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m6A modulates neuronal functions and sex determination in Drosophila | Nature Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain
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            and JavaScript. Advertisement View all journals Saved research Search Log in Content Explore content About the journal Publish with us Subscribe Sign up for alerts RSS feed nature articles article Article Published: 30 November 2016 m 6 A modulates neuronal functions and sex determination in Drosophila Tina Lence 1 , Junaid Akhtar 1 , Marc Bayer 1 , Katharina Schmid 2 , Laura Spindler 3 , Cheuk Hei Ho 4 , Nastasja Kreim 1 , Miguel A. Andrade-Navarro 1 , 5 , Burkhard Poeck 3 , Mark Helm 2 & … Jean-Yves Roignant 1 Show authors Nature volume 540 , pages 242–247 ( 2016 ) Cite this article Save article View saved research 25k Accesses 557 Citations 89 Altmetric Metrics details Abstract N 6 -methyladenosine RNA (m 6 A) 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 m 6 A 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 m 6 A effector in these processes. Altogether, our work substantially extends our knowledge of m 6 A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal. This is a preview of subscription content, access via your institution Access options Access through your institution Subscribe to this journal Receive 52 print issues and online access $199.00 per year only $3.83 per issue Learn more Buy this article Purchase on SpringerLink Instant access to the full article PDF. USD 39.95 Prices may be subject to local taxes which are calculated during checkout Additional access options: Log in Learn about institutional subscriptions Read our FAQs Contact customer support Figure 1: Drosophila m 6 A methyltransferase complex is enriched in the nervous system. Figure 2: m 6 A controls alternative splicing via YT521-B. Figure 3: YT521-B and the methyltransferase complex control fly behaviour. Figure 4: YT521-B and the methyltransferase complex regulate Sxl splicing. Figure 5: Nito is a novel member of the methyltransferase complex. Similar content being viewed by others Regulatory roles of N6-methyladenosine (m 6 A) methylation in RNA processing and non-communicable diseases Article 05 June 2024 mRNA m 6 A detection Article 12 December 2024 Mettl3 -dependent m 6 A modification attenuates the brain stress response in Drosophila Article Open access 14 September 2022 Explore related subjects Discover the latest articles and news in related subjects. Developmental neurogenesis RNA modification Accession codes Primary accessions Gene Expression Omnibus GSE79297 References Motorin, Y. & Helm, M. RNA nucleotide methylation. Wiley Interdiscip . Rev. RNA 2 , 611–631 (2011) CAS PubMed Google Scholar Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m 6 A RNA methylation. Nature Rev. Genet. 15 , 293–306 (2014) Article CAS Google Scholar Meyer, K. D. & Jaffrey, S. R. The dynamic epitranscriptome: N 6 -methyladenosine and gene expression control. Nature Rev. Mol. Cell Biol. 15 , 313–326 (2014) Article CAS Google Scholar Dominissini, D. et al. Topology of the human and mouse m 6 A RNA methylomes revealed by m 6 A-seq. Nature 485 , 201–206 (2012) Article ADS CAS Google Scholar Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149 , 1635–1646 (2012) Article CAS Google Scholar Schwartz, S. et al. High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Cell 155 , 1409–1421 (2013) Article CAS Google Scholar Li, Y. et al. Transcriptome-wide N 6 -methyladenosine profiling of rice callus and leaf reveals the presence of tissue-specific competitors involved in selective mRNA modification. RNA Biol. 11 , 1180–1188 (2014) Article Google Scholar Lin, S. & Gregory, R. I. Methyltransferases modulate RNA stability in embryonic stem cells. Nature Cell Biol. 16 , 129–131 (2014) Article CAS Google Scholar Schwartz, S. et al. