Double-stranded RNA-mediated gene silencing of cysteine proteases (falcipain-1 and -2) of Plasmodium falciparum

Abstract

Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double-stranded RNA of cysteine protease genes (falcipain-1 and -2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double-stranded (ds)RNAs are reminiscent of the effects observed upon administering E-64, a cysteine protease inhibitor. The parasites treated with falcipain's dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs ≈ 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence-specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.