Abstract
Malaria pathogenesis results from the asexual replication of Plasmodium falciparum within human red blood cells, which relies on a precisely timed cascade of gene expression over a 48-h life cycle. Although substantial post-transcriptional regulation of this hardwired program has been observed, it remains unclear how these processes are mediated on a transcriptome-wide level. To this end, we identified mRNA modifications in the P. falciparum transcriptome and performed a comprehensive characterization of N6-methyladenosine (m6A) over the course of blood-stage development. Using mass spectrometry and m6A RNA sequencing, we demonstrate that m6A is highly developmentally regulated, exceeding m6A levels known in any other eukaryote. We characterize a distinct m6A writer complex and show that knockdown of the putative m6A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m6A. In accordance, we find an inverse correlation between m6A methylation and mRNA stability or translational efficiency. We further identify two putative m6A-binding YTH proteins that are likely to be involved in the regulation of these processes across the parasite's life cycle. Our data demonstrate unique features of an extensive m6A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.