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Reports 8 , 284–296 (2014) Article CAS Google Scholar Wang, X. et al. N 6 -methyladenosine-dependent regulation of messenger RNA stability. Nature 505 , 117–120 (2014) Article ADS Google Scholar Wang, Y. et al. N 6 -methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nature Cell Biol. 16 , 191–198 (2014) Article CAS Google Scholar Alarcón, C. R. et al. HNRNPA2B1 is a mediator of m 6 A-dependent nuclear RNA processing events. Cell 162 , 1299–1308 (2015) Article Google Scholar Liu, N. et al. N 6 -methyladenosine-dependent RNA structural switches regulate RNA–protein interactions. Nature 518 , 560–564 (2015) Article ADS CAS Google Scholar Meyer, K. D. et al. 5′ UTR m 6 A promotes Cap-independent translation. Cell 163 , 999–1010 (2015) Article CAS Google Scholar Wang, X. et al. N 6 -methyladenosine modulates messenger RNA translation efficiency. Cell 161 , 1388–1399 (2015) Article CAS Google Scholar Zhou, J. et al. Dynamic m 6 A mRNA methylation directs translational control of heat shock response. Nature 526 , 591–594 (2015) Article ADS CAS Google Scholar Wang, X. & He, C. Reading RNA methylation codes through methyl-specific binding proteins. RNA Biol. 11 , 669–672 (2014) CAS Google Scholar Liu, J. et al. A METTL3–METTL14 complex mediates mammalian nuclear RNA N 6 -adenosine methylation. Nature Chem. Biol. 10 , 93–95 (2014) Article ADS CAS Google Scholar Ping, X. L. et al. Mammalian WTAP is a regulatory subunit of the RNA N 6 -methyladenosine methyltransferase. Cell Res. 24 , 177–189 (2014) Article CAS Google Scholar Jia, G. et al. N 6 -methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nature Chem. Biol. 7 , 885–887 (2011) CAS Google Scholar Fu, Y. et al. FTO-mediated formation of N 6 -hydroxymethyladenosine and N 6 -formyladenosine in mammalian RNA. Nature Commun. 4 , 1798 (2013) Article ADS Google Scholar Hess, M. E. et al. The fat mass and obesity associated gene ( Fto ) regulates activity of the dopaminergic midbrain circuitry. Nature Neurosci. 16 , 1042–1048 (2013) Article CAS Google Scholar Zheng, G. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49 , 18–29 (2013) Article CAS Google Scholar Batista, P. J. et al. m 6 A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15 , 707–719 (2014) Article CAS Google Scholar Geula, S. et al. m 6 A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347 , 1002–1006 (2015) Article ADS CAS Google Scholar Hongay, C. F. & Orr-Weaver, T. L. Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis. Proc. Natl Acad. Sci. USA 108 , 14855–14860 (2011) Article ADS CAS Google Scholar Zhong, S. et al. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell 20 , 1278–1288 (2008) Article CAS Google Scholar Bodi, Z. et al. Adenosine methylation in Arabidopsis mRNA is associated with the 3′ end and reduced levels cause developmental defects. Front. Plant Sci. 3 , 48 (2012) Article CAS Google Scholar Shah, J. C. & Clancy, M. J. IME4 , a gene that mediates MAT and nutritional control of meiosis in Saccharomyces cerevisiae . Mol. Cell. Biol. 12 , 1078–1086 (1992) Article CAS Google Scholar Clancy, M. J., Shambaugh, M. E., Timpte, C. S. & Bokar, J. A. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N 6 -methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene. Nucleic Acids Res. 30 , 4509–4518 (2002) Article CAS Google Scholar Agarwala, S. D., Blitzblau, H. G., Hochwagen, A. & Fink, G. R. RNA methylation by the MIS complex regulates a cell fate decision in yeast. PLoS Genet. 8 , e1002732 (2012) Article CAS Google Scholar Wang, P., Doxtader, K. A. & Nam, Y. Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases. Mol. Cell 63 , 306–317 (2016) Article CAS Google Scholar Wang, X. et al. Structural basis of N 6 -adenosine methylation by the METTL3–METTL14 complex. Nature 534 , 575–578 (2016) Article ADS CAS Google Scholar Gabel, H. W. et al. Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature 522 , 89–93 (2015) Article ADS CAS Google Scholar Götz, K. G. Visual guidance in Drosophila . Basic Life Sci. 16 , 391–407 (1980) PubMed Google Scholar Strauss, R., Hanesch, U., Kinkelin, M., Wolf, R. & Heisenberg, M. No-bridge of Drosophila melanogaster : portrait of a structural brain mutant of the central complex. J. Neurogenet. 