The m6A pathway facilitates sex determination in Drosophila

The conserved modification N-6-methyladenosine (m(6)A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m(6)A pathway. We first apply miCLIP to map m(6)A across embryogenesis, characterize its m(6)A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m(6)A factors. While m(6)A factors with additional roles in splicing are lethal, m(6)A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m(6)A facilitates the master female determinant Sxl, since multiple m(6)A components enhance female lethality in Sxl sensitized backgrounds. The m(6)A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m(6)A target, and female-specific Sxl splicing is compromised in multiple m(6)A pathway mutants. YT521-B is a dominant m(6)A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m(6)A pathway.