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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.

References

  1. Baltz, A. G. et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol. Cell 46, 674–690 (2012).

    CAS  PubMed  Google Scholar 

  2. Castello, A. et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406 (2012).

    CAS  PubMed  Google Scholar 

  3. Castello, A. et al. System-wide identification of RNA-binding proteins by interactome capture. Nat. Protoc. 8, 491–500 (2013).

    CAS  PubMed  Google Scholar 

  4. Kwon, S. C. et al. The RNA-binding protein repertoire of embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1122–1130 (2013).

    CAS  PubMed  Google Scholar 

  5. Beckmann, B. M. et al. The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs. Nat. Commun. 6, 10127 (2015).

    CAS  PubMed  Google Scholar 

  6. Liao, Y. et al. The cardiomyocyte RNA-binding proteome: links to intermediary metabolism and heart disease. Cell Rep. 16, 1456–1469 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Liepelt, A. et al. Identification of RNA-binding proteins in macrophages by interactome capture. Mol Cell Proteomics 15, 2699–2714 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Matia-Gonzalez, A. M., Laing, E. E. & Gerber, A. P. Conserved mRNA-binding proteomes in eukaryotic organisms. Nat. Struct. Mol. Biol. 22, 1027–1033 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Reichel, M. et al. In planta determination of the mRNA-binding proteome of Arabidopsis etiolated seedlings. Plant Cell 28, 2435–2452 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sysoev, V. O. et al. Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila. Nat. Commun. 7, 12128 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Despic, V. et al. Dynamic RNA-protein interactions underlie the zebrafish maternal-to-zygotic transition. Genome Res. 27, 1184–1194 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Nandan, D. et al. Comprehensive identification of mRNA-binding proteins of Leishmania donovani by interactome capture. PLoS ONE 12, e0170068 (2017).

    PubMed  PubMed Central  Google Scholar 

  13. Bach-Pages, M. et al. Discovering the RNA-binding proteome of plant leaves with an improved rna interactome capture method. Biomolecules 10, 661 (2020).

    CAS  PubMed Central  Google Scholar 

  14. Perez-Perri, J. I. et al. Discovery of RNA-binding proteins and characterization of their dynamic responses by enhanced RNA interactome capture. Nat. Commun. 9, 4408 (2018).

    PubMed  PubMed Central  Google Scholar 

  15. Garcia-Moreno, M. et al. System-wide profiling of RNA-binding proteins uncovers key regulators of virus infection. Mol. Cell 74, 196–211 e111 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Rogell, B. et al. Specific RNP capture with antisense LNA/DNA mixmers. RNA 23, 1290–1302 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Ong, S. E. & Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660 (2006).

    CAS  PubMed  Google Scholar 

  18. Hughes, C. S. et al. Ultrasensitive proteome analysis using paramagnetic bead technology. Mol. Syst. Biol. 10, 757 (2014).

    PubMed  Google Scholar 

  19. Hughes, C. S. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 14, 68–85 (2019).

    CAS  PubMed  Google Scholar 

  20. Lukong, K. E., Chang, K. W., Khandjian, E. W. & Richard, S. RNA-binding proteins in human genetic disease. Trends Genet. 24, 416–425 (2008).

    CAS  PubMed  Google Scholar 

  21. Castello, A., Fischer, B., Hentze, M. W. & Preiss, T. RNA-binding proteins in Mendelian disease. Trends Genet. 29, 318–327 (2013).

    CAS  PubMed  Google Scholar 

  22. Moore, S., Jarvelin, A. I., Davis, I., Bond, G. L. & Castello, A. Expanding horizons: new roles for non-canonical RNA-binding proteins in cancer. Curr. Opin. Genet. Dev. 48, 112–120 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Garcia-Moreno, M., Jarvelin, A. I. & Castello, A. Unconventional RNA-binding proteins step into the virus-host battlefront. Wiley Interdiscip. Rev. RNA 9, e1498 (2018).

