RNA triphosphatase-mediated mRNA capping is essential for maintaining transcript homeostasis and the survival of Toxoplasma gondii.

RNA modifications are crucial for gene expression in eukaryotes; however, the regulatory role of 5' 7-methylguanosine (mG) cap, the first modification of mRNA, remains unknown in the protozoan parasite Toxoplasma gondii. Here, we show that the mRNA capping machinery of Toxoplasma consists of three distinct enzymes: RNA triphosphatase, guanylyltransferase, and guanine-N7-methyltransferase, which together add mG cap to RNA, recognized by cap-binding protein, TgeIF4E. Biochemical and genetic studies show that among three capping enzymes, RNA triphosphatase (TgCet) is unique and a member of the tunnel family of metal-dependent phosphohydrolases, which is structurally and mechanistically distinct from the human RNA triphosphatase. Using conditional knockdown, we show that TgCet is essential for mRNA capping, and its depletion generates widespread changes in mG-capped transcripts, resulting in the complete arrest of parasite replication both in culture and in mouse host, thereby protecting them from lethal infection. Finally, the therapeutic potential of TgCet was evaluated using two compounds, Myricetin and 3,4-dicaffeoylquinic acid, reported to inhibit Trypanosoma Cet enzyme. However, only Myricetin demonstrated selective inhibition of TgCet activity and effectively blocked parasite growth in culture. Overall, this study highlights the essential role of TgCet-mediated mRNA capping, establishing RNA triphosphatase as a promising drug target for Toxoplasma infection.