m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma 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. Knockdown of the m6A writer components METTL3 and WTAP resulted in diminished m6A marks 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. Loss of METTL3, WTAP, or YTH1 led to a defect in transcript 3’-end formation. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3’-ends.

Epigenetic and Epitranscriptomic Gene Regulation in Plasmodium falciparum and How We Can Use It against Malaria

“…This contrasts with what has been described in other apicomplexan parasites, like Toxoplasma gondii , in which the m6A modification is in the 3′ end of the mRNA. In that parasite, this modification has been linked to mRNA maturation and 3′ end polyadenylation [ 140 , 141 ]. Another modification, the mC5 has been associated to transcript stabilisation in gametocytes.…”

Epitranscriptomics in parasitic protists: Role of RNA chemical modifications in posttranscriptional gene regulation

“…falciparum , the mammalian ortholog of METTL3 has been identified and designated as PfMT-A70 (PF3D7_0729500) [ 70 ]. The writer complex in this organism has not been characterized as well as in other protozoan species like Toxoplasma , in which the additional proteins VIRMA and TGGTI_275990 have been identified [ 71 ]. HHPHRED webservers have made strong homology associations between WTAP and TGGTI_275990 although it remains unannotated [ 71 , 72 ].…”

“…Compelling experiments performed by independent groups further explored m 6 A’s importance in the viability of transcripts facilitated by appropriate 3′ end processing in T . gondii [ 71 , 94 ]. A knockdown study of proteins paramount for m 6 A installment (METTL3 and WTAP) resulted in a complete arrest of parasite replication and impairs appropriate 3′ end formation, which makes perfect sense being that m 6 A distribution along the transcriptome was primarily found to reside near 3′ transcript ends [ 71 ].…”

Epitranscriptome profiling of spleen mRNA m6A methylation reveals pathways of host responses to malaria parasite infection

“…In addition to Plasmodium , m 6 A enrichment was reported in 342 transcripts of Trypanosoma brucei including transcripts encoding variant surface glycoproteins that are essential for the survival of the parasites ( 22 ). In Toxoplasma gondii , m 6 A has been reported as a critical mRNA modification widespread across multiple stages of the parasite’s life cycle and is essential for parasite viability and development ( 23 , 24 ). Emerging evidence has provided new insights into the roles of RNA epitranscriptome in parasite biology and the treatment of the associated diseases ( 25 ).…”