The m⁶A pathway facilitates sex determination in Drosophila.

The conserved modification N⁶-methyladenosine (m⁶A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m⁶A pathway. We first apply miCLIP to map m⁶A across embryogenesis, characterize its m⁶A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m⁶A factors. While m⁶A factors with additional roles in splicing are lethal, m⁶A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m⁶A facilitates the master female determinant Sxl, since multiple m⁶A components enhance female lethality in Sxl sensitized backgrounds. The m⁶A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m⁶A target, and female-specific Sxl splicing is compromised in multiple m⁶A pathway mutants. YT521-B is a dominant m⁶A 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 m⁶A pathway.

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