The mA pathway facilitates sex determination in Drosophila.

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