Functional characterisation and identification of a core interactome of Plasmodium regulatory protein Alba4

Plasmodium spp. parasites have complex lifecycles that rely on strict regulation of gene and protein expression. Current evidence suggests that P. falciparum has a relatively limited repertoire of canonical transcriptional regulators and RNA interference (RNAi)-associated small RNAs compared to model eukaryotes, potentially reflecting a greater reliance on RNA binding protein (RBP)-mediated post-transcriptional regulation (PTR). Among these RBPs, the acetylation lowers binding affinity (Alba) family of DNA/RNA-binding proteins has been implicated in RNA metabolism and regulation of mRNA stability and translation. In Plasmodium spp. parasites, the Alba family has expanded to include up to six members, with sequence divergence and structural modifications beyond the conserved Alba domain leading to diversification of family members, some of which appear restricted to Plasmodium and related parasites. In this study, we were unable to truncate PfAlba4, but successfully generated an endogenously tagged PfAlba4 parasite line, representing the first successful instance of endogenous tagging of a PfAlba family protein. Utilising this transgenic line, we confirm ubiquitous expression throughout blood stage development and its predominantly cytosolic localisation. Conditional loss of PfAlba4 through the knock-sideways approach demonstrated an important role for PfAlba4 in blood stage development and proteomics analysis defined a protein interaction network of 67 significantly enriched proteins, with notable enrichment of proteins potentially involved in RNA processing and regulation. Further characterisation of one of these proteins, the uncharacterised protein PF3D7_0706500, revealed a partially overlapping network of proximal proteins enriched for RNA-regulatory factors, suggesting potentially shared roles in PTR. RNA Immunoprecipitation-Sequencing (RIP-seq) identified 692 PfAlba4 associated transcripts, with an enrichment of a pyrimidine-rich motif in their 3' untranslated regions, suggesting a potential sequence-dependent mechanism of RNA recognition. Integration of proteomic and RIP-Seq datasets in late blood stage parasites revealed a consistent association with RNA processing and regulatory pathways, implicating PfAlba4 in post-transcriptional regulation. Together these findings establish PfAlba4 as an important component of the RNA-regulatory landscape in P. falciparum and highlight the importance of PfAlba proteins for malaria parasite blood stage development.