Hakai is required for stabilizing core components of the m6A mRNA methylation machinery

N6-methyladenosine RNA (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions, including circadian clock, metabolism and embryonic stem cell differentiation. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila. Its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerisation and interaction with other members of the m6A machinery, while its catalytic activity seems dispensable. Finally, we demonstrate that the loss of Hakai destabilizes the level of several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Thus, our work adds new functional and molecular insights into the mechanism of the m6A mRNA writer complex.

N6-甲基腺嘌呤RNA（m6A）是信使RNA（messenger RNA, mRNA）上含量最为丰富的内部修饰，可影响信使RNA代谢的几乎所有步骤，并参与包括生物钟调控、代谢过程以及胚胎干细胞分化在内的多种生物学功能。此前已有研究在植物与哺乳动物细胞中发现，E3泛素连接酶（E3 ubiquitin ligase）Hakai可与m6A甲基化复合物的组分形成复合物，但其确切功能仍有待进一步探究。本研究证实，Hakai是果蝇（Drosophila）中甲基转移酶复合物的保守组分。敲低Hakai会导致m6A水平降低，并改变依赖m6A的生物学功能，其中包括性别决定。研究表明，Hakai的泛素化结构域是其发生二聚化以及与m6A甲基化复合物其他成员相互作用所必需的，而其催化活性似乎并非不可或缺。最后，本研究证实，Hakai的缺失会使甲基转移酶复合物的多个亚基稳定性受损，进而导致其蛋白水平降低，最终造成m6A修饰的沉积障碍。综上，本研究为m6A信使RNA写入复合物的作用机制提供了全新的功能与分子层面的研究见解。