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An RNA map predicting Nova-dependent splicing regulation

Nature volume 444, pages 580586 (30 November 2006) | Download Citation

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Abstract

Nova proteins are neuron-specific alternative splicing factors. We have combined bioinformatics, biochemistry and genetics to derive an RNA map describing the rules by which Nova proteins regulate alternative splicing. This map revealed that the position of Nova binding sites (YCAY clusters) in a pre-messenger RNA determines the outcome of splicing. The map correctly predicted Nova’s effect to inhibit or enhance exon inclusion, which led us to examine the relationship between the map and Nova’s mechanism of action. Nova binding to an exonic YCAY cluster changed the protein complexes assembled on pre-mRNA, blocking U1 snRNP (small nuclear ribonucleoprotein) binding and exon inclusion, whereas Nova binding to an intronic YCAY cluster enhanced spliceosome assembly and exon inclusion. Assays of splicing intermediates of Nova-regulated transcripts in mouse brain revealed that Nova preferentially regulates removal of introns harbouring (or closest to) YCAY clusters. These results define a genome-wide map relating the position of a cis-acting element to its regulation by an RNA binding protein, namely that Nova binding to YCAY clusters results in a local and asymmetric action to regulate spliceosome assembly and alternative splicing in neurons.

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Acknowledgements

We thank T. Eom and D. Licatalosi for help with breeding Nova1–/–Nova2–/– mice and providing mouse brain; P. Ariel for technical assistance; M. Babu, C. Smith, J. Valcarcel, G. Yeo, D. Licatalosi, J. Darnell, S. Xie, and S. W. Chi for critically reading the manuscript; D. Karolchik and J. Jackson for help with the UCSC Genome Bioinformatics tools; J. Okano for the hnRNP K expression construct; A. Krainer and L. Manche for help with in vitro splicing assays; K. Dredge for DNA constructs; D. Black for sharing unpublished results; and M. Konarska and members of the laboratory for discussions. Supported by the NIH (R.B.D.) and the Howard Hughes Medical Institute, the tumour immunology program of Cancer Research Institute (J.U.) and a Human Frontiers Science Program Fellowship (M.R.). R.B.D. is an Investigator of the Howard Hughes Medical Institute. Author Contributions J.U. bioinformatically defined the RNA map, and predicted and analysed Nova-target exons and splicing intermediates; G.S. performed in vitro studies of the mechanisms of Nova action and its effects on spliceosome assembly; A.M. purified PCR products for sequencing; M.R. characterized the Nova1–/–Nova2–/– mice; X.W. wrote the sequence analysis programs; B.T. and T.G. provided the database of alternative exons; B.J.B. provided depleted extracts; and R.B.D. supervised all studies. The manuscript was prepared by J.U., G.S. and R.B.D., with the participation of all authors.

Author information

Author notes

    • Jernej Ule
    •  & Giovanni Stefani

    These authors contributed equally to this work.

    • Jernej Ule
    • , Giovanni Stefani
    • , Bahar Taneri
    •  & Terry Gaasterland

    Present addresses: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK (J.U.); Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06511, USA (G.S.); UCSD, Scripps Institution of Oceanography, Scripps Genome Center, 8750 Biological Grade, La Jolla, California 92037, USA (B.T., T.G.); Eastern Mediterranean University, Famagusta, Cyprus (B.T.).

Affiliations

  1. Howard Hughes Medical Institute,

    • Jernej Ule
    • , Giovanni Stefani
    • , Aldo Mele
    • , Matteo Ruggiu
    •  & Robert B. Darnell
  2. Laboratory of Molecular Neuro-Oncology,

    • Jernej Ule
    • , Giovanni Stefani
    • , Aldo Mele
    • , Matteo Ruggiu
    •  & Robert B. Darnell
  3. Biocomputing, Information Technology, and,

    • Xuning Wang
  4. Laboratory of Computational Genomics, The Rockefeller University, 1230 York Ave,, New York, New York 10021, USA

    • Bahar Taneri
    •  & Terry Gaasterland
  5. Banting and Best Department of Medical Research, University of Toronto, Ontario M5G 1L6, Canada

    • Benjamin J. Blencowe

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Corresponding author

Correspondence to Robert B. Darnell.

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https://doi.org/10.1038/nature05304

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