title: m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.
abstract: N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that includes METTL3 (methyltransferase-like 3), METTL14, WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. Here we show that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. We further show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.|School of Life Science, Faculty of Health and Life Sciences, Coventry University, Coventry CV1 5FB, UK.
authors.forename: Irmgard U
authors.initials: IU
authors.lastname: Haussmann
authors.affiliation: School of Biosciences, Plant Science Division, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Bodi
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Sanchez-Moran
authors.affiliation: School of Veterinary Medicine and Sciences, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Mongan
authors.affiliation: School of Biosciences, Plant Science Division, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK.
authors.forename: Nathan
authors.initials: N
authors.lastname: Archer
authors.affiliation: School of Biosciences, Plant Science Division, University of Nottingham, Sutton Bonington, Loughborough LE12 5RD, UK.
authors.forename: Rupert G
authors.initials: RG
authors.lastname: Fray
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Matthias
authors.initials: M
authors.lastname: Soller
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D004303:Dosage Compensation, Genetic; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D005260:Female; D007438:Introns / Q000235:genetics; D008297:Male; D008780:Methyltransferases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*; D012727:Sex Characteristics; D019849:Sex Determination Processes / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C067777:Sxl protein, Drosophila; D014157:Transcription Factors; C000616957:Ythdc1 protein, Drosophila; C094166:msl-2 protein, Drosophila; C010223:N-methyladenosine; D008780:Methyltransferases; C000616958:Mettl3 protein, Drosophila; D000241:Adenosine