Screening of <i>Drosophila melanogaster</i> RNA m⁶A modification pathway factors.

<i>N</i>⁶-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 <i>Ythdc1</i> was overexpressed in <i>Drosophila melanogaster</i> eye imaginal discs. Our results showed that overexpression of <i>Ythdc1</i> 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 <i>Drosophila</i> strain overexpressing <i>Ythdc1</i> 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.