5-methylcytosine modification by <i>Plasmodium</i> NSUN2 stabilizes mRNA and mediates the development of gametocytes.

Abstract

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 <i>Plasmodium</i> 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 (<i>Plasmodium yoelii</i>) and human (<i>Plasmodium falciparum</i>) 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 <i>Plasmodium</i> transcripts and that a <i>Plasmodium</i> ortholog of NSUN2 is a major mRNA m⁵C methyltransferase in malaria parasites. Upon knockout of <i>P. yoelii nsun2</i> (<i>pynsun2</i>), 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 <i>nsun2</i> 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 <i>Plasmodium</i>, our findings demonstrate a major role for NSUN2-mediated m⁵C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.