Plasmodium falciparum accounts for 95% of the nearly 600,000 estimated deaths from malaria annually. Multiple rounds of asexual replication during the intra-erythrocytic developmental cycle (IDC) stage of P. falciparum results in the pathology associated with malaria. Each cycle of the IDC is approximately 48 hours and begins with the parasite invading a new erythrocyte. Completion of the IDC requires precise regulation of gene expression. mRNA modifications — such as N6‑methyladenosine (m6A) — are known to regulate mRNA export, abundance and translational efficiency in yeast and mammalian cells.
Like other eukaryotes m6A is the most prevalent modification in P. falciparum mRNA. Adenosine is converted to m6A by the nuclear protein complex known as the m6A writer. Orthologs of the human writer components such as METTL3 (PF3D7_0729500; annotated as PfMT-A70 in P. falciparum genomes) have been detected in P. falciparum. In our lab, we use the rapamycin inducible knock sideways system to mislocalise PfMT-A70 away from the nucleus to the parasite plasma membrane to study the effects of m6A.
Using nanopore direct RNA sequencing we have observed that knock sideways of PfMT-A70 depletes m6A in mRNA and changes in the abundance of some mRNA. The consequence of this altered mRNA biology on protein synthesis has not been studied. The aim of this study was to study the effects of PfMT-A70 knock sideways on protein abundance using label‑free quantitative proteomics.
Parasites that were 28±2 (28HPI) and 36±2 (36HPI) hours post invasion of a new erythrocyte were used for the analysis. Peptides for 1,548 and 2,117 were detected for the 28HPI and 36HPI time-points respectively. A small proportion of these (6.9% and 11.8% for each time-point respectively) had statistically significant changes to their abundance following 8 hours of knock sideways.