Technical Report

iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution

  • Nature Structural & Molecular Biology volume 17, pages 909915 (2010)
  • doi:10.1038/nsmb.1838
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Abstract

In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C. Despite their abundance, however, it remained unclear whether these particles control pre-mRNA processing. Here, we developed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. iCLIP data show that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. hnRNP particles assemble on both introns and exons but remain generally excluded from splice sites. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an 'RNA map' indicates how the positioning of hnRNP particles determines their effect on the inclusion of alternative exons. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes.

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Acknowledgements

The authors thank all members of the Ule laboratory for experimental assistance and discussion, A. Klug, K. Nagai, M. Babu, S. Eustermann, N. McGlincy, D. Daujotyte and O. Rossbach for fruitful discussions and comments on the manuscript, J. Hadfield and N. Matthews for high-throughput sequencing and B. Rhead and B. Raney for modifying the UCSC Genome Browser BedGraph format. This work was supported by the European Research Council grant 206726-CLIP and Human Frontiers Science Program grant RGP0024 to J.U. and a Long-term Human Frontiers Science Program fellowship to J.K.

Author information

Author notes

    • Julian König
    •  & Kathi Zarnack

    These authors contributed equally to this work.

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

    • Julian König
    • , Melis Kayikci
    •  & Jernej Ule
  2. European Molecular Biology Laboratory-European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, UK.

    • Kathi Zarnack
    •  & Nicholas M Luscombe
  3. Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia.

    • Gregor Rot
    • , Tomaž Curk
    •  & Blaž Zupan
  4. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK.

    • Daniel J Turner
  5. European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.

    • Nicholas M Luscombe

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Contributions

J.K. and J.U. designed the iCLIP protocol and J.K. carried out iCLIP, microarray and PCR experiments; D.J.T. performed high-throughput sequencing; G.R., T.C. and B.Z. mapped the iCLIP sequence reads to genome and evaluated random barcodes; K.Z. and N.M.L. analyzed the sequence and positioning of hnRNP C cross-link sites and the function of hnRNP particles; M.K. developed the ASPIRE3 software to analyze splice-junction microarray data and generated the RNA map; J.K., K.Z. and J.U. wrote the manuscript.

Competing interests

The authors have applied for a patent for iCLIP.

Corresponding author

Correspondence to Jernej Ule.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–9, Supplementary Tables 1–3 and Supplementary Methods