m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability ofToxoplasma gondii

Abstract

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3’-boundary of mRNAs within the consensus sequence, YGCAUGCR. Knockdown of the m6A writer enzyme METTL3 resulted in diminished m6A marks, loss of a target transcript, and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3’-end processing of pre-mRNAs. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3’-ends.Author SummaryToxoplasma gondii is a parasite of medical importance that causes disease upon immuno-suppression. Uncovering essential pathways that the parasite uses for its basic biological processes may reveal opportunities for new anti-parasitic drug therapies. Here, we describe the machinery that Toxoplasma uses to modify specific adenosine residues within its messenger RNAs (mRNA) by N6-adenosine methylation (m6A). We discovered that m6A mRNA methylation is prevalent in multiple stages of the parasite life cycle and is required for parasite replication. We also establish that m6A plays a major role in the proper maturation of mRNA. Two proteins that bind m6A modifications on mRNA associate with factors responsible for the cleavage and final processing steps of mRNA maturation. Since all of the machinery is conserved from plants to Toxoplasma and other related parasites, we propose that this system operates similarly in these organisms.

A potent HDAC inhibitor blocks Toxoplasma gondii tachyzoite growth and profoundly disrupts parasite gene expression

“…T. gondii has been shown to lack detectable DNA methylation at the tachyzoite stage [31]. Therefore, it is most likely that this inhibitor at this high concentration may target other enzymatic activities, such as RNA methylation, that have been shown to be essential for parasite survival [32,33]. The cytotoxic effect on host cells at 10 µM (Table 2) may also explain the results presented in Figure 1 for these two compounds.…”