8 , 125–155 (1992) Article CAS Google Scholar Clough, E. & Oliver, B. Genomics of sex determination in Drosophila. Brief. Funct . Genomics 11 , 387–394 (2012) CAS Google Scholar Granadino, B., Campuzano, S. & Sánchez, L. The Drosophila melanogaster fl(2)d gene is needed for the female-specific splicing of Sex-lethal RNA. EMBO J. 9 , 2597–2602 (1990) Article CAS Google Scholar Hilfiker, A., Amrein, H., Dübendorfer, A., Schneiter, R. & Nöthiger, R. The gene virilizer is required for female-specific splicing controlled by Sxl, the master gene for sexual development in Drosophila . Development 121 , 4017–4026 (1995) CAS PubMed Google Scholar Patil, D. P. et al. m 6 A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537 , 369–373 (2016) Article ADS CAS Google Scholar Kondo, S. & Ueda, R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila . Genetics 195 , 715–721 (2013) Article CAS Google Scholar Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23 , 2947–2948 (2007) Article CAS Google Scholar Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32 , 1792–1797 (2004) Article CAS Google Scholar Astigarraga, S., Hofmeyer, K., Farajian, R. & Treisman, J. E. Three Drosophila liprins interact to control synapse formation. J. Neurosci. 30 , 15358–15368 (2010) Article CAS Google Scholar Bahadorani, S. & Hilliker, A. J. Cocoa confers life span extension in Drosophila melanogaster . Nutr. Res. 28 , 377–382 (2008) Article CAS Google Scholar Oktaba, K. et al. Dynamic regulation by polycomb group protein complexes controls pattern formation and the cell cycle in Drosophila . Dev. Cell 15 , 877–889 (2008) Article CAS Google Scholar Littleton, J. T., Bellen, H. J. & Perin, M. S. Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse. Development 118 , 1077–1088 (1993) CAS PubMed Google Scholar Bluhm, A., Casas-Vila, N., Scheibe, M. & Butter, F. Reader interactome of epigenetic histone marks in birds. Proteomics 16 , 427–436 (2016) Article CAS Google Scholar Kellner, S. et al. Absolute and relative quantification of RNA modifications via biosynthetic isotopomers. Nucleic Acids Res. 42 , e142 (2014) Article Google Scholar Deng, X. et al. Widespread occurrence of N 6 -methyladenosine in bacterial mRNA. Nucleic Acids Res. 43 , 6557–6567 (2015) Article CAS Google Scholar Poeck, B., Triphan, T., Neuser, K. & Strauss, R. Locomotor control by the central complex in Drosophila –An analysis of the tay bridge mutant. Dev. Neurobiol. 68 , 1046–1058 (2008). 10.1002/dneu.20643 Article CAS Google Scholar Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29 , 15–21 (2013) Article CAS Google Scholar Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9 , R137 (2008) Article Google Scholar Yu, G., Wang, L. G. & He, Q. Y. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31 , 2382–2383 (2015) Article CAS Google Scholar Download references 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 Authors and Affiliations Institute of Molecular Biology (IMB), Mainz, 55128, Germany Tina Lence, Junaid Akhtar, Marc Bayer, Nastasja Kreim, Miguel A. Andrade-Navarro & Jean-Yves Roignant Institute of Pharmacy and Biochemistry, Johannes Gutenberg University of Mainz, Mainz, 55128, Germany Katharina Schmid & Mark Helm Institute of Zoology III (Neurobiology), Johannes Gutenberg University of Mainz, Mainz, 55128, Germany Laura Spindler & Burkhard Poeck Department of Cell Biology, Kimmel Center for Biology and Medicine of the Skirball Institute, NYU School of Medicine, 540 First Avenue, New York, 10016, New York, USA Cheuk Hei Ho Faculty of Biology, Johannes Gutenberg University of Mainz, Mainz, 55128, Germany Miguel A. Andrade-Navarro Authors Tina Lence View author publications Search author on: PubMed Google Scholar Junaid Akhtar View author publications Search author on: PubMed Google Scholar Marc Bayer View author publications Search author on: PubMed Google Scholar Katharina Schmid View author publications Search author on: PubMed Google Scholar Laura Spindler View author publications Search author on: PubMed Google Scholar Cheuk Hei Ho View author publications Search author on: PubMed Google Scholar Nastasja Kreim View author publications Search author on: PubMed Google Scholar Miguel A. Andrade-Navarro View author publications Search author on: PubMed Google Scholar Burkhard Poeck View author publications Search author on: PubMed Google Scholar Mark Helm View author publications Search author on: PubMed Google Scholar Jean-Yves Roignant View author publications Search author on: PubMed Google Scholar 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 m 6 A 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. Corresponding author Correspondence to Jean-Yves Roignant . Ethics declarations Competing interests The authors declare no competing financial interests. Extended data figures and tables Extended Data Figure 1 Characterization of the Drosophila m 6 A methyltransferase complex. a , Calibration curve for m 6 A nucleoside versus stable isotope labelled