GSE97004
The m6A pathway facilitates sex determination in Drosophila
N6-methyladenosine (m6A) has recently emerged as a widespread and conserved RNA modification that modulates messenger RNA (mRNA) processing and activity. As most m6A studies have been conducted in cultured cells, we established Drosophila as a model system that combines molecular and genomic analyses of m6A with investigations of organismal biology. We first apply miCLIP sequencing to map m6A modifications at single-nucleotide resolution during Drosophila embryogenesis. We next characterize Drosophila components of the m6A "writer" methyltransferase complex [MTC factors, METTL3/IME4, METTL14, FL(2)D and Nito], and characterize m6A-binding properties of the YTH domain-containing proteins, YT521-B and CG6422. We complement this by generating a collection of mutants in m6A writer and reader components. While Drosophila orthologs of mammalian MTC factors that have additional roles in splicing are lethal, our mutants in METTL and YTH factors are viable but present specific developmental and behavioral defects. We note that several mammalian m6A factors were originally identified as Drosophila regulators of Sxl splicing, which generates the master determinant of female identity. While our mutants in dedicated writer and reader machinery are not required to accumulate Sxl protein in the ovary, maternal loss of m6A writers and readers collaborates with reduction of Sxl to induce female lethality. Our data indicate Sxl is directly regulated by the m6A pathway, since miCLIP data show Sxl is a substantial target of intronic m6A in early embryos, in addition to bearing exonic m6A. Consistent with this, female-specific Sxl splicing is defective in m6A pathway mutants. YT521-B appears to be the major effector of m6A for Sxl regulation, as it shows much stronger genetic interactions with Sxl than CG6422 and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit for studying the m6A pathway in Drosophila reveals a major biological utilization in establishing sex-specific splicing. Overall design: UV-crosslinking and immunoprecipitation with m6A-antibodies was used to map m6A sites with single nucleotide resolution