ABSTRACT
RNAi has emerged recently as one of the most promising tools in studying gene function (summarized in Chapter 1). It was initially believed to be based on an evolutionary defense mechanism of silencing RNA in eukaryotes to protect their genomes from exogenous (viral) and endogenous (transposon) elements. Related mechanisms with small RNAs are involved in cell homeostasis by regulating translation (Caenorhabditis elegans) and structure of heterochromatic domains in the genome. In many organisms, including C. elegans, Drosophila, zebra fish, and Xenopus, RNAi is activated by the presence of foreign dsRNA in the cell which triggers sequence-specific mRNA degradation.1 The dsRNA is enzymatically cleaved with the help of Dicer RNase III to give rise to small interfering RNA duplexes (siRNA). These siRNAs, usually 21 to 25 nucleotides in length and characterized by a two-nucleotide 3'-overhang, form a complex with a nuclease complex, the RNA-induced silencing complex (RISC), where they become unwound and aid in the homology-dependent degradation of the target RNA. Similar processes occur in plants as posttranscriptional gene silencing and cosuppression and quelling in fungi and algae.2 Plasmodium was the first intracellular organism in which RNAi was successfully demonstrated.3 Subsequent studies have shown that small RNAs may be used and that siRNAs are generated, but the mechanism of RNA silencing in Plasmodium has yet to be elucidated.4,5
Members of the genus Plasmodium, a single-celled protozoan, are the etiologic agents of malaria. There are more than 250 million cases of malaria annually. Plasmodium falciparum is the species responsible for the highest mortality, which claims the lives of 1 to 2 million people each year.6 Resistance to antimalarial drugs is increasing, and multiple drug resistance has been observed. Hence, there is a great need for new antimalarial drugs and the development of an effective vaccine. The complete genome sequence of P. falciparum was released in October 2002.7 This is a landmark achievement as the first protozoan genome completely sequenced and it presented a particular challenge with its A/T rich genome (80.6% A/T). The genome of the mosquito vector for human malaria, Anopheles gambiae, has also been sequenced.8 With the human genome sequence completed, the three genomes in the triad of malaria are now known. Additionally, a rodent malaria species has been sequenced and incomplete genome sequences for four other Plasmodium species are publicly available, permitting comparative genomic studies. Combined with microarray and proteomic data on P. falciparum, there is a plethora of information for design of RNAi studies.911
