Harnessing RNA modifications in therapeutics has enabled breakthroughs in vaccines. Despite their importance in RNA stability and translation, the mechanisms of RNA modifications are poorly understood. N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotes and changes translational efficiency or reduces mRNA stability. We used a knock-sideways system to disrupt the methyltransferase which writes m6A to RNA in the human malaria parasite Plasmodium falciparum. 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 RNA-sequencing to study differential methylation at multiple points during the P. falciparum lifecycle.
We were able to detect 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. Nanopore direct RNA sequencing targets mRNA, although we also detected other transcript types including rRNAs, snoRNAs and tRNAs. Methyltransferases preferentially write m6A to RRACH motifs, yet we detected non-RRACH methylation. Conserved modification patterns can be representative of biological significance. We found the majority of m6A occurred in regions where there were no conserved patterns, and some RRACH motifs were consistently methylated while others were not. We detected genes that were differentially expressed after disrupting the methyltransferase, however there was no clear link between differentially expressed and differentially methylated transcripts.
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.