Proximity interaction analysis of the Plasmodium falciparum putative ubiquitin ligase PfRNF1 reveals a role in RNA regulation

Abstract

Some proteins have acquired both ubiquitin ligase activity and RNA-binding properties and are therefore known as RNA-binding Ubiquitin ligases (RBULs). These proteins provide a link between the RNA metabolism and the ubiquitin proteasome system (UPS). The UPS is a crucial protein surveillance system of eukaryotes primarily involved in the selective proteolysis of proteins which are covalently marked with ubiquitin through a series of steps involving ubiquitin E1 activating, E2 conjugating and E3 ligating enzymes. The UPS also regulates other key cellular processes such as cell cycle, proliferation, cell differentiation, transcription and signal transduction. While RBULs have been characterized in other organisms, little is known about their role in Plasmodium falciparum, the causative agent of the deadliest human malaria, malaria tropica. In this study, we characterized a previously identified putative P. falciparum RING finger E3 ligase PfRNF1. We show that the protein is highly expressed in sexual stage parasites and mainly present in immature male gametocytes. Using proximity interaction studies with parasite lines expressing PfRNF1 tagged with the Biotin ligase BirA, we identified an interaction network of PfRNF1 in both the asexual blood stages and gametocytes composed mainly of ribosomal proteins, RNA-binding proteins including translational repressors such DOZI, CITH, PUF1 and members of the CCR4-NOT complex, as well as proteins of the UPS such as RPN11, RPT1 and RPT6. Our interaction network analysis reveals PfRNF1 as a potential RNA-binding E3 ligase which links RNA dependent processes with protein ubiquitination to regulate gene expression. ImportanceRBULs provide a link between RNA-mediated processes with the ubiquitin system. Only a few RBULs have been identified and none has been characterized in the malaria parasite P. falciparum. In this study, we unveiled the interactome of the putative P. falciparum E3 ligase PfRNF1. We show that PfRNF1 interacts with both proteins of the ubiquitin system as well as RNA-binding proteins therefore indicating that it is a putative RBUL which links RNA regulation with the ubiquitin system in P. falciparum.