Harnessing RNA modifications in therapeutics has enabled major breakthroughs in vaccine development. Despite their importance in RNA stability and translation, the mechanisms of RNA modifications are poorly understood. N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes and changes translational efficiency or reduces mRNA stability. We studied this modification in Plasmodium falciparum by disrupting the methyltransferase that makes m6A with a knock-sideways mislocalisation system. Recent developments in sequencing by Oxford Nanopore Technologies (ONT) have enabled the direct detection of m6A in RNA. We therefore disrupted the methyltransferase and used Nanopore direct RNA-sequencing to study differential methylation at multiple points during the P. falciparum asexual lifecycle.
We detected differentially methylated transcripts after mislocalising the methyltransferase, confirming the utility of both the knock-sideways system and Nanopore RNA-sequencing in studying m6A in P. falciparum. Moreover, we observed differential gene expression after disrupting the methyltransferase and found removing m6A affected the 3’ UTR length in transcripts. We observed that different genes have varying numbers of canonical m6A sites, which were positionally associated with polyadenylation sites. Our work shows Nanopore RNA-seq can be used to detect m6A abundance and location in P. falciparum, proving it a valuable technique for studying the impact of RNA modifications on parasite biology.