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

Nature Structural & Molecular Biology volume 19, pages 441–449 (2012)Cite this article

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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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Figure 1: The Drosophila EJC assembles on oskar mRNAs ~20–24 nt upstream of splice junctions in vitro.
Figure 2: The oskar coding region and oskar intron 1 splicing are necessary for mRNA posterior localization.
Figure 3: 28 nucleotides flanking the first oskar intron promote mRNA localization to the posterior pole.
Figure 4: Integrity of the SOLE proximal stem is required for posterior transport of oskar mRNA.
Figure 5: An intact SOLE is not required for pre-mRNA splicing or EJC deposition.
Figure 6: Tracking of motile wild-type and SOLE mutant oskar RNPs in living oocytes.

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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.

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Author notes

  1. Sanjay Ghosh and Virginie Marchand: These authors contributed equally to this work.

Authors and 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.

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Correspondence to Anne Ephrussi.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–3 and Supplementary Methods (PDF 14024 kb)

Supplementary Video 1

Motility of oskΔi(2,3) RNPs in living stage-9 oocyte (AVI 4849 kb)

Supplementary Video 2

Motility of oskMS2 RNPs in living stage-9 oocyte (AVI 4744 kb)

Supplementary Video 3

Motility of colchicine treated oskΔi(2,3) RNPs in living stage 9 oocyte (AVI 4814 kb)

Supplementary Video 4

Motility of oskΔi(1,2,3) RNPs in living stage-9 oocyte (AVI 4836 kb)

Supplementary Video 5

Motility of oskPSLz RNPs in living stage-9 oocyte (AVI 4864 kb)

Supplementary Video 6

Motility of oskPSLzc RNPs in living stage-9 oocyte (AVI 3502 kb)

Supplementary Video 7

Viability and development of a dissected stage 8-9 egg chamber expressing oskΔi(2,3)*GFP (AVI 10803 kb)

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Ghosh, S., Marchand, V., Gáspár, I. et al. Control of RNP motility and localization by a splicing-dependent structure in oskar mRNA. Nat Struct Mol Biol 19, 441–449 (2012). https://doi.org/10.1038/nsmb.2257

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