Abstract
N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. 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 and human cells. 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 dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.
Highlights
N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions
M6A on mRNA is deposited co-transcriptionally by a conserved multiprotein complex that can be divided into two stable sub-complexes: the heterodimer METTL3/METTL14 known as m6A-METTL Complex (MAC) that contains the catalytic activity, and the m6A-METTL Associated Complex (MACOM)
We recently showed that two conserved sub-complexes, MAC and MACOM interact to deposit m6A on mRNA in flies and mice[24]
Summary
N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. 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. We show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. M6A on mRNA is deposited co-transcriptionally by a conserved multiprotein complex that can be divided into two stable sub-complexes: the heterodimer METTL3/METTL14 known as m6A-METTL Complex (MAC) that contains the catalytic activity, and the m6A-METTL Associated Complex (MACOM). HAKAI was identified as one of the strongest WTAP interactors in mammalian cells[38] and as part of an evolutionary conserved protein complex including WTAP, VIRMA and ZC3H1339. We identified Hakai among the top enriched proteins in our Spenito (Nito) interactome in Drosophila S2R+ cells, suggesting its evolutionary conserved role within the m6A pathway[24]
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