The serine-arginine-rich protein PfSR-X2 modulates human malaria parasite gene expression during the...

Malaria, caused by Plasmodium parasites, remains a major parasitic disease worldwide. Post-transcriptional gene regulation is essential for the intraerythrocytic development of Plasmodium falciparum, yet the functions of many parasite RNA-binding proteins remain poorly understood. Serine/arginine-rich proteins are important regulators of RNA metabolism in eukaryotes. In this study, we investigated the function of the SR-related protein PfSR-X2, encoded by PF3D7_0319500, during the asexual blood-stage development of P. falciparum.

PfSR-X2 was characterized by sequence analysis, structural prediction, expression profiling, and subcellular localization assays. A glucosamine-inducible glmS-based conditional knockdown parasite line was generated using CRISPR-Cas9-mediated genome editing. The effects of PfSR-X2 depletion on parasite growth, developmental progression, gene expression, and alternative splicing were examined by growth assays, Giemsa-stained blood smears, and RNA sequencing. RNA immunoprecipitation followed by sequencing was further performed to identify PfSR-X2-associated transcripts.

PfSR-X2 was expressed throughout the intraerythrocytic developmental cycle and localized to both nuclear and cytoplasmic compartments. Attempts to disrupt pfsr-x2 were unsuccessful, suggesting that PfSR-X2 is required for asexual blood-stage growth. Conditional depletion of PfSR-X2 resulted in reduced parasite proliferation and impaired merozoite production. Transcriptomic analysis revealed marked stage-specific changes in gene expression after PfSR-X2 knockdown, particularly affecting genes involved in RNA metabolism, antigenic variation, host-parasite interactions, and ApiAP2-associated regulatory pathways. The rif multigene family was strongly perturbed, whereas the overall var transcriptional pattern was largely unchanged. In contrast, alternative splicing changes were limited and were mainly detected at the schizont stage. RNA immunoprecipitation sequencing identified a defined subset of PfSR-X2-associated transcripts, including transcripts encoding antigenically variant proteins and ApiAP2 transcription factors.

These findings identify PfSR-X2 as an essential RNA-associated regulator in P. falciparum. Rather than acting as a global splicing factor, PfSR-X2 appears to contribute mainly to stage-specific post-transcriptional regulation of transcripts associated with parasite development, antigenic variation, and transcriptional regulatory networks.