Abstract
BackgroundIn many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.ResultsUsing the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility.ConclusionsThis work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.
Highlights
In many eukaryotic cells, double-stranded RNA triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence
The genes selected were TbPFR2 and FLA1; TbPFR2 encodes one of the two major components of the paraflagellar rod and is necessary for flagellum motility [13]; FLA1 encodes a protein required for flagellum attachment to the cell body [50]
We demonstrated that RNAi-mediated silencing of a gene by targeting its UTRs is useful in studies where the loss of function resulting from this silencing must be complemented with the expression of an RNAiresistant copy of the silenced gene, in order to demonstrate that the phenotype is due to silencing of that gene, and not to inactivation of another one
Summary
Double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. The presence of long dsRNA (>50 base pairs) triggers the activation of sequence-unspecific interferonrelated pathways [27,28,29]. To circumvent this difficulty, researchers resorted to the transfection of small interfering RNAs [16] or in vivo synthesis of small hairpin RNAs, which were demonstrated to produce gene-specific silencing [27,30,31]; reviewed in [32,33]
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