Sense–antisense RNA duplexes mediate stress-responsive translational control in Plasmodium falciparum

Abstract

Plasmodium falciparum, the primary cause of human malaria, relies on tightly coordinated gene-expression programs to adapt to host-derived stress despite possessing a limited repertoire of canonical transcription factors. Antisense long noncoding RNAs have emerged as important regulators of parasite biology, including virulence gene regulation and sexual commitment; however, their prevalence, origin, and broader functional significance remain poorly understood. Here, we demonstrate that antisense transcription is a widespread, reproducible, and regulated feature of the P. falciparum transcriptome rather than a by-product of pervasive euchromatic transcription. Environmental stress, including febrile temperature exposure and artemisinin treatment, extensively remodeled antisense transcription, particularly at loci associated with virulence and stress adaptation, promoting widespread sense–antisense RNA duplex formation. Functional analyses of two stress-responsive chromatin regulators, PfGCN5 and PfHDAC1, identified as antisense-expressing loci, revealed that increased antisense expression elevated steady-state mRNA abundance while reducing cognate protein levels. Mechanistically, sense–antisense RNA duplex formation stabilized complementary transcripts but suppressed translation. Integrated transcriptomic, RNA–RNA duplex profiling, ribosome sequencing, and proteomic analyses further showed that duplex-enriched transcripts exhibit reduced ribosome occupancy and reduced protein abundance, accompanied by localized antisense enrichment near transcription end sites and altered ribosome distribution consistent with impaired translational engagement. Collectively, our findings identify an antisense RNA–ribosome regulatory axis that couples RNA duplex formation to adaptive translational control, providing a previously unrecognized mechanism underlying stress adaptation and posttranscriptional gene regulation in P. falciparum.