Screening of Drosophila melanogaster RNA m⁶A modification pathway factors

N⁶-methyladenosine (m⁶A), one of the most prevalent mRNA modifications, plays crucial roles during animal and plant development and in various physiological and pathological processes. Previous studies have characterized m⁶A methyltransferase complexes, demethylases, and m⁶A-binding proteins, but as a relatively new epitranscriptomic pathway, it is likely that new m⁶A components remain to be discovered. To explore the effects of m⁶A modification on tissues and organs, the m⁶A reader Ythdc1 was overexpressed in Drosophila melanogaster eye imaginal discs. Our results showed that overexpression of Ythdc1 leads to ectopic expression of Sxl in males, the rough eye in both males and females, and the activation of JNK signaling and apoptotic pathway. In order to screen m⁶A modifiers using the rough eye phenotype, a stable Drosophila strain overexpressing Ythdc1 was further constructed. By screening of more than 1,500 RNAi lines, several repressors and enhancers that may be involved in m⁶A modification were successfully identified. These genes are less studied in m⁶A pathway, and therefore we further verified them and conducted preliminary mechanistic analyses on them. In summary, this study identified multiple potential factors of the m⁶A modification pathway, expanded our understanding of the m⁶A modification network, and provided ideas and directions for exploring new regulatory mechanisms of this important pathway.