Letter | Published:

hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1

Nature volume 519, pages 491494 (26 March 2015) | Download Citation

Abstract

The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors1. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs). Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3′ untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions. We also discover a duplex spanning 858 nucleotides in the 3′ UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.

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Acknowledgements

We wish to thank S. Granneman and C. Sibley for discussions on the development of hiCLIP protocol; K. Zarnack, N. Haberman, C. Ravarani and B. Lang for assistance with bioinformatic analyses; D. Daujotyte and P. Lukavsky for sharing the STAU1 plasmid and helping in setting up the project; L. Maquat for sharing the ARF1 SBS plasmid; the genomic team at the Cancer Research UK Cambridge Institute for Illumina HiSeq sequencing, and M. Babu Mohan and Ule group members for support and comments on the manuscript. This work was supported by funding from Human Frontier Science Program (RGP0024/2008-C), European Research Council (206726-CLIP and 617837-Translate) and Medical Research Council (U105185858) to J.U., Cancer Research UK and UCL to N.M.L., a Wellcome Trust Joint Investigator Award to N.M.L. and J.U. (103760/Z/14/Z), the Nakajima Foundation fellowship and MRC Centenary Early Career Award to Y.S.

Author information

Author notes

    • Alessandra Vigilante
    •  & Elodie Darbo

    These authors contributed equally to this work.

Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK

    • Yoichiro Sugimoto
    • , Andrea D’Ambrogio
    •  & Jernej Ule
  2. Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK

    • Alexandra Zirra
    • , Cristina Militti
    • , Andrea D’Ambrogio
    •  & Jernej Ule
  3. Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK

    • Alessandra Vigilante
    • , Elodie Darbo
    •  & Nicholas M. Luscombe
  4. UCL Genetics Institute, Department of Genetics, Evolution and Environment, University College London, Gower Street, London WC1E 6BT, UK

    • Alessandra Vigilante
    •  & Nicholas M. Luscombe
  5. Okinawa Institute of Science & Technology, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa 904-0495, Japan

    • Nicholas M. Luscombe

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Contributions

Y.S. and J.U. developed the hiCLIP protocol and designed the project; Y.S. performed the hiCLIP, mRNA-seq, ribosome profiling experiments; Y.S., A.V., E.D. and N.M.L. designed and performed bioinformatic analyses; A.D., C.M. and A.Z. performed the reporter assays. Y.S., A.D., N.M.L. and J.U. wrote the manuscript. All authors contributed to the analysis and interpretation of the data.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Nicholas M. Luscombe or Jernej Ule.

The sequence data and scripts are publicly available from ArrayExpress (E-MTAB-2937, E-MTAB-2940, E-MTAB-2941) and (https://github.com/jernejule/STAU1_hiCLIP).

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a discussion of challenges of the experimental methods available to study RNA structures in vivo.

Excel files

  1. 1.

    Supplementary Table 1

    This table contains mapping statistics of high-throughput sequencing data.

  2. 2.

    Supplementary Table 2

    This table contains genomic positions and sequences of STAU1 target mRNA duplexes that are located in CDS or 3'UTR.

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DOI

https://doi.org/10.1038/nature14280

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