internal standard from digested Escherichia coli RNA. Areas under the curve (AUCs) are taken from MS/MS chromatograms. Amounts of 1–500 fmol m 6 A were evaluated. b , Calibration curve for external calibration of adenosine (A) nucleoside from 2–500 pmol. AUCs are extracted from chromatograms generated by ultraviolet detection. c , Phylogenetic analysis of METTL3 homologues. Each Drosophila ( D.m ) sequence clusters with the corresponding human ( H.s ), Danio rerio ( D.r ) and fungal orthologue. Fungi probably lost ancestral versions of individual methyltransferases with these families, with Schizosaccharomyces pombe ( S.p ) keeping only one orthologue (METTL4) and Saccharomyces cerevisiae ( S.c ) keeping the two other (METTL3 and METTL14). METTL3, METTL14 and METTL4 orthologues are indicated in green, blue and purple, respectively. See Methods for details about the tree construction. d , Box plots of average expression (rpkm) for all genes expressed by at least 1 rpkm in different conditions. Red dots indicate the position of m 6 A components in comparison to other expressed genes. Bottom, relative expression of target genes upon different knockdowns (KDs). The mean ± s.d. of three technical measurements from three biological replicates is shown. e , Relative vir mRNA expression and levels of m 6 A in mRNA during Drosophila development. Number of hours post-fertilization for different embryo, larval and pupal stages is indicated on the x axis. vir expression correlates with m 6 A levels. The mean ± s.d. of three technical measurements from three biological replicates is shown. f , LC–MS/MS quantification of m 6 A levels in either control samples or in mRNA extracts depleted for the indicated proteins. Vir depletion affects m 6 A levels to the same extent as Fl(2)d knockdown. The mean ± s.d. of three technical measurements from three biological replicates is shown for fl(2)d and mean ± s.d. of three technical measurements from two biological replicates for vir . g , Co-immunoprecipitation of Myc–Vir with HA–Ime4 and HA–Fl(2)d. Extracts from S2R+ cells expressing HA-tagged proteins either with Myc alone or with Myc–Vir were immunoprecipitated using Myc-specific beads. Expression of indicated proteins was monitored by western blot analysis using anti-Myc and anti-HA antibodies. RNaseT1 treatment before immunoprecipitation is indicated at the bottom. h , Co-immunoprecipitation studies were carried out with lysates prepared from S2R+ cells co-expressing Myc–dMettl14 and HA–Ime4 upon control (Ctr) or Fl(2)d knockdown. For control experiments, S2R+ cells were transfected with Myc alone and HA–Ime4. Lysates were immunoprecipitated using anti-Myc antibody and then detected with anti-Myc and anti-HA antibodies. Knockdown of Fl(2)d weakens the interaction between Ime4 and dMettl14. i , Western blots showing Ime4 and dMettl14 protein expression levels in extracts from indicated genotypes. Tubulin is used as a loading control. Extended Data Figure 2 m 6 A quantification, MeRIP-seq validation and sequence features of m 6 A sites in Drosophila mRNA. a , Scatter plot of counts per million (CPM) values for intersected MeRIP peaks. The peaks have at least a support of 3 CPM in one of the replicates. b , qPCR validation of MeRIP peaks. Enrichment is calculated over a negative region in the Rpl15 transcript. The mean ± s.d. of three technical measurements from two replicates is shown. c , Sequence motifs enriched in a fraction of m 6 A peaks, analysis performed by Homer. Extended Data Figure 3 Significant fold changes and correspondence between biological replicates of RNA-seq data. a , Average versus mean–difference plots (MA-plots) show the moderated estimation of fold change and dispersion for RNA-seq data in the different knockdown conditions (adjusted P value < 0.05). The significant values are highlighted in red. b , Spearman sample-to-sample correlation based on gene expression profiles. c , Spearman sample-to-sample correlation based on splicing levels. d , Empirical cumulative distribution function (ECDF) plot of fold changes (log2) upon Ime4/dMettl14 double knockdown over control separated between m 6 A targets and non-targets. Values are shown between −0.5 and 0.5. The distributions were compared using Wilcoxon rank sum test ( P value = 9.9 × 10 −4 ). e , Fold change upon Ime4/dMettl14 double knockdown versus control separated into genes