m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination

N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins1-7. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that include METTL3 (Methyltransferase-like 3), METTL14, WTAP (Wilms tumour 1 associated protein) and KIAA1429. Drosophila has corresponding homologues named dIME4 and dKAR4 (Inducer of meiosis-4 and Karyogamy protein-4), and Female-lethal(2)d (Fl(2)d) and Virilizer (Vir)8-12. In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing (AS) of the sex determination factor Sex-lethal (Sxl)13. However, the functions of m6A in introns in the regulation of AS remain uncertain3. Here we show that m6A is absent in mRNA of Drosophila lacking dIME4. In contrast to mouse and plant knock-out models5,7,14, Drosophila dIME4 null mutants remain viable, though flightless and show a sex bias towards maleness. This is because m6A is required for female-specific AS of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal2 (msl-2) to prevent dosage compensation normally occurring in males. We further show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies dIME4 mutants. Loss of m6A also affects AS of additional genes, predominantly in the 5’UTR, and has global impacts on the expression of metabolic genes. Requirement of m6A and its reader YT521-B for female-specific Sxl AS reveal that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.