Nucleotide-resolution mapping of RNA N6-methyladenosine (m 6 A) modifications and comprehensive analysis of global polyadenylation events in mRNA 3’ end processing in malaria pathogen Plasmodium falciparum

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA, yet its distribution and potential involvement in mRNA 3′-end processing in the malaria parasite Plasmodium falciparum remain poorly understood. Here, we combined nucleotide resolution DART-seq, Nanopore DRS, MeRIP-seq and complementary 3′-end sequencing approaches to investigate the relationship between m6A deposition and mRNA 3′-end formation across the parasite transcriptome. DART-seq identified 2,330 reproducible m6A sites in 1,195 genes, revealing stage-associated differences in the epitranscriptome and preferential enrichment of m6A within 3′ UTRs. Independent MeRIP-seq analysis further supported the reproducibility of these methylation patterns. mRNA 3′-end mapping revealed that cleavage and polyadenylation frequently occur within exceptionally adenosine-rich sequence regions. Integration of Nanopore DRS-derived m6A stoichiometry with experimentally defined poly(A) sites revealed preferential m6A enrichment at canonical DRACH motifs approximately 30–50 nucleotides upstream of transcript cleavage sites. Notably, single-molecule DRS analysis demonstrated differential m6A occupancy at shared transcript positions between proximal and distal alternative polyadenylation isoforms of the same gene, with lower occupancy in distal isoforms near proximal cleavage sites. Moreover, m6A enrichment was consistently observed upstream of the utilized poly(A) site in individual transcripts, suggesting an association between m6A deposition and alternative 3′-end formation. Distinct spatial associations of RNA secondary structure and m6A with poly(A) site regions were observed. Together, these findings provide a transcriptome-wide and single-molecule view of m6A modification patterns in relation to native poly(A) sites in P. falciparum, and the data are consistent with a model in which m6A is associated with and may contribute to 3′-end processing.