Abstract
RNA interference (RNAi) describes an epigenetic gene silencing reaction by which gene-specific double-stranded RNA acts as a trigger to induce the ribonucleolytic degradation of homologous transcripts. RNAi in African trypanosomes has been shown to be involved in regulating the transcript abundance of retroposons, and the process currently represents the method of choice in gene function studies of the parasite. However, little is known concerning the mechanistic and structural aspects of the processing reaction. This is in part due to the absence of a trypanosome-specific RNAi in vitro system. Here we demonstrate that both the Dicer and the RNA-induced silencing complex steps of the RNAi reaction pathway can be monitored in vitro using cell-free trypanosome extracts. The two in vitro activities and the generated small interfering RNAs (siRNAs) are characterized by features known from other organisms, and we demonstrate that chemically as well as enzymatically synthesized siRNAs are functional in the parasite. Thus, the transfection of synthetic siRNAs can be used to rapidly monitor gene knockdown phenotypes in Trypanosoma brucei, which should be helpful in genome-wide, RNAi-based screening experiments.
Highlights
In animals, double-stranded RNA1 has been shown to induce a gene-specific silencing reaction known as RNA interference (RNAi) [2, 3]
The phenomenon is characterized by the ribonucleolytic degradation of the double-stranded RNA (dsRNA) into 21–26-ntlong RNA molecules, which have been termed small interfering RNAs. siRNAs represent trans-acting specificity determinants of the RNAi reaction pathway, which results in the degradation of homologous transcripts
SiRNAs have been identified in African trypanosomes [37], the technique has so far not been used in the protozoan parasite
Summary
Double-stranded RNA (dsRNA) has been shown to induce a gene-specific silencing reaction known as RNA interference (RNAi) [2, 3]. SiRNAs represent trans-acting specificity determinants of the RNAi reaction pathway, which results in the degradation of homologous transcripts. The reaction pathway has been shown to be widely conserved, and as a consequence, has become a powerful experimental tool to study gene knockdown or loss-of-function phenotypes in many organisms [3, 15,16,17]. For recombinant human Dicer, it was shown that the dsRNA cleavage is an endonucleolytic reaction [25]. The thermodynamic stability at either end of the siRNA determines which of the two strands of the symmetric molecule remains in the RISC complex and guides the recognition of the mRNA via base pairing [1, 32]. The mRNA is endonucleolytically cleaved by the endonuclease Slicer [4, 5], and the resulting mRNA fragments are likely subjected to nonspecific degradation processes in the cytoplasm
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