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The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease

Nature Structural & Molecular Biology volume 23, pages 705713 (2016) | Download Citation

Abstract

Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exuperantia (Exu) is a putative exonuclease (EXO) associated with bcd and required for its localization. We present the crystal structure of Exu, which reveals a dimeric assembly with each monomer consisting of a 3′-5′ EXO-like domain and a sterile alpha motif (SAM)-like domain. The catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and RNA binding. We show that Exu binds RNA directly in vitro, that the SAM-like domain is required for RNA binding activity and that Exu binds a structured element present in the bcd 3′ untranslated region with high affinity. Through structure-guided mutagenesis, we show that Exu dimerization is essential for bcd localization. Our data demonstrate that Exu is a noncanonical RNA-binding protein with EXO-SAM-like domain architecture that interacts with its target RNA as a homodimer.

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Referenced accessions

References

  1. 1.

    & Spatial regulation of translation through RNA localization. F1000 Biol. Rep. 4, 16 (2012).

  2. 2.

    & Dendritic protein synthesis, synaptic plasticity, and memory. Cell 127, 49–58 (2006).

  3. 3.

    & Dendritic mRNA: transport, translation and function. Nat. Rev. Neurosci. 8, 776–789 (2007).

  4. 4.

    & Translational control of localized mRNAs: restricting protein synthesis in space and time. Nat. Rev. Mol. Cell Biol. 9, 971–980 (2008).

  5. 5.

    , , & Remote control of gene function by local translation. Cell 157, 26–40 (2014).

  6. 6.

    & Localization, anchoring and translational control of oskar, gurken, bicoid and nanos mRNA during Drosophila oogenesis. Fly (Austin) 3, 15–28 (2009).

  7. 7.

    et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).

  8. 8.

    et al. Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. eLife 4, e05003 (2015).

  9. 9.

    Germline cysts: communes that work. Cell 72, 649–651 (1993).

  10. 10.

    & The villin-like protein encoded by the Drosophila quail gene is required for actin bundle assembly during oogenesis. Cell 78, 291–301 (1994).

  11. 11.

    & Seeing is believing: the bicoid morphogen gradient matures. Cell 116, 143–152 (2004).

  12. 12.

    & Organization of anterior pattern in the Drosophila embryo by the maternal gene bicoid. Nature 334, 120–125 (1986).

  13. 13.

    , & Determination of anteroposterior polarity in Drosophila. Science 238, 1675–1681 (1987).

  14. 14.

    & The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54, 95–104 (1988).

  15. 15.

    , , , & Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. Development 107 (suppl.), 13–19 (1989).

  16. 16.

    et al. The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 7, 1749–1756 (1988).

  17. 17.

    , , & Direct observation of regulated ribonucleoprotein transport across the nurse cell/oocyte boundary. Mol. Biol. Cell 18, 2254–2263 (2007).

  18. 18.

    , , & Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during drosophila oogenesis. Dev. Cell 3, 685–696 (2002).

  19. 19.

    & A gradient of bicoid protein in Drosophila embryos. Cell 54, 83–93 (1988).

  20. 20.

    , , & Rescue of bicoid mutant Drosophila embryos by Bicoid fusion proteins containing heterologous activating sequences. Nature 342, 149–154 (1989).

  21. 21.

    , , , & Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266, 1996–1999 (1994).

  22. 22.

    & Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila. Dev. Biol. 113, 443–448 (1986).

  23. 23.

    , & In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule-dependent axis specification pathway. Cell 106, 35–46 (2001).

  24. 24.

    , , & Microtubules, the ER and Exu: new associations revealed by analysis of mini spindles mutations. Mech. Dev. 126, 289–300 (2009).

  25. 25.

    & Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature 369, 400–403 (1994).

  26. 26.

    , & A sponge-like structure involved in the association and transport of maternal products during Drosophila oogenesis. J. Cell Biol. 139, 817–829 (1997).

