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J. Biol. Chem., Vol. 280, Issue 21, 20573-20579, May 27, 2005

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In Vitro Synthesized Small Interfering RNAs Elicit RNA Interference in African Trypanosomes

AN IN VITRO AND IN VIVO ANALYSIS^*

Alexander Best, Lusy Handoko*, Elke Schlüter, and H. U. Göringer

From the Department of Microbiology and Genetics, Darmstadt University of Technology, 64287 Darmstadt, Germany

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.

Received for publication, December 23, 2004 , and in revised form, March 16, 2005.

^* This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure showing a time course experiment of a synthetic siRNA with a cell-free T. Brucei extract.

Present address: Institut für Biochemie, Biozentrum der Universität Würzburg, Am Hubland, 97074 Würzburg

An International Research Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Microbiology and Genetics, Darmstadt University of Technology, Schnittspahnstr. 10, 64287 Darmstadt, Germany. Tel.: 6151-162855; Fax: 6151-165640; E-mail: goringer{at}hrzpub.tu-darmstadt.de.

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				H. Shi, C. Tschudi, and E. Ullu An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei RNA, December 1, 2006; 12(12): 2063 - 2072. [Abstract] [Full Text] [PDF]