Biomedical subjects

Mickaël Durand-Dubief

Publications and source records attributed to Mickaël Durand-Dubief.

3 recordsLinked to original sources

PubMed2005

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.

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PubMed2003

TbAGO1, an argonaute protein required for RNA interference, is involved in mitosis and chromosome segregation in Trypanosoma brucei.

BACKGROUND: RNA silencing processes are widespread in almost all eukaryotic organisms. They have various functions including genome protection, and the control of gene expression, development and heterochromatin formation. RNA interference (RNAi) is the post-transcriptional destruction of RNA, which is mediated by a ribonucleoprotein complex that contains, among several components, RNA helicases and Argonaute proteins. RNAi is functional in trypanosomes, protozoan parasites that separated very early from the main eukaryotic lineage and exhibit several intriguing features in terms of the control of gene expression. In this report, we investigated the functions of RNAi in Trypanosoma brucei. RESULTS: By searching through genome databases, novel Argonaute-like proteins were identified in several protozoa that belong to the kinetoplastid order, a group of organisms that diverged early from the main eukaryotic lineage. T. brucei possesses two Argonaute-like genes termed TbAGO1 and TbPWI1. Dual transient transfection assays suggest that TbAGO1, but not TbPWI1, is involved in RNAi. The entire coding region of TbAGO1 was deleted by double gene knockout. TbAGO1-/- cells turned out to be completely resistant to RNAi generated either by transfected double-stranded RNA or by expression of an inverted repeat. TbAGO1-/- cells were viable but showed a dramatically reduced growth rate. This was probably due to defects in mitosis and abnormal chromosome segregation as revealed by in situ analysis. The RNAi and growth phenotypes were complemented by the inducible expression of a GFP::TbAGO1 fusion protein that revealed the cytoplasmic location of the protein. CONCLUSIONS: The requirement of TbAGO1 for RNAi in trypanosomes demonstrates the evolutionary ancient involvement of Argonaute proteins in RNAi silencing processes. RNAi-deficient TbAGO1-/- cells showed numerous defects in chromosome segregation and mitotic spindle assembly. We propose a working hypothesis in which RNAi would be involved in heterochromatin formation at the centromere and therefore in chromosome segregation.

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PubMed2003

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.

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