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

Abstract

Abstract The protozoan parasite Toxoplasma gondii is thought to rely on RNA processing to accomplish the differential gene expression needed during life cycle stage transitions. Here, we show how RNA capping, the first major pre-mRNA processing event, safeguards transcript homeostasis in Toxoplasma. A functional RNA capping system of Toxoplasma consists of separate RNA triphosphatase, guanylyltransferase, and guanine-N7-methyltransferase enzymes, which together add 5’ 7-methylguanosine (m7G) cap to RNA. The in vitro generated capped RNAs bind to the Toxoplasma translation initiator factor, eIF4E, and are translated to protein in the transfected parasites. Biochemical and genetic characterization demonstrates that among three capping enzymes, triphosphatase (TgRT) is unique and a member of the tunnel family of metal-dependent phosphohydrolases, structurally and mechanistically unrelated to the human cysteine-phosphatase-type RNA triphosphatase. We show that TgRT is essential for pre-mRNA capping and parasite growth through inducible conditional knockdown. TgRT perturbation leads to global diminished m7G-capped transcripts, as demonstrated by cap-seq, which resulted in the complete arrest of parasite replication in the culture and the mouse host, protecting them from lethal infection. Overall, this study shows the essential role of TgRT-mediated mRNA capping for parasite survival, thereby presenting RNA triphosphatase as an attractive target for Toxoplasma infection.