Fig. 2. Hakai is required to maintain proper levels of m⁶A methylation.

a Flybase JBrowse view of the Hakai gene locus. Hakai has four transcripts due to alternative splicing that generates two long and two short protein isoforms. The positions of three independent shRNAs, Hakai sgRNA, and the protein region used to generate a Hakai antibody are indicated. b Sequencing results showing frameshift indels in Hakai^SH2 and Hakai^SH4 flies generated by CRISPR/Cas9-mediated mutagenesis. The targeted genomic DNA sequence is underlined and the NGG PAM sequence is in bold type. c–c” Hakai and Fl(2)d antibody staining in WT wing discs showing a high degree of co-localization. Hakai antibody staining was strongly reduced in Hakai^SH2 (d) or Hakai^SH4 (e) homozygous mutant wing discs. The experiments in c–e were repeated at least twice independently with similar results, and each time around 30 wing discs for any genotype were examined. Scale bars: 10 μm. f Quantifications of m⁶A relative to A in mRNA extracted from male adult flies by LC-MS. Compared to yw and w¹¹¹⁸ controls, m⁶A levels dropped to about 30% in Mettl3 or Mettl14 mutants and to <50% in Hakai^SH2 or Hakai^SH4 mutants. g Quantifications of m⁶A/A, m¹A/A, m⁵C/C, and ac⁴C/C levels in mRNA extracted from yw and w¹¹¹⁸ male flies, Drosophila S2 cells, and human HeLa cells. Note the substantial difference between fly and human m⁶A levels. f, g Data are presented as mean ± SD from three biological replicates. Source data are provided as a Source Data file.