RNA-Binding Proteins and RNA-Dependent Proteins - An Emerging Role for RNAs in Plasmodium Biology

Abstract Nearly half of the world’s population lives in countries where malaria is endemic. Plasmodium falciparum, the causative agent of the most severe form of human malaria, is responsible for 95% of malaria deaths worldwide. The project’s main goals are to identify RNA-binding proteins (RBPs) and RNA-dependent proteins (RDPs) in the pathogen; elucidate their importance in parasite development; and finally identify novel pathways that can be targeted to kill the parasite. Ribonucleoprotein complexes are composed of RNA, RBPs, and RDPs and have been shown to play fundamental roles in RNA regulation in eukaryotic organisms. However, in the human malaria parasite, P. falciparum, identification and characterization of these proteins are particularly limited. Our lab has used several approaches including an unbiased proteome-wide approach, called R-DeeP, a novel method based on sucrose density gradient ultracentrifugation, to successfully identify not only RBPs but also RDPs in the asexual stages of the P. falciparum life cycle. Using quantitative analysis by mass spectrometry as well as a combination of computational and molecular approaches, we identified over 800 RDPs, including 500+ proteins not yet associated with RNA. We also reconstruct Plasmodium multiprotein complexes based on their co-segregation and their RNA-dependence. One RDP candidate was functionally characterized and validated as interacting in complex with various Plasmodium non-coding transcripts, including var genes and several ap2 transcription factors. Overall, our novel genome-wide proteomic and molecular approaches provided the first snapshot of the Plasmodium protein-protein interaction networks in the presence and absence of RNA in the asexual cycle of the parasite. No data were however acquired for parasite sexual differentiation, a stage essential for the transmission of the pathogen to the mosquito vector. We therefore now propose to use our novel R-DeeP methodology to not only identify and reconstruct Plasmodium multiprotein complexes based on their RNA-dependence during the parasite sexual differentiation but also validate their potential as new therapeutic targets. We have therefore designed the following two specific aims. In AIM 1, we will adapt and expend our unbiased proteome-wide approach to identify RDPs from early and late gametocytes. Our data will also be used to determine RNA dependent multiprotein complexes that are specific to these parasite sexual stages. In AIM 2, we will use a combination of CRISPR-Cas9 genome editing tools as well as functional genomics approaches to validate the role of RDPs candidates that are involved in sexual differentiation. The results of this work will transform our understanding of the role of RNA-dependent protein complexes (RDPCs) in the parasite biology and identify novel intervention strategies that may interrupt the development of malaria parasites.