Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei.
In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.
Durand-Dubief
Mickaël
M
Unité INSERM U565 & CNRS UMR8646, Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, 43 rue Cuvier, 75231 Paris Cedex 05, France.
Kohl
Linda
L
Bastin
Philippe
P
D000818:Animals; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D008957:Models, Genetic; D010641:Phenotype; D015800:Protozoan Proteins / Q000032:analysis; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism
D015800:Protozoan Proteins; D012330:RNA, Double-Stranded; C045519:paraflagellar rod proteins, Trypanosoma brucei