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Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein

Nature Protocols volume 12, pages 611–637 (2017)Cite this article

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Abstract

The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing. Mapping of the sequenced arms to a reference transcriptome identifies the exact locations of duplexes. hiCLIP data can directly identify all types of RNA duplexes bound by RBPs, including those that are challenging to predict computationally, such as intermolecular and long-range intramolecular duplexes. Moreover, the use of an adaptor that links the two arms of the RNA duplex permits hiCLIP to unambiguously identify the duplexes. Here we describe in detail the procedure for a hiCLIP experiment and the subsequent streamlined data analysis with an R package, 'hiclipr' (https://github.com/luslab/hiclipr/). Preparation of the library for high-throughput DNA sequencing takes ∼7 d and the basic bioinformatic pipeline takes 1 d.

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Figure 1: Overview of high-throughput methods for studying in vivo RNA structures.
Figure 2: Schematic overview of the hiCLIP procedure.
Figure 3: Comparison of data generated using this protocol with the original pipeline.
Figure 4: Example genome browser view.
Figure 5: Quality control of hiCLIP by autoradiography.
Figure 6: Adaptor preparation (adenylation and purification).
Figure 7: PCR cycle number optimization for cDNA library amplification.

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European Nucleotide Archive

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Acknowledgements

We thank F. Agostini (Luscombe laboratory) for helpful advice on the 'hiclipr' package; C. Militti (Ule laboratory) for valuable comments on the manuscript; F. Lee and I. Ruiz de los Mozos (Ule laboratory) for testing the 'hiclipr' package; and all the members of the Ule and Luscombe laboratories for providing fruitful discussions throughout the study. The hiCLIP project was supported by funding from the European Research Council (617837-Translate) to J.U., a Wellcome Trust Joint Investigator Award to J.U. and N.M.L. (103760/Z/14/Z), the Nakajima Foundation Fellowship and an MRC Centenary Early Career Award to Y.S., a Wellcome Trust PhD Training Fellowship for Clinicians to A.M.C., and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002), and the Wellcome Trust (FC001002).

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Authors and Affiliations

  1. Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK

    Yoichiro Sugimoto & Jernej Ule

  2. The Francis Crick Institute, London, UK

    Yoichiro Sugimoto, Anob M Chakrabarti, Nicholas M Luscombe & Jernej Ule

  3. Department of Genetics, Evolution and Environment, UCL Genetics Institute, University College London, London, UK

    Anob M Chakrabarti & Nicholas M Luscombe

  4. Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, Japan

    Nicholas M Luscombe

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  1. Yoichiro Sugimoto
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Contributions

Y.S. and J.U. conceived the hiCLIP protocol; Y.S. and A.M.C. wrote and described the software for data analysis; A.M.C. developed the 'hiclipr' package; Y.S., A.M.C., N.M.L., and J.U. wrote the manuscript; and J.U. and N.M.L. supervised the project.

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Correspondence to Jernej Ule.

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Sugimoto, Y., Chakrabarti, A., Luscombe, N. et al. Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein. Nat Protoc 12, 611–637 (2017). https://doi.org/10.1038/nprot.2016.188

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