without m 6 A peaks (non-targets) or containing m 6 A peaks within the CDS (CDS) or within a 300-bp window around the start or stop codon. Only genes considered for differential expression testing according to DESeq2 default filters are shown. f , Representation of differentially spliced events in the different knockdowns. Selection of 5′ alternative splice sites and increase in intron retention are the two most enriched classes. Classification of splicing changes upon knockdown of the unrelated EJC component eIF4AIII is shown for comparison. Extended Data Figure 4 Gene ontology term enrichment analysis. a–e , Significant GO terms (adjusted P value < 0.05) of differentially expressed genes in Ime4 knockdown ( a ), dMettl14 knockdown ( b ), Fl(2)d knockdown ( c ), CG6422 knockdown ( d ) and YT521-B knockdown ( e ) cells versus control S2R+ cells. Analysis was performed using the Bioconductor package of GOstats. Extended Data Figure 5 m 6 A nuclear components control fl(2)d splicing. a , UCSC Genome Browser screenshots of fl(2)d showing normalized RNA-seq data from control and indicated knockdown samples in S2R+ cells. The gene architecture of fl(2)d is shown at the top, with thin blue boxes representing the 5′ and 3′ UTRs, thick blue boxes representing the CDS, and thin lines representing introns. Exon numbers are indicated at the top. Signals are displayed as RPM. b , Usage of different 5′ splice sites in exon 1 of fl(2)d transcript and skipping of exon 2 upon different knockdowns. Analysis by semi-quantitative RT–PCR using primers in exon 1 and 3 (red arrows in the scheme). Quantification is indicated underneath the gel. ss1, splice site 1; ss2, splice site 2; ss3, splice site3. Extended Data Figure 6 Characterization of Drosophila YTH components. a , Phylogenetic analysis. Sequences from Ustilago hordei (a basidiomycota fungi) were used, in the absence of appropriate sequences from S. cerevisiae , and worked as outliers for each cluster to show the separation between the two major groups. b , Relative expression of YT521-B and CG6422 transcripts and levels of m 6 A in mRNA during Drosophila development. Number of hours post-fertilization for different embryo, larval and pupal stages is indicated on the x axis. The mean ± s.d. of three technical measurements from three biological replicates is shown. c , Dot-blot assay using biotinylated probe from prolactin transcripts with or without m 6 A RNA modification. Protein extracts from S2R+ cells transfected with either Myc–GFP or Myc–YT521-B were analysed for binding specificity to the crosslinked probes. Left, methylene-blue staining of crosslinked probes. Right, immunostaining using anti-Myc or anti-m 6 A antibody. YT521-B protein shows the same enrichment to the methylated probe as anti-m 6 A antibody. d , Pull-down using biotinylated m 6 A probe from prolactin transcripts and protein extracts from S2R+ cells transfected with either Myc–GFP or Myc–YT521-B. The same probe lacking the methylation was used as a negative control. Left, western blot using anti-Myc antibody. Right, dot blot using anti-Strep-HRP antibody. The binding of Myc–YT521-B is increased with the methylated probe. Three independent experiments show similar results. e , Walking behaviour in Buridan’s paradigm in heterozygous and transheterozygous YT521-B mutants. Left, median angular displacements from the direct approach to one of the stripes. Right, median fraction of time spent walking during a 15 min test period (Kruskal–Wallis analysis of variance with a Bonferroni correction). Fifteen female flies per genotype were used in both assays. NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. Extended Data Figure 7 Genetic characterization of Ime4 , dMettl14 and YT521-B . a–c , Top, Ime4 ( a ), dMettl14 ( b ) and YT521-B ( c ) loci with indicated deletions. Bottom, loss of function for Ime4 and dMettl14 were monitored by western blot using respective endogenous antibodies, while anti-Tubulin antibody was used as a loading control. To analyse YT521-B deletion, PCR using genomic DNA from heterozygous or homozygous YT521-B ΔN mutant flies was loaded on agarose gel. d , Scheme of the dMettl14 protein showing the conserved MT-A70 domain. The frameshift position caused by the guide RNA-induced deletions and the molecular nature of the allele are indicated below. e , Representative confocal images of muscle-6/7 NMJ synapses of abdominal hemisegment A2 for the indicated genotypes labelled with anti-DLG (magenta), anti-Synaptotagmin (green) and HRP (red) to reveal the synaptic vesicles and the neuronal membrane. Bottom, quantification of normalized bouton number (total number of boutons/muscle surface area (μm 2 × 1,000)) and normalized Synaptotagmin area (total Synaptotagmin-positive area (μm 2 )/muscle surface area (μm 2 × 1,000)) of NMJ 6/7 in A3 of the indicated genotypes. Error bars show mean ± s.e.m. P values were determined with a Student’s t -test. The number of boutons and of active zones per boutons are increased upon Ime4 knockout. MSA, muscle surface area. Extended Data Figure 8 Ime4 mutant flies have reduced locomotion and shortened lifespan but apparent normal ovarian development. a , Ovarian immunostaining of indicated genotypes. DAPI (blue) stains nucleus, Vasa protein (Vasa) (green) shows the germ cells and Orb protein (Orb) (red) the oocyte. Only one oocyte per egg chamber is seen in control and mutant ovaries, arguing against encapsulation defects. b , Survival curves of adult Drosophila . The lifespan of Ime4 Δcat mutant flies (purple) and Ime4 Δcat mutant flies expressing Ime4 cDNA ubiquitously (green) were quantified for both females and males. c , Walking behaviour in Buridan’s paradigm in Ime4 Δcat mutant flies or Ime4 Δcat mutant flies expressing Ime4 cDNA ubiquitously ( Tub -GAL4), in neurons ( elav -GAL4) or in muscles ( 24B -GAL4). Left, median angular displacements from the direct approach to one of the stripes. Right, median fraction of time spent walking during a 15 min test period. Fifteen female flies per genotype. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 (Kruskal–Wallis analysis of variance with a Bonferroni correction). Extended Data Figure 9 m 6 A components fine-tune the sex determination pathway via YT521-B. a , Quantification of RT–qPCR experiments from RNA extracts of whole females using primers spanning exons 2 and 4 (top), as well as exons 2 and 3 (bottom) to quantify the levels of the Sxl female and male isoforms, respectively. The mean ± s.d. of three technical measurements from two biological replicates is shown. b , Top, msl-2 genome architecture with thin blue boxes representing the 5′ and 3′ UTRs, thick blue boxes representing the CDSs and thin lines representing introns. Arrowheads display the position of primers used for quantification. Bottom, spliced isoforms for msl-2 were monitored by RT–PCR and PCR extracts were loaded on agarose gel. The quantification of three biological replicates is shown below as mean ± s.d. c , Top, tra genome architecture with thin blue boxes representing the 5′ and 3′ UTRs, thick blue boxes representing the CDSs and thin lines representing introns. Arrowheads display the position of primers used for quantification. Bottom, spliced isoforms for tra were monitored by RT–PCR and PCR extracts were loaded on agarose gels. The quantification of three biological replicates is shown below as mean ± s.d. L, long isoform; S, short isoform. d , Table indicating the percentage of males and females hatching for the indicated genotypes. Ime4 interacts genetically with Sxl to control female survival. e , Bar chart showing the number of differentially spliced genes upon knockout (KO) of Ime4 and YT521-B in adult females. f , Venn diagram showing the overlap of targets in the indicated knockout. g , Pie charts showing distribution of splicing events in the different knockout conditions. Intron retention is overrepresented upon knockout of Ime4 and YT521-B in vivo . Extended Data Figure 10 RNA interference screen identifies Nito as a new member of the methyltransferase complex. a , SILAC-coupled mass spectrometry analysis using YT521-B–Myc as a bait. Scatterplot of normalized forward versus inverted reverse experiments plotted on a log 2 scale. The threshold was set to a twofold enrichment (blue dashed line). Proteins in the top right quadrant are enriched in both duplicates. b , mRNA quantification of fl(2)d isoforms after knockdown of potential YT521-B-interacting proteins. Three proteins, Hrb27C, Qkr58E-1 and Nito, in addition to m 6 A components, control fl(2)d splicing in the same direction. Data points of three technical replicates are shown. c–f , mRNA quantification of m 6 A-regulated transcripts including Hairless ( a ), Aldh-III ( b ), CG8929 ( c ), hts ( d ) upon