualization, writing—original draft and writing—review and editing. M.P.: conceptualization, methodology, investigation, visualization and writing—original draft. M.E.: writing—original draft. V.D.: writing—original draft. R.F.H.: writing—original draft. M.R.: writing—original draft. A.E.W.: writing—original draft, project administration and funding acquisition. K.S.L.: conceptualization, writing—original draft, project administration and funding acquisition.

Corresponding authors

Correspondence to Eneko Villanueva or Kathryn S. Lilley.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Andre Gerber, Markus Hafner and David Tollervey for their contribution to the peer review of this work.

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Related links

Key reference using this protocol:

Queiroz, R. M. L. et al. Nat. Biotechnol. 37, 169–178 (2019): https://doi.org/10.1038/s41587-018-0001-2

Supplementary information

Supplementary Information

Complementary information about how to do the following (includes Supplementary Figs 1–4): Check if the media absorbs light at 254 nm. Check if the media contains RNases. Optimize UV cross-linking in non-human models. Do extra controls for organisms with a cell wall. Solubilize the interface. Apply linear models to identify changes in RNA binding. Set up the Nano-flow LC method for LC–MS/MS. Table 1: Method editor parameters for SPS-MS3 mass spectrometry data acquisition. Table 2: Method editor parameters for CHOPIN MS data acquisition. Supplementary References

Reporting Summary

Supplementary Dataset 1

An R markdown notebook tutorial (‘Identify_changes_in_RNA_binding.Rmd’) demonstrating how to apply linear models to identify changes in RNA binding using the data presented in ref. 5. Also includes R markdown notebooks to generate Fig. 7 plots from ref. 5 data.

Supplementary Video 1

Video showing how to clean and precipitate OOPS interfaces.

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Villanueva, E., Smith, T., Queiroz, R.M.L. et al. Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS). Nat Protoc 15, 2568–2588 (2020). https://doi.org/10.1038/s41596-020-0344-2

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