5-methylcytosine (m⁵C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m⁵C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m⁵C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (Plasmodium yoelii) and human (Plasmodium falciparum) malaria parasites. While different representations of m⁵C-modified mRNAs are associated with the different stages, the abundance of the m⁵C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m⁵C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m⁵C methyltransferase in malaria parasites. Upon knockout of P. yoelii nsun2 (pynsun2), marked reductions of m⁵C 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 nsun2 gene in the knockout parasites rescued the gametocyte production phenotype as well as m⁵C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m⁵C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m⁵C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.

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