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 N 6-methyladenosine (m 6 A) over the course of blood-stage development. Using mass spectrometry and m 6 A RNA sequencing, we demonstrate that m 6 A is highly developmentally regulated, exceeding m 6 A levels known in any other eukaryote. We characterize a distinct m 6 A writer complex and show that knockdown of the putative m 6 A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m 6 A. In accordance, we find an inverse correlation between m 6 A methylation and mRNA stability or translational efficiency. We further identify two putative m 6 Abinding 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 m 6 A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.

Erythroid cells, specifically red blood cells (RBCs), are constantly exposed to highly reactive radicals during cellular gaseous exchange. Such exposure often exceeds the cells' innate anti-oxidant defense systems, leading to progressive damage and eventual senescence. One of the contributing factors to this process are alterations to hemoglobin conformation and globin binding to red cell cytoskeleton. However, in addition to the aforementioned changes, it is possible that oxidative damage induces critical changes to the erythrocyte cytoskeleton and corresponding bio-mechanical and nano-structural properties of the red cell membrane. To quantitatively characterize how oxidative damage accounts for such changes, we employed single-cell manipulation techniques such as micropipette aspiration and atomic force microscopy (AFM) on RBCs. These investigations demonstrated visible morphological changes upon chemically induced oxidative damage (using hydrogen peroxide, diamide, primaquine bisphosphate and cumene hydroperoxide). Our results provide previously unavailable observations on remarkable changes in red cell cytoskeletal architecture and membrane stiffness due to oxidative damage. Furthermore, we also demonstrate that a pathogen that infects human blood cells, Plasmodium falciparum was unable to penetrate through the oxidant-exposed RBCs that have damaged cytoskeleton and stiffer membranes. This indicates the importance of bio-physical factors pertinent to aged RBCs and it's relevance to malaria infectivity.

Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR‐based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 (“dCas9”) was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl‐CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene‐associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin‐associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.