Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA

Abstract

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the a-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.

Mitochondrial Initiation Factor 2 of Trypanosoma brucei Binds Imported Formylated Elongator-type tRNAMet

“…In Vivo Depletion of IF2 by RNAi-Due to the availability of a tightly controlled tetracycline-inducible expression system (29), RNAi has become the method of choice to disrupt gene function in T. brucei (22,23). Thus, to investigate the function of IF2 by RNAi, we prepared a construct that contained a defined sequence of the IF2 gene in the sense, as well as the antisense, direction.…”

“…In the presence of tetracycline, the RNA was expressed and formed a stem loop that eventually led to the specific degradation of the IF2 mRNA. There have been two types of stuffer sequences used in stem loop constructs, one of trypanosomal origin as described previously (22) and another one of mouse origin (23). Surprisingly, it was shown that the efficiency of RNAi was generally higher when the mouse sequence was used.…”

“…Two versions of the plasmid were prepared. In the first one (TbIF2-Tb), the sense and antisense sequences were separated by a fragment corresponding to 690 nucleotides of the trypanosomal spliced leader sequence (22), whereas in the second one (TbIF2-Mm), a 439-bp fragment, corresponding to positions 341-779 of the mouse Pex11b mRNA, was used as a spacer (23). Using the mouse sequence as a spacer yielded a more efficient down-regulation of the targeted mRNA than if the T. brucei sequence was used.…”

Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei

“…The expression of Lsm proteins was silenced by the two approaches. The Lsm8 was silenced by producing dsRNA in the form of a stem-loop structure, as described under "Experimental Procedures" (35,37). For silencing the Lsm3 gene, PCR was used to amplify the gene that was cloned into the pZJM vector carrying the two T7 opposing promoters (35).…”

Composition of the editing complex ofTrypanosoma brucei

“…Regulatable TbMP48 sequences to produce interfering dsRNAi were introduced into both the bloodstream and procyclic stage cell lines, as described (Shi et al 2000;Wang et al 2000). We were unable to produce null mutants of TbMP52 and thus produced mutant BFs in which we introduced a regulatable TbMP52 allele and then deleted both endogenous alleles in BFs as described by Wirtz et al (1999).…”