  27. 27.

    & In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway. Development 125, 3655–3666 (1998).

  28. 28.

    , , & Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128, 3233–3242 (2001).

  29. 29.

    et al. Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes. J. Cell Biol. 148, 427–440 (2000).

  30. 30.

    , , & Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz. J. Cell Biol. 163, 1197–1204 (2003).

  31. 31.

    , , & The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains. Nucleic Acids Res. 25, 5110–5118 (1997).

  32. 32.

    , & RNA recognition by 3′-to-5′ exonucleases: the substrate perspective. Biochim. Biophys. Acta 1779, 256–265 (2008).

  33. 33.

    & Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia. Development 131, 5897–5907 (2004).

  34. 34.

    , & exl protein specifically binds BLE1, a bicoid mRNA localization element, and is required for one phase of its activity. Proc. Natl. Acad. Sci. USA 92, 10787–10791 (1995).

  35. 35.

    et al. The crystal structure of TREX1 explains the 3′ nucleotide specificity and reveals a polyproline II helix for protein partnering. J. Biol. Chem. 282, 10537–10543 (2007).

  36. 36.

    et al. Structural basis for RNA trimming by RNase T in stable RNA 3′-end maturation. Nat. Chem. Biol. 7, 236–243 (2011).

  37. 37.

    & Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

  38. 38.

    & A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. USA 90, 6498–6502 (1993).

  39. 39.

    & Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. Nucleic Acids Res. 29, 1017–1026 (2001).

  40. 40.

    & The 3′ 5′ exonucleases. Nat. Rev. Mol. Cell Biol. 3, 364–376 (2002).

  41. 41.

    et al. Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins. Nat. Methods 11, 1064–1070 (2014).

  42. 42.

    , & The temporal and spatial distribution pattern of maternal exuperantia protein: evidence for a role in establishment but not maintenance of bicoid mRNA localization. EMBO J. 10, 4259–4266 (1991).

  43. 43.

    , , , & Sequence-specific recognition of RNA hairpins by the SAM domain of Vts1p. Nat. Struct. Mol. Biol. 13, 168–176 (2006).

  44. 44.

    & RNA recognition by the Vts1p SAM domain. Nat. Struct. Mol. Biol. 13, 177–178 (2006).

  45. 45.

    , , , , & Shape-specific recognition in the structure of the Vts1p SAM domain with RNA. Nat. Struct. Mol. Biol. 13, 160–167 (2006).

  46. 46.

    & Secondary structure of the 3′ UTR of bicoid mRNA. Biochimie 86, 91–104 (2004).

  47. 47.

    bicoid mRNA localization signal: phylogenetic conservation of function and RNA secondary structure. Development 110, 161–171 (1990).

  48. 48.

    & Cis-acting sequences responsible for anterior localization of bicoid mRNA in Drosophila embryos. Nature 336, 595–598 (1988).

  49. 49.

    & Redundant RNA recognition events in bicoid mRNA localization. RNA 3, 1413–1420 (1997).

  50. 50.

    , , & RNA regulatory element BLE1 directs the early steps of bicoid mRNA localization. Development 118, 1233–1243 (1993).

  51. 51.

    , , & Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor. Genes Dev. 23, 1546–1558 (2009).

  52. 52.

    , , & A′-form RNA helices are required for cytoplasmic mRNA transport in Drosophila. Nat. Struct. Mol. Biol. 17, 703–709 (2010).

  53. 53.

    et al. The exuperantia gene is required for Drosophila spermatogenesis as well as anteroposterior polarity of the developing oocyte, and encodes overlapping sex-specific transcripts. Genetics 126, 607–617 (1990).

  54. 54.

    , , , & Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate. Nat. Cell Biol. 6, 427–435 (2004).

  55. 55.

    et al. Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists. RNA 21, 833–839 (2015).

