The role of m6A modification on mRNA processing in Drosophila melanogaster
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Abstract
Over 170 RNA modifications can decorate various RNA molecules and represent one of the layers of gene regulation. N6-methyladenosine (m6A) is among the most prevalent modifications in eukaryotic mRNA and is implicated in nearly every step of mRNA biogenesis. m6A installation is accomplished by a large protein complex, whose exact composition was long unknown. This modification can be recognised by the so-called YTH domain-containing m6A reader proteins that are main mediators of m6A functions. Using molecular biology techniques and high-throughput sequencing experiments this study shows that the m6A writer complex in D. melanogaster consists of seven subunits that are conserved in higher eukaryotes. Methyltransferase-like protein 3 (Mettl3) carries catalytic activity and forms a heterodimer with the Methyltransferase-like protein 14 (Mettl14). In addition, five accessory proteins were found to be required for efficient target methylation: Fl(2)d, Vir, Nito, Flacc, and Hakai. Flacc was shown to promote the interaction between Fl(2)d and Nito, whereas Hakai maintained the stability of Vir, Fl(2)d and Flacc. Analysis of m6A distribution along mRNA revealed enrichment of modification within 5` UTR regions in an A-rich RRACH sequence motif. Functionally, loss of m6A altered alternative splicing. Among aberrantly spliced transcripts was Sex lethal (Sxl), the master regulator of sex determination and dosage compensation pathways in D. melanogaster, which implicated m6A in the modulation of these processes. Characterisation of m6A readers identified the nuclear protein Ythdc1 as one of the key mediators of m6A functions in D. melanogaster. By exploring the importance of m6A modification during fly development, this work revealed high levels of m6A during early embryogenesis, at the onset of pupation, as well as in heads and ovaries of adult flies. Notably, fly mutants lacking Mettl3 or Mettl14 had reduced lifespan and were flightless. In addition, flies lacking m6A displayed severe locomotion defects due to altered neuronal functions and loss of Ythdc1 resembled the loss of m6A writer components.