Functional complementation of RNA interference mutants in trypanosomes.
BACKGROUND
In 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.


RESULTS
Using 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.


CONCLUSIONS
This 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.
Rusconi
Filippo
F
UMR5153 CNRS, USM0503 MNHN, U565 INSERM-57, rue Cuvier, P.B 26 - F-75231, Paris Cedex 05, France. rusconi@mnhn.fr
Durand-Dubief
Mickaël
M
Bastin
Philippe
P
D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D005407:Flagella / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D020868:Gene Silencing; D018390:Gene Targeting; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D008957:Models, Genetic; D009068:Movement; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins; D034622:RNA Interference; D012323:RNA Processing, Post-Transcriptional; D012330:RNA, Double-Stranded / Q000737:chemistry; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000378:metabolism
D015800:Protozoan Proteins; D012330:RNA, Double-Stranded