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Eukaryotic Cell, December 2004, p. 1445-1453, Vol. 3, No. 6
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.6.1445-1453.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Selection and Characterization of RNA Interference-Deficient Trypanosomes Impaired in Target mRNA Degradation

Huafang Shi,1 Nathalie Chamond,1 Christian Tschudi,1,2 and Elisabetta Ullu1,3*

Departments of Internal Medicine,1 Cell Biology,3 Epidemiology and Public Health, Yale Medical School, New Haven, Connecticut2

Received 20 July 2004/ Accepted 2 September 2004

Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with {alpha}-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei. Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).

* Corresponding author. Mailing address: Department of Internal Medicine, Yale Medical School, BCMM 136D, 295 Congress Ave., Box 9812, New Haven, CT 06536-8012. Phone: (203) 785-3563. Fax: (203) 785-7329. E-mail: elisabetta.ullu{at}yale.edu.

Eukaryotic Cell, December 2004, p. 1445-1453, Vol. 3, No. 6
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.6.1445-1453.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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