knockdown of indicated components. Nito controls m 6 A splicing events. The quantification of three technical replicates from two biological experiments is shown as mean ± s.d. g , Co-immunoprecipitation studies were carried out with lysates prepared from S2R+ cells co-expressing Myc–Qkr58E-1, Myc–Hrb27C and HA–YT521-B. For control, S2R+ cells were transfected with Myc alone and HA–YT521-B. Myc-containing proteins were immunoprecipitated using a Myc antibody and then immunoblotted with anti-Myc and anti-HA antibodies. h , Co-immunoprecipitation of Myc–Qkr58E-1 with HA–YT521-B with or without RNaseT1. Extracts from S2R+ cells expressing HA–YT521-B either with Myc control or with Myc–Qkr58E-1 were immunoprecipitated using Myc-specific beads. Expression of indicated proteins was monitored by immunoblotting using anti-Myc and anti-HA antibodies. i , Relative nito mRNA expression and levels of m 6 A in mRNA during Drosophila development. Number of hours post-fertilization for different embryo, larval and pupal stages is indicated on the x axis. nito expression correlates with m 6 A levels. The mean ± s.d. of three technical measurements from three biological replicates are shown. j , Relative expression of indicated transcripts upon control, Nito and Vir knockdown. Vir and Nito knockdowns do not reduce expression of other components of the methlytransferase complex. The mean ± s.d. of three technical measurements from two biological replicates is shown. Supplementary information Supplementary Figure 1 (download PDF ) Uncropped scans with protein size indications in kDa and DNA size indicators in bp. (PDF 5074 kb) Supplementary Table 1 (download XLS ) Analysis of the transcriptome wide m6A profile in S2R+ cells. (XLS 612 kb) Supplementary Table 2 (download XLS ) Fl(2)d KD versus LacZ KD in S2R+ cells. (XLS 626 kb) Supplementary Table 3 (download XLS ) Ime4 KD versus LacZ KD in S2R+ cells (XLS 164 kb) Supplementary Table 4 (download XLS ) Ime4/dMettl14 KD versus LacZ KD in S2R+ cells. (XLS 369 kb) Supplementary Table 5 (download XLS ) dMettl14 KD versus LacZ KD in S2R+ cells. (XLS 92 kb) Supplementary Table 6 (download XLS ) Ime4 KO (ImeDcat) in adult heads of 1-2 day old females. (XLS 455 kb) Supplementary Table 7 (download XLSX ) Comparison of transcripts affected upon Ime4 KO in adult heads with transcripts containing m6A peak(s) in S2R+ cells. (XLSX 64 kb) Supplementary Table 8 (download XLSX ) Candidate proteins enriched by 2-fold in the YT521-Myc IP pull down in S2R+ cells. (XLSX 84 kb) Supplementary Table 9 (download XLSX ) Primers used for dsRNA synthesis. (XLSX 102 kb) Supplementary Table 10 (download XLSX ) Mass transitions and QQQ parameters of the monitored modifications. (XLSX 33 kb) PowerPoint slides PowerPoint slide for Fig. 1 (download PPT ) PowerPoint slide for Fig. 2 (download PPT ) PowerPoint slide for Fig. 3 (download PPT ) PowerPoint slide for Fig. 4 (download PPT ) PowerPoint slide for Fig. 5 (download PPT ) Rights and permissions Reprints and permissions About this article Cite this article Lence, T., Akhtar, J., Bayer, M. et al. m 6 A modulates neuronal functions and sex determination in Drosophila . Nature 540 , 242–247 (2016). https://doi.org/10.1038/nature20568 Download citation Received : 24 March 2016 Accepted : 21 October 2016 Published : 30 November 2016 Issue date : 08 December 2016 DOI : https://doi.org/10.1038/nature20568 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy shareable link to clipboard Provided by the Springer Nature SharedIt content-sharing initiative Access through your institution Buy or subscribe Editorial Summary m 6 A modulates neural activity One of the most pervasive modifications found in messenger RNAs is N 6 -methyladenosine (m 6 A). Complementary studies from the laboratories of Jean-Yves Roignant and Matthias Soller describe how this modification can affect Drosophila neuronal function and behaviour. They identify the complex that catalyses this modification, as well as proteins that recognize it. They find that the modification affects alternative splicing of the Sex-lethal transcript, amongst other genes, and therefore alters gene expression during sex determination. show all Associated content m<sup>6</sup>A potentiates <i>Sxl</i> alternative pre-mRNA splicing for robust <i>Drosophila</i> sex determination Irmgard U. 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