Phosphoproteomics of ATR Signaling in Prophase I of Mouse Meiosis

During mammalian meiosis, the ATR kinase plays crucial roles in the coordination of DNA repair, meiotic sex chromosome inactivation and checkpoint signaling. Despite the importance of ATR in meiosis, the meiotic ATR signaling network remains largely unknown. Here we defined ATR signaling during prophase I in mice. Quantitative analysis of phosphoproteomes obtained after genetic ablation of the ATR-activating 9-1-1 complex or chemical inhibition of ATR revealed over 12,000 phosphorylation sites, of which 863 phosphorylation sites were dependent on both 9-1-1 and ATR. ATR and 9-1-1-dependent signaling was enriched for S/T-Q and S/T-X-X-K motifs and included proteins involved in DNA damage signaling, DNA repair, and piRNA and mRNA metabolism. We find that ATR targets the RNA processing factors SETX and RANBP3 and regulate their localization to the sex body. Overall, our analysis establishes a comprehensive map of ATR signaling in spermatocytes and highlights potential meiotic-specific actions of ATR during prophase I.