Role of Hakai in m⁶A modification pathway in Drosophila

N6-methyladenosine (m⁶A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. Here we show that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m⁶A writer components, and Hakai mutants exhibit typical m⁶A pathway defects in Drosophila, such as lowered m⁶A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m⁶A modification is mostly distributed in 5' UTRs in Drosophila, in contrast to the mammalian system. Interestingly, we demonstrate that m⁶A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m⁶A writer. Together, our work not only advances the understanding of mechanism and regulation of the m⁶A writer complex, but also provides insights into how Sxl cooperate with the m⁶A pathway to control its own splicing.