Article

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

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Abstract

Protein–RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein–RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova–RNA interactions in 3′ untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein–RNA interactions in vivo.

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Acknowledgements

The authors are grateful to members of the Darnell and Ule laboratories and J. Richter for critical discussions and review of the manuscript, B. Friedman for suggesting the use of exon arrays, F. Allain for communicating unpublished results, and M. Suarez-Farinas for help with statistics. This work was supported by NIH R01 NS34389 (R.B.D.) and the Howard Hughes Medical Institute. R.B.D. is an HHMI Investigator.

Author Contributions D.D.L. and R.B.D. wrote the paper. D.D.L., A.M. and J.J.F. performed the biochemical and CLIP experiments. J.U. and M.K. developed ASPIRE2 and analysed exon junction array data. D.D.L., S.W.C., X.W. and R.B.D. did bioinformatic analysis. D.D.L, J.C.D. and R.B.D. analysed the data. T.A.C., A.C.S. and J.E.B. developed the exon junction microarray.

Author information

Affiliations

  1. Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute,

    • Donny D. Licatalosi
    • , Aldo Mele
    • , John J. Fak
    • , Sung Wook Chi
    • , Jennifer C. Darnell
    •  & Robert B. Darnell
  2. Biocomputing, Information Technology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA

    • Xuning Wang
  3. MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK

    • Jernej Ule
    •  & Melis Kayikci
  4. Expression Research, Affymetrix, Inc., Santa Clara, California 95051, USA

    • Tyson A. Clark
    • , Anthony C. Schweitzer
    •  & John E. Blume

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Competing interests

T.A.C., A.C.S. and J.E.B. are employees of Affymetrix, Inc.

Corresponding author

Correspondence to Robert B. Darnell.

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    Supplementary Information

    This file contains Supplementary Methods, Supplementary Figures 1-9 with Legends, Supplementary References, and Supplementary Tables 1-2 with Supplementary References

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