Establishment of split-TurboID in Plasmodium falciparum facilitates identification of a PfAlba2 protein interaction network during late blood stage development

Malaria parasites of the genus Plasmodium undergo complex multi-host lifecycles that require precise regulation of gene expression. Unlike model eukaryotes, Plasmodium spp. genomically encodes a limited repertoire of canonical transcriptional regulators, but possess a substantial complement of RNA-binding proteins (RBPs), highlighting the importance of post-transcriptional regulatory mechanisms in regulating parasite gene expression. Among these RBPs are a family of DNA/RNA-binding proteins termed 'Alba' (acetylation lowers binding affinity) of which six have so far been identified in these parasites. Here, we investigated the localisation, functional importance and molecular associations of P. falciparum Alba protein PfAlba2 using two different endogenously tagged parasite lines. Using a GFP-FKBP tagged line, we demonstrated that PfAlba2 is expressed throughout asexual blood stage development and primarily localised in the cytosol. Conditional mislocalisation experiments indicated that PfAlba2 contributes to normal parasite growth. To overcome challenges in generating a miniTurboID proximity labelling line, we established a custom split-TurboID proximity labelling system for use in P. falciparum to characterise the PfAlba2 proxiome during asexual blood stage development. The split-TurboID system facilitated identification of 101 enriched PfAlba2 proximal proteins in schizont stage development, with significant enrichment for proteins involved in translation initiation, including two additional Alba family members, PfAlba1 and PfAlba4. Complementary RNA immunoprecipitation sequencing (RIP-seq) identified 380 PfAlba2-associated transcripts with significant enrichments including cytosolic ribosomes, rRNA-processing factors, PfAlba1, and egress and invasion-related transcripts (PfRALP1, PfRON4, PfRH1, PfRH5). Together, these findings implicate PfAlba2 in RNA metabolism and translational regulation, suggesting an important regulatory function for this protein during parasite blood stage development.