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

<h4>Background</h4>Malaria, caused by <i>Plasmodium</i> parasites, remains a major parasitic disease worldwide. Post-transcriptional gene regulation is essential for the intraerythrocytic development of <i>Plasmodium falciparum</i>, 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 <i>P. falciparum</i>.<h4>Methods</h4>PfSR-X2 was characterized by sequence analysis, structural prediction, expression profiling, and subcellular localization assays. A glucosamine-inducible <i>glmS</i>-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.<h4>Results</h4>PfSR-X2 was expressed throughout the intraerythrocytic developmental cycle and localized to both nuclear and cytoplasmic compartments. Attempts to disrupt <i>pfsr-x2</i> 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 <i>rif</i> multigene family was strongly perturbed, whereas the overall <i>var</i> 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.<h4>Conclusion</h4>These findings identify PfSR-X2 as an essential RNA-associated regulator in <i>P. falciparum</i>. 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.