The P. falciparum orthologue of Male Development Protein 3 is a Male-associated regulator of translation initiation or enhancement [RNA-Seq]

Sex determination and development of Plasmodium gametocytes is critical to the transmission of malaria parasites and involves a complex interplay of multiple molecular factors. Here, we characterized the P. falciparum gene syntenic to P. berghei MD3- pf3d7_0315600. This protein contains a non-classical CCCH zinc-finger domain and Lysine-Proline rich domain associated with ribosome receptors, suggesting a role in mRNA regulation. Disruption of PfMD3 causes a pronounced defect in male gametogenesis which severely decreases parasite transmission to mosquitoes but does not affect female gamete production. We show that abrogation of PfMD3 significantly decreases the expression of male gamete markers on the transcriptome and proteome level but also affects regulators of translation. We further investigated the function of PfMD3-3xHA using in vivo using crosslinked RNA-Protein interaction capture followed by RNA-sequencing (CLIP-seq) to determine its RNA targets and IP-MS to determine protein interactors. We found 143 bound mRNA transcripts in early-stage gametocytes and 86 transcripts in late-stage gametocytes including arid/md4 andp230, not1-g, spm3 and morc mRNA bound at both stages. Interestingly, these CLIP targets decrease significantly in the ΔPfMD3 proteome, suggesting a role for PfMD3 in translational processing. IP-MS using a PfMD3-3xHA-tagged line further confirmed this role, showing an enrichment of proteins involved in mRNA metabolism. We then propose that PfMD3 functions as a Male Associated Regulator of Translation Initiation or Enhancement (MARTIE) in the human malaria parasite P. falciparum. To investigate the function of PfMD3, we established a clonal genetic knockout line of Plasmodium falciparum parasites (PfMD3_KO clone 1) in the NF54e wild type background. We then performed RNA-sequencing (RNA-seq) for PfMD3 knockout clone 1 in Plasmodium falciparum NF54e parasites in stage II-III, stage IV and stage V gametocytes (2 biological replicates each) and co-cultured NF54e wild type parasites