5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes

5-methylcytosine (m C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of parasites. Here, we characterize the landscape of m C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent ( ) and human ( ) malaria parasites. While different representations of m C-modified mRNAs are associated with the different stages, the abundance of the m C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m C modifications confer stability to the transcripts and that a ortholog of NSUN2 is a major mRNA m C methyltransferase in malaria parasites. Upon knockout of ( ), marked reductions of m C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the gene in the knockout parasites rescued the gametocyte production phenotype as well as m C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m C profiles for two species of , our findings demonstrate a major role for NSUN2-mediated m C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.