Abstract
The exon junction complex (EJC) is a central effector of the fate of mRNAs, linking nuclear processing to mRNA transport, translation and surveillance. However, little is known about its transcriptome-wide targets. We used cross-linking and immunoprecipitation methods coupled to high-throughput sequencing (CLIP-seq) in human cells to identify the binding sites of the DEAD-box helicase eIF4AIII, an EJC core component. CLIP reads form peaks that are located mainly in spliced mRNAs. Most expressed exons harbor peaks either in the canonical EJC region, located ~24 nucleotides upstream of exonic junctions, or in other noncanonical regions. Notably, both of these types of peaks are preferentially associated with unstructured and purine-rich sequences containing the motif GAAGA, which is a potential binding site for EJC-associated factors. Therefore, EJC positions vary spatially and quantitatively between exons. This transcriptome-wide mapping of human eIF4AIII reveals unanticipated aspects of the EJC and broadens its potential impact on post-transcriptional regulation.
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Acknowledgements
We thank C. Tomasetto and J. Stévenin (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France) and J. Marie (Centre de Génétique Moléculaire, Gif-sur-Yvette, France) and E. Izaurralde (Max Planck Institute, Tübingen, Germany) for antibodies, C. Thermes and E. van Dijk (Centre de Génétique Moléculaire, Gif-sur-Yvette, France) at the IMAGIF platform for Illumina deep sequencing and J. Ule (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) for technical advice. We acknowledge our laboratory for helpful advice, comments and discussions and A. Morillon (Institut Curie, Paris, France) for carefully reading the manuscript. We also thank M. Gratigny and A. Louis (Institut de Biologie de l'Ecole Normale Supérieure, Paris, France) for bioinformatic advice. This work was supported in part by the Centre National de la Recherche Scientifique (ATIP programme blanc 2008 to H.L.H. and ANR-07-JCJC-0097-01 to L.P.), the Institut National de la Santé et de la Recherche Médicale (INSERM; J.S.), the Agence Nationale de la Recherche (2008-BLAN-0323 to H.L.H.) and the Fondation pour la Recherche Médicale (J.S. and H.L.H.). Z.W. is supported by a European Molecular Biology Organization long-term postdoctoral fellowship.
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O.L.T., Y.A., L.P. and J.S. contributed to the development of the CLIP protocol. J.S. performed CLIP experiments. V.M. and H.R.C. performed the bioinformatic analyses. Z.W. performed RIP experiments. E.M. performed protein coimmunoprecitation assays. I.B. purified the antibodies. J.S. and H.L.H. conceived the project. H.L.H. provided resources. J.S. and H.L.H. wrote the paper.
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Saulière, J., Murigneux, V., Wang, Z. et al. CLIP-seq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex. Nat Struct Mol Biol 19, 1124–1131 (2012). https://doi.org/10.1038/nsmb.2420
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DOI: https://doi.org/10.1038/nsmb.2420
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