    PubMed  PubMed Central  Google Scholar 

  24. Castello, A., Hentze, M. W. & Preiss, T. Metabolic enzymes enjoying new partnerships as RNA-binding proteins. Trends Endocrinol. Metab. 26, 746–757 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Hentze, M. W., Castello, A., Schwarzl, T. & Preiss, T. A brave new world of RNA-binding proteins. Nat. Rev. Mol. Cell Biol. 19, 327–341 (2018).

    CAS  PubMed  Google Scholar 

  26. Horos, R. et al. The small non-coding vault RNA1-1 acts as a riboregulator of autophagy. Cell 176, 1054–1067 e1012 (2019).

    CAS  PubMed  Google Scholar 

  27. Choudhury, N. R. et al. RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination. BMC Biol. 15, 105 (2017).

    PubMed  PubMed Central  Google Scholar 

  28. Jiao, A. L. & Slack, F. J. RNA-mediated gene activation. Epigenetics 9, 27–36 (2014).

    CAS  PubMed  Google Scholar 

  29. Ivanyi-Nagy, R. et al. The RNA interactome of human telomerase RNA reveals a coding-independent role for a histone mRNA in telomere homeostasis. eLife https://doi.org/10.7554/eLife.40037 (2018).

  30. Kumari, P. & Sampath, K. cncRNAs: bi-functional RNAs with protein coding and non-coding functions. Semin. Cell Dev. Biol. 47-48, 40–51 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Castello, A. et al. Comprehensive identification of RNA-binding domains in human cells. Mol. Cell 63, 696–710 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Castello, A. et al. Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap. Nat. Protoc. 12, 2447–2464 (2017).

    CAS  PubMed  Google Scholar 

  33. Backlund, M. et al. Plasticity of nuclear and cytoplasmic stress responses of RNA-binding proteins. Nucleic Acids Res. 48, 4725–4740 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Bunnik, E. M. et al. The mRNA-bound proteome of the human malaria parasite Plasmodium falciparum. Genome Biol. 17, 147 (2016).

    PubMed  PubMed Central  Google Scholar 

  35. Wessels, H. H. et al. The mRNA-bound proteome of the early fly embryo. Genome Res. 26, 1000–1009 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Kilchert, C. et al. System-wide analyses of the fission yeast poly(A)(+) RNA interactome reveal insights into organization and function of RNA-protein complexes. Genome Res. https://doi.org/10.1101/gr.257006.119 (2020).

  37. Bao, X. et al. Capturing the interactome of newly transcribed RNA. Nat. Methods 15, 213–220 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Huang, R., Han, M., Meng, L. & Chen, X. Capture and identification of RNA-binding proteins by using click chemistry-assisted RNA-interactome capture (CARIC) strategy. J Vis. Exp. https://doi.org/10.3791/58580 (2018).

  39. Asencio, C., Chatterjee, A. & Hentze, M. W. Silica-based solid-phase extraction of cross-linked nucleic acid-bound proteins. Life Sci. Alliance 1, e201800088 (2018).

    PubMed  PubMed Central  Google Scholar 

  40. Shchepachev, V. et al. Defining the RNA interactome by total RNA-associated protein purification. Mol. Syst. Biol. 15, e8689 (2019).

    PubMed  PubMed Central  Google Scholar 

  41. Trendel, J. et al. The human RNA-binding proteome and its dynamics during translational arrest. Cell 176, 391–403 e319 (2019).

    CAS  PubMed  Google Scholar 

  42. Queiroz, R. M. L. et al. Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS). Nat. Biotechnol. 37, 169–178 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Urdaneta, E. C. et al. Purification of cross-linked RNA-protein complexes by phenol-toluol extraction. Nat. Commun. 10, 990 (2019).

    PubMed  PubMed Central  Google Scholar 

  44. Burger, K. et al. 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response. RNA Biol. 10, 1623–1630 (2013).

    PubMed  PubMed Central  Google Scholar 

  45. Muckenthaler, M. U., Rivella, S., Hentze, M. W. & Galy, B. A red carpet for iron metabolism. Cell 168, 344–361 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Altelaar, A. F. et al. Benchmarking stable isotope labeling based quantitative proteomics. J. Proteomics 88, 14–26 (2013).

