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Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap

Abstract

This protocol is an extension to: Nat. Protoc. 8, 491–500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013

RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein–RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein–RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

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Figure 1: Schematic representation of RBDmap.
Figure 2: RBDmap quality controls and data analysis.
Figure 3: From cell culture to lysis.
Figure 4: Analysis of individual RBDmap protein profiles.

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Acknowledgements

Our colleagues Bernd Fischer and Kathrin Eichelbaum unexpectedly passed away during the time in which this manuscript was revised. We dedicate this work to their memory. We thank B. Beckmann and the Hentze group for helpful discussions. A.C. was funded by MRC Career Development Award no. MR/L019434/1. M.W.H. acknowledges support from ERC Advanced Grant ERC-2011-ADG_20110310 and the Virtual Liver Network of the German Ministry for Science and Education. C.K.F. was supported by an EMBO postdoctoral fellowship (LTF1006-2013).

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Authors

Contributions

A.C., B.F. and M.W.H. conceived and designed the project. A.C., R.H. and A.-M.A. carried out the experimental work. C.K.F., S.F. and J.K. performed the proteomic analyses. B.F., A.I.J, T.C., A.C., C.K.F., J.K. and M.W.H. performed the data analyses. A.C. and M.W.H. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Alfredo Castello or Matthias W Hentze.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

RBDmap data analysis pipeline A detailed example analysis workflow for RBDmap data based on R packages described in refs. 9, 29, 30. (PDF 1578 kb)

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Castello, A., Frese, C., Fischer, B. 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). https://doi.org/10.1038/nprot.2017.106

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