Article | Published:

Control of RNP motility and localization by a splicing-dependent structure in oskar mRNA

Nature Structural & Molecular Biology volume 19, pages 441449 (2012) | Download Citation

Abstract

oskar RNA localization to the posterior pole of the Drosophila melanogaster oocyte requires splicing of the first intron and the exon junction complex (EJC) core proteins. The functional link between splicing, EJC deposition and oskar localization has been unclear. Here we demonstrate that the EJC associates with oskar mRNA upon splicing in vitro and that Drosophila EJC deposition is constitutive and conserved. Our in vivo analysis reveals that splicing creates the spliced oskar localization element (SOLE), whose structural integrity is crucial for ribonucleoprotein motility and localization in the oocyte. Splicing thus has a dual role in oskar mRNA localization: assembling the SOLE and depositing the EJC required for mRNA transport. The SOLE complements the EJC in formation of a functional unit that, together with the oskar 3′ UTR, maintains proper kinesin-based motility of oskar mRNPs and posterior mRNA targeting.

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Acknowledgements

We thank H. Le Hir (Institut de Biologie de l'Ecole Normale Supérieure, Paris, France) for his gift of TAP-tagged EJC proteins; R. Lührmann (Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany) and D. Rio (University of California, Berkeley, USA) for Kc cell nuclear extracts; J. Ellenberg (the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany) for the gift of λN-GFP and boxB reporter vectors; D. St Johnston (The Wellcome Trust, Cancer Research UK Gurdon Institute, Cambridge, UK) for the oskMS2-GFP; D. Brunner (Institute of Molecular Life Sciences, University of Zürich, Switzerland) for the UASp-EB1-mCherry fly stocks; the EMBL GeneCore Facility for DNA sequencing, the EMBL Advanced Light Microscopy Facility for the use of microscopes, and A.-M. Voie and S. Müller for fly transgenesis. We also thank A. Cyrklaff for assistance with embryo collection, S.-J. Fan for the gift of the pUASpλN-GFP plasmid, M. Jeske for inspiration in naming of transgenes, A. Obrdlik, M. Hentze and I. Telley for critical discussions and comments on the manuscript. S.G. was supported in part by a Deutsche Forschungsgemeinschaft SPP grant (DFG EP 37/1-3) to A.E.; V.M. by a fellowship from the Fondation pour la Recherche Médicale; and I.G. by an EMBL Interdisciplinary Postdoctoral fellowship (EIPOD) and an EMBO Long-term Fellowship.

Author information

Author notes

    • Sanjay Ghosh
    •  & Virginie Marchand

    These authors contributed equally to this work.

Affiliations

  1. Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

    • Sanjay Ghosh
    • , Virginie Marchand
    • , Imre Gáspár
    •  & Anne Ephrussi

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Contributions

S.G. generated the transgenes and carried out all aspects of the in situ analysis of mRNA localization. V.M. generated Drosophila embryo nuclear extracts, established the in vitro splicing assay, and performed the RNase protection experiments and immunoprecipitations. I.G. performed the time-lapse imaging of live oocytes and the statistical analyses. S.G., V.M., I.G. and A.E. conceived the experiments, analyzed the data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Anne Ephrussi.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7, Supplementary Tables 1–3 and Supplementary Methods

Videos

  1. 1.

    Supplementary Video 1

    Motility of oskΔi(2,3) RNPs in living stage-9 oocyte

  2. 2.

    Supplementary Video 2

    Motility of oskMS2 RNPs in living stage-9 oocyte

  3. 3.

    Supplementary Video 3

    Motility of colchicine treated oskΔi(2,3) RNPs in living stage 9 oocyte

  4. 4.

    Supplementary Video 4

    Motility of oskΔi(1,2,3) RNPs in living stage-9 oocyte

  5. 5.

    Supplementary Video 5

    Motility of oskPSLz RNPs in living stage-9 oocyte

  6. 6.

    Supplementary Video 6

    Motility of oskPSLzc RNPs in living stage-9 oocyte

  7. 7.

    Supplementary Video 7

    Viability and development of a dissected stage 8-9 egg chamber expressing oskΔi(2,3)*GFP

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DOI

https://doi.org/10.1038/nsmb.2257

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