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

Plasmodium falciparum, the deadliest malaria-causing parasite, challenges eradication efforts due to drug resistance. To date, mRNA translation remains an unexplored therapeutic target. Most mRNAs contain unusually long 5'UTR and multiple upstream AUGs. How the parasite overcomes scanning distance and uORF constraints is unknown. Using Ribo-seq, TIS-seq, and long-read data, we mapped translation initiation sites, determined translation efficiency, and re-annotated the 5'UTRs. We identified actively translated uORFs in 81% of mRNAs, predominantly initiated by unspecified AUG, while inactive uORFs are enriched with inhibitory AUG contexts. Notably, initiation within CDS through leaky scanning is also highly prevalent. Surprisingly, mRNAs with long 5'UTR and active uORFs exhibit higher translation efficiency compared to those lacking uORFs. This is facilitated by a specific spacing of active uORFs and peptide length, optimizing scanning distance, ribosome density, and reinitiation. Remarkably, one-hour exposure to the eIF1-eIF4G1 inhibitor i14G1-12 reduced parasite cell 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 Overall design: To obtain a genome-wide, quantitative view of translation in P. falciparum, and under treamtent with the eIF1-eIF4G1 inhibitor i14G1-12, we performed Ribosome Profiling on DMSO and i14G1-12 treated cultures of trophozoite-stage parasites. To assess translation efficiency (TE), we used cycloheximide (CHX), a translation elongation inhibitor that stalls all translating ribosomes. Measurement of total mRNA abundance from the same samples was used for normalization. Two independent replicates were assigned for each library (CHX, HAR, total RNA) in each treatment group (DMSO, i14G1-12), adding up to a total of 12 samples.