2021 FDA Science Forum
Identifying the Human Malaria Parasite RNA-Protein Interactome Using Enhanced Crosslinking Technology
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Contributing OfficeCenter for Biologics Evaluation and Research
Abstract
Progress in the prevention and control of malaria is threatened by the emergence of multidrug resistance and lack of a long-term protective vaccine against the causative agent, Plasmodium spp. This creates a serious need for the development of novel antimalarial and transmission-blocking strategies. However, identifying promising targets of intervention within the complex lifecycle of Plasmodium parasites has been challenging as the molecular drivers of developmental progression remain incompletely characterized. It has been well established that post-transcriptional regulation is essential to the Plasmodium growth and parasite transmission, making RNA-binding proteins (RBPs) attractive targets of intervention. Yet, due to the lack of effective in vivo methodologies, characterization of RNA-protein interactions and their role in Plasmodium development has been limited. Here, we optimize the parameters of photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) (PMID: 22658674) for use in identifying RBPs involved in the P. falciparum RNA-protein interactome. To accomplish this, we utilize a transgenic P. falciparum strain that is genetically altered to salvage thiol-modified uracil (PMID: 28416533), enabling photoactivated covalent crosslinking and capture of zero-distant RBPs bound to thiolated RNA. Two distinct RNA purification methods are employed to isolate crosslinked RNA-RBP complexes, capturing either RBPs interacting with any RNA species or mRNAs. Following protein-RNA complex capture, mass spectrometry and novel sequencing techniques are used to identify both protein and cognate RNA-interacting motifs based upon thiolated-RNA footprinting. Preliminary proteomic analysis has identified both previously reported and novel RBP candidates enriched in samples crosslinked via the PAR-CL method, which confirms the validity of this method in Plasmodium and demonstrates the possibility of its increased sensitivity compared to conventional crosslinking methods. Our study provides a new and powerful tool to analyze RNA-protein interaction in P. falciparum throughout parasite development and transmission, which may facilitate the identification of novel therapeutic target candidates.
