The organization principles of P. falciparum translation initiation features and their potential as drug targets.

Plasmodium (P.) falciparum, the deadliest malaria-causing parasite, challenges eradication efforts due to drug resistance. The regulation of mRNA translation as a therapeutic target remains unexplored. Most mRNAs contain unusually long 5'UTR and multiple upstream AUGs. How the parasite overcomes scanning distance and multiple uORF constraints is unknown. We employed the eIF1-eIF4G1 inhibitor i14G1-12, Ribo-seq, TIS-seq, and long-read data on P. falciparum trophozoites. Re-annotation of 5'UTRs, initiation site mapping, and translation efficiency (TE) analysis revealed extended 5'UTR length in hundreds of genes and actively translated uORFs in 81% of mRNAs. Active uORFs were predominantly initiated with AUG lacking specific context, while inactive uORFs were enriched with inhibitory AUG contexts. Notably, initiation within coding sequence through leaky scanning is also highly prevalent. Surprisingly, mRNAs with long 5'UTR and active uORFs exhibit higher TE than those without. We demonstrate that this is facilitated by a specific spacing of active uORFs and peptide length, optimizing scanning distance, ribosome density, and reinitiation. Remarkably, short-term i14G1-12 treatment reduced parasite viability and caused translational repression by enhancing leaky scanning and disturbing the unique arrangement of uORFs. Collectively, our findings uncovered the unusual translation regulatory features of P. falciparum and highlighted the therapeutic potential of targeting these mechanisms.