    CAS  PubMed  Google Scholar 

  47. Hogrebe, A. et al. Benchmarking common quantification strategies for large-scale phosphoproteomics. Nat. Commun. 9, 1045 (2018).

    PubMed  PubMed Central  Google Scholar 

  48. Dimayacyac-Esleta, B. R. et al. Rapid high-pH reverse phase StageTip for sensitive small-scale membrane proteomic profiling. Anal. Chem. 87, 12016–12023 (2015).

    CAS  PubMed  Google Scholar 

  49. Greenberg, J. R. Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids Res. 6, 715–732 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. He, C. et al. High-resolution mapping of RNA-Binding regions in the nuclear proteome of embryonic stem cells. Mol. Cell 64, 416–430 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    CAS  PubMed  Google Scholar 

  52. Niu, L. et al. Modified TCA/acetone precipitation of plant proteins for proteomic analysis. PLoS ONE 13, e0202238 (2018).

    PubMed  PubMed Central  Google Scholar 

  53. Sielaff, M. et al. Evaluation of FASP, SP3, and iST protocols for proteomic sample preparation in the low microgram range. J. Proteome Res. 16, 4060–4072 (2017).

    CAS  PubMed  Google Scholar 

  54. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  PubMed  Google Scholar 

  55. Villen, J. & Gygi, S. P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat. Protoc. 3, 1630–1638 (2008).

    PubMed  PubMed Central  Google Scholar 

  56. Krijgsveld, J., Gauci, S., Dormeyer, W. & Heck, A. J. In-gel isoelectric focusing of peptides as a tool for improved protein identification. J. Proteome Res. 5, 1721–1730 (2006).

    CAS  PubMed  Google Scholar 

  57. Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).

    CAS  PubMed  Google Scholar 

  58. Gierlinski, M., Gastaldello, F., Cole, C. & Barton, G. J. Proteus: an R package for downstream analysis of MaxQuant output. Preprint at bioRxiv https://doi.org/10.1101/416511 (2018).

  59. Lazar, C., Gatto, L., Ferro, M., Bruley, C. & Burger, T. Accounting for the multiple natures of missing values in label-free quantitative proteomics data sets to compare imputation strategies. J. Proteome Res. 15, 1116–1125 (2016).

    CAS  PubMed  Google Scholar 

  60. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    CAS  PubMed  Google Scholar 

  61. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    PubMed  PubMed Central  Google Scholar 

  62. Harsha, H. C., Molina, H. & Pandey, A. Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat. Protoc. 3, 505–516 (2008).

    CAS  PubMed  Google Scholar 

  63. Anantharaman, V. & Aravind, L. The PRC-barrel: a widespread, conserved domain shared by photosynthetic reaction center subunits and proteins of RNA metabolism. Genome Biol. 3, RESEARCH0061 (2002).

    PubMed  PubMed Central  Google Scholar 

  64. Dennis, G. Jr. et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol. 4, P3 (2003).

    PubMed  Google Scholar 

  65. Strein, C., Alleaume, A. M., Rothbauer, U., Hentze, M. W. & Castello, A. A versatile assay for RNA-binding proteins in living cells. RNA 20, 721–731 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Konig, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

    PubMed  PubMed Central  Google Scholar 

  67. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat. Methods 13, 508–514 (2016).

    PubMed  PubMed Central  Google Scholar 

  69. Van Nostrand, E. L. et al. A large-scale binding and functional map of human RNA-binding proteins. Nature 583, 711–719 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Jarvelin, A. I., Noerenberg, M., Davis, I. & Castello, A. The new (dis)order in RNA regulation. Cell Commun. Signal 14, 9 (2016).

    PubMed  PubMed Central  Google Scholar 

  71. Arif, A. et al. The GAIT translational control system. Wiley Interdiscip. Rev. RNA https://doi.org/10.1002/wrna.1441 (2018).

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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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