  56. 56.

    et al. PIWI slicing and EXD1 drive biogenesis of nuclear piRNAs from cytosolic targets of the mouse piRNA pathway. Mol. Cell 61, 138–152 (2016).

  57. 57.

    et al. Crystal structure and activity of the endoribonuclease domain of the piRNA Pathway factor Maelstrom. Cell Rep. 11, 366–375 (2015).

  58. 58.

    & An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila. Genes Dev. 11, 423–435 (1997).

  59. 59.

    & 'De-evolution' of Drosophila toward a more generic mode of axis patterning. Int. J. Dev. Biol. 47, 497–503 (2003).

  60. 60.

    et al. Bicoid occurrence and Bicoid-dependent hunchback regulation in lower cyclorrhaphan flies. Evol. Dev. 10, 413–420 (2008).

  61. 61.

    et al. ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics 19, 163–164 (2003).

  62. 62.

    , , , & Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer. J. Struct. Biol. 175, 178–188 (2011).

  63. 63.

    Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

  64. 64.

    Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

  65. 65.

    & Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

  66. 66.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  67. 67.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  68. 68.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  69. 69.

    et al. mzML: a community standard for mass spectrometry data. Mol Cell Proteomics 10, R110.000133 (2011).

  70. 70.

    et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).

  71. 71.

    et al. TOPP: the OpenMS proteomics pipeline. Bioinformatics 23, e191–e197 (2007).

  72. 72.

    et al. OpenMS: an open-source software framework for mass spectrometry. BMC Bioinformatics 9, 163 (2008).

  73. 73.

    et al. Open mass spectrometry search algorithm. J. Proteome Res. 3, 958–964 (2004).

  74. 74.

    & MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

  75. 75.

    & Female sterile mutations on the second chromosome of Drosophila melanogaster. I. Maternal effect mutations. Genetics 121, 101–117 (1989).

  76. 76.

    & Kinesin light chain-independent function of the Kinesin heavy chain in cytoplasmic streaming and posterior localisation in the Drosophila oocyte. Development 129, 5473–5485 (2002).

  77. 77.

    , & NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

  78. 78.

    & Preparing early whole-mount Drosophila embryos for immunostaining. CSH Protoc. (2006).

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Acknowledgements

We wish to thank the MPI-Martinsried Crystallization Facility. We also thank the staff at the Swiss Light Source synchrotron for assistance during data collection, S. Grüner, E. Khazina and V. Ahl for assistance with MALLS measurements, and D. Hildebrand and N. Weiss for assistance with antibody production. We thank A. Cook, E. Lorentzen and E. Conti for discussion and critical reading of the manuscript. This project received funding from the Max Planck Gesellschaft, the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013), ERC grant agreement no. 310957 and the Deutsche Forschungsgemeinschaft (SFB860 to K.K. and H.U., and BO3588/2-1 to F.B.).

Author information

Author notes

    • Katharina Veith
    •  & Katharina Kramer

    Present addresses: Institute for Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany (K.V.) and Max Planck Institute for Plant Breeding Research, Köln, Germany (K.K.).

Affiliations

  1. Max Planck Institute for Developmental Biology, Tübingen, Germany.

    • Daniela Lazzaretti
    • , Katharina Veith
    • , Uwe Irion
    •  & Fulvia Bono
  2. Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

    • Katharina Kramer
    •  & Henning Urlaub
  3. Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany.

    • Katharina Kramer
    •  & Henning Urlaub
  4. Max Planck Institute of Biochemistry, Martinsried, Germany.

    • Claire Basquin

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Contributions

Biochemical, biophysical and crystallization work was performed by D.L. and K.V.; fly work was performed by D.L. and U.I.; K.K. and H.U. carried out the MS analysis; D.L. and C.B. analyzed FA data; F.B. solved the structures and supervised the project. F.B., D.L. and U.I. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Fulvia Bono.

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https://doi.org/10.1038/nsmb.3254

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