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

Abstract

SummaryMalaria 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 doublestranded (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.

Knockdown of Aminopeptidase-N from Helicoverpa armigera Larvae and in Transfected Sf21 Cells by RNA Interference Reveals Its Functional Interaction with Bacillus thuringiensis Insecticidal Protein Cry1Ac

“…The truncated fragment of H. armigera haapn1 was subcloned in pGEM-Te and used for the preparation of dsRNA. As a control, the gene for falcipain of P. falciparum cloned in pGEM-T was used as described earlier (18). The pGEM-Te-cloned fragments were amplified by PCR using vector-specific universal and reverse primers (Promega).…”

“…The pGEM-Te-cloned fragments were amplified by PCR using vector-specific universal and reverse primers (Promega). The PCR product was purified (Qiagen GmbH) and used as DNA template for dsRNA preparation after the in vitro transcription procedure described by us (18). The T7 and SP6 RNA polymerases (MBI Fermentas) were used to generate single strand sense RNA and antisense RNA, respectively, from the DNA.…”

RNA Interference with Special Reference to Combating Viruses of Crustacea

“…To date, RNAi related events have been recognised in almost all eukaryotic animals including algae [202], yeast [151], protozoans [11,27,94,118], plants [43,196], insects [2,17,26,77,100], fish [29,131] and in mammals [38,48,121,198].…”

The transcription machinery and the molecular toolbox to control gene expression in Toxoplasma gondii and other protozoan parasites

“…In T. brucei, a combination of efficient RNAi and a tight tet-regulated transcription is routinely applied for large scale analysis of genome function. In contrast, the efficiency of RNAi in apicomplexans is still a matter of debate [85,86], even if previous and more recent studies have reported the successful use of antisense/ribozyme in T. gondii [87][88][89] and some studies have suggested that the mechanism of RNAi can operate in the malaria parasites [90][91][92].…”