Nucleotide-resolution Mapping of RNA N6-Methyladenosine (m6A) modifications and comprehensive analysis of global polyadenylation events in mRNA 3' end processing in malaria pathogen Plasmodium falciparum.

Plasmodium falciparum is an obligate human parasite of the phylum Apicomplexa and is the causative agent of the most lethal form of human malaria. Although N6-methyladenosine modification is thought to be one of the major post-transcriptional regulatory mechanisms for stage-specific gene expression in apicomplexan parasites, the precise base position of m6A in mRNAs or noncoding RNAs in these parasites remains unknown. Here, we report global nucleotide-resolution mapping of m6A residues in P. falciparum using DART-seq technology, which quantitatively displayed a stage-specific, dynamic distribution pattern with enrichment near mRNA 3' ends. In this process we identified 894, 788, and 1,762 m6A-modified genes in Ring, Trophozoite and Schizont stages respectively, with an average of 5-7 m6A sites per-transcript at the individual gene level. Notably, several genes involved in malaria pathophysiology, such as KAHRP, ETRAMPs, SERA and stress response genes, such as members of Heat Shock Protein (HSP) family are highly enriched in m6A and therefore could be regulated by this RNA modification. Since we observed preferential methylation at the 3' ends of P. falciparum transcripts and because malaria polyadenylation specificity factor PfCPSF30 harbors an m6A reader 'YTH' domain, we reasoned that m6A might play an important role in 3'-end processing of malaria mRNAs. To investigate this, we used two complementary high-throughput RNA 3'-end mapping approaches, which provided an initial framework to explore potential roles of m6A in the regulation of alternative polyadenylation (APA) during malaria development in human hosts.