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

Nature Structural & Molecular Biology volume 17, pages 909–915 (2010)Cite this article

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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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Figure 1: iCLIP identifies hnRNP C cross-link nucleotides on RNAs.
Figure 2: The genomic location of hnRNP C cross-link nucleotides.
Figure 3: hnRNP C binds uridine tracts with a defined spacing.
Figure 4: The RNA map relates hnRNP particle positioning to splicing regulation.
Figure 5: iCLIP data predict exons that are silenced by hnRNP C.
Figure 6: A model of hnRNP C tetramer binding at silenced and enhanced alternative exons.

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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.

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Author notes

  1. Julian König and Kathi Zarnack: These authors contributed equally to this work.

Authors and 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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  1. Julian König
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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.

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Correspondence to Jernej Ule.

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The authors have applied for a patent for iCLIP.

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Supplementary Figures 1–9, Supplementary Tables 1–3 and Supplementary Methods (PDF 5874 kb)

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König, J., Zarnack, K., Rot, G. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17, 909–915 (2010). https://doi.org/10.1038/nsmb.1838

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