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m6A modulates neuronal functions and sex determination in Drosophila

Nature 540, 242–247 (08 December 2016) | Download Citation

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Abstract

N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. Here we carry out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. We identify the member of the split ends protein family, Spenito, as a novel bona fide subunit of the methyltransferase complex. We further demonstrate important roles of this complex in neuronal functions and sex determination, and implicate the nuclear YT521-B protein as a main m6A effector in these processes. Altogether, our work substantially extends our knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.

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Acknowledgements

We would like to thank the Bloomington Drosophila Stock Center for fly reagents; the Drosophila Genomics Resource Center at Indiana University for plasmids; The Developmental Studies Hybridoma Bank and the Lehmann laboratory for antibodies; M. Soller for sharing unpublished data; members of the J.-Y.R. laboratory for helpful discussion; A. Dold and V. Morin for experimental help; the IMB Genomics; the IMB Genomics, Proteomics and Bioinformatics Core facilities for support; and Bioinformatics Core facilities for support; and R. Ketting, N. Soshnikova, R. Strauss, J. Treisman and K. Zarnack for critical reading of the manuscript. Research in the laboratory of J.-Y.R. is supported by the Marie Curie CIG 334288 and the Deutsche Forschungsgemeinschaft (DFG) SPP1935 grant RO 4681/4-1. L.S. is funded by the Rhineland-Palatinate program Gene RED. The project was also supported by a DFG grant (HE 3397/13-1, SPP1784) to M.H.

Author information

Affiliations

  1. Institute of Molecular Biology (IMB), 55128 Mainz, Germany

    • Tina Lence
    • , Junaid Akhtar
    • , Marc Bayer
    • , Nastasja Kreim
    • , Miguel A. Andrade-Navarro
    •  & Jean-Yves Roignant

  2. Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, 55128 Mainz, Germany

    • Katharina Schmid
    •  & Mark Helm

  3. Institute of Zoology III (Neurobiology), Johannes Gutenberg University of Mainz, 55128 Mainz, Germany

    • Laura Spindler
    •  & Burkhard Poeck

  4. Kimmel Center for Biology and Medicine of the Skirball Institute, NYU School of Medicine, Department of Cell Biology, 540 First Avenue, New York, New York 10016, USA

    • Cheuk Hei Ho

  5. Faculty of Biology, Johannes Gutenberg University of Mainz, 55128 Mainz, Germany

    • Miguel A. Andrade-Navarro

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Contributions

T.L. and J.-Y.R. conceived the idea. T.L. designed and performed the experiments. J.A. performed the YT521-B RNA immunopreciptation experiment and M.B. generated the YT521-B allele. K.S. and M.H. performed the LC–MS/MS quantification of m6A levels. L.S. and B.P. carried out the Buridan analysis. C.H.H. performed NMJ staining and analysis. M.A.A.-N. performed the phylogenetic analysis. N.K. performed the computational analysis. T.L. and J.-Y.R. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jean-Yves Roignant.

Extended data

Extended data figures

  1. 1.

    Characterization of the Drosophila m6A methyltransferase complex.

  2. 2.

    m6A quantification, MeRIP-seq validation and sequence features of m6A sites in Drosophila mRNA.

  3. 3.

    Significant fold changes and correspondence between biological replicates of RNA-seq data.

  4. 4.

    Gene ontology term enrichment analysis.

  5. 5.

    m6A nuclear components control fl(2)d splicing.

  6. 6.

    Characterization of Drosophila YTH components.

  7. 7.

    Genetic characterization of Ime4, dMettl14 and YT521-B.

  8. 8.

    Ime4 mutant flies have reduced locomotion and shortened lifespan but apparent normal ovarian development.

  9. 9.

    m6A components fine-tune the sex determination pathway via YT521-B.

  10. 10.

    RNA interference screen identifies Nito as a new member of the methyltransferase complex.

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Uncropped scans with protein size indications in kDa and DNA size indicators in bp.

Excel files

  1. 1.

    Supplementary Table 1

    Analysis of the transcriptome wide m6A profile in S2R+ cells.

  2. 2.

    Supplementary Table 2

    Fl(2)d KD versus LacZ KD in S2R+ cells.

  3. 3.

    Supplementary Table 3

    Ime4 KD versus LacZ KD in S2R+ cells

  4. 4.

    Supplementary Table 4

    Ime4/dMettl14 KD versus LacZ KD in S2R+ cells.

  5. 5.

    Supplementary Table 5

    dMettl14 KD versus LacZ KD in S2R+ cells.

  6. 6.

    Supplementary Table 6

    Ime4 KO (ImeDcat) in adult heads of 1-2 day old females.

  7. 7.

    Supplementary Table 7

    Comparison of transcripts affected upon Ime4 KO in adult heads with transcripts containing m6A peak(s) in S2R+ cells.

  8. 8.

    Supplementary Table 8

    Candidate proteins enriched by 2-fold in the YT521-Myc IP pull down in S2R+ cells.

  9. 9.

    Supplementary Table 9

    Primers used for dsRNA synthesis.

  10. 10.

    Supplementary Table 10

    Mass transitions and QQQ parameters of the monitored modifications.

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DOI

https://doi.org/10.1038/nature20568

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