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Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture

Nature Protocols volume 16, pages 27–60 (2021)Cite this article

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Abstract

Interactions between RNA-binding proteins (RBPs) and RNAs are critical to cell biology. However, methods to comprehensively and quantitatively assess these interactions within cells were lacking. RNA interactome capture (RIC) uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes. Recent advances have empowered RIC to quantify RBP responses to biological cues such as metabolic imbalance or virus infection. Enhanced RIC exploits the stronger binding of locked nucleic acid (LNA)-containing oligo(dT) probes to poly(A) tails to maximize RNA capture selectivity and efficiency, profoundly improving signal-to-noise ratios. The subsequent analytical use of SILAC and TMT proteomic approaches, together with high-sensitivity sample preparation and tailored statistical data analysis, substantially improves RIC’s quantitative accuracy and reproducibility. This optimized approach is an extension of the original RIC protocol. It takes 3 d plus 2 weeks for proteomics and data analysis and will enable the study of RBP dynamics under different physiological and pathological conditions.

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Fig. 1: Schematic representation of main steps of eRIC.
Fig. 2: Overview of the proteomic workflow.
Fig. 3: Assessment of relative RNA binding by total proteomics and RIC.
Fig. 4: eRIC quality controls.
Fig. 5: Statistical analysis of proteomic data.

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Data availability

Raw and processed proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009789. Original data were generated in ref. 15 and re-analyzed to generate Figs. 3 and 5, which illustrate typical results from the analytical workflow.

Code availability

An R markdown file, including the code to perform analysis for a SILAC-labeled experiment, is provided as Supplementary Software (a .rmd file) and as the Supplementary Note, including exemplary output in .pdf format.

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Acknowledgements

We thank M. Rettel and N. Shuai for fruitful discussions on sample preparation for mass spectrometry and data analysis, respectively. A.C. was funded by MRC Career Development Award MR/L019434/1, MRC grant MR/R021562/1, and John Fell Funds from the University of Oxford. W.K. was funded by a Marie Sklodowska-Curie fellowship (DLV-842067).

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Author notes

  1. These authors contributed equally: Joel I. Perez-Perri, Marko Noerenberg.

Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Joel I. Perez-Perri & Matthias W. Hentze

  2. Department of Biochemistry, University of Oxford, Oxford, UK

    Marko Noerenberg, Wael Kamel, Caroline E. Lenz, Shabaz Mohammed & Alfredo Castello

  3. MRC–University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow, Scotland, UK

    Marko Noerenberg & Alfredo Castello

  4. Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, UK

    Shabaz Mohammed

  5. The Rosalind Franklin Institute, Oxfordshire, UK

    Shabaz Mohammed

  6. Molecular Medicine Partnership Unit (MMPU), Heidelberg, Germany

    Matthias W. Hentze

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Contributions

J.I.P.-P., M.N., S.M., M.W.H., and A.C. conceived and designed the protocol. J.I.P.-P. and M.N. carried out the experimental work. C.E.L., M.N., W.K. and S.M. performed the proteomic analyses. J.I.P.-P., M.N., S.M., M.W.H., and A.C. performed the data analyses. J.I.P.-P., M.N., W.K., M.W.H., and A.C. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Matthias W. Hentze or Alfredo Castello.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Xing Chen, Jingyi Hui and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Perez-Perri, J. I. et al. Nat. Commun. 9, 4408 (2018): https://doi.org/10.1038/s41467-018-06557-8

Garcia-Moreno, M. et al. Mol. Cell 74, 196–211.e11 (2019): https://doi.org/10.1016/j.molcel.2019.01.017

Sysoev, V. O. et al. Nat. Commun. 7, 12128 (2016): https://doi.org/10.1038/ncomms12128

Castello, A. et al. Cell 149, 1393–406 (2012): https://doi.org/10.1016/j.cell.2012.04.031

Protocol to which this paper is an extension

Castello, A. et al. Nat. Protoc. 8, 491–500 (2013): https://doi.org/10.1038/nprot.2013.020

This protocol is an extension to: Nat. Protoc. 8, 491–500 (2013): https://doi.org/10.1038/nprot.2013.020

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Perez-Perri, J.I., Noerenberg, M., Kamel, W. et al. Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture. Nat Protoc 16, 27–60 (2021). https://doi.org/10.1038/s41596-020-00404-1

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