No abstract

An inducible expression system was developed for the protozoan parasite Trypanosoma brucei. Transgenic trypanosomes expressing the tetracycline repressor of Escherichia coli exhibited inducer (tetracycline)-dependent expression of chromosomally integrated reporter genes under the control of a procyclic acidic repetitive protein (PARP) promoter bearing a tet operator. Reporter expression could be controlled over a range of four orders of magnitude in response to tetracycline concentration, a degree of regulation that exceeds those exhibited by other eukaryotic repression-based systems. The tet repressor-controlled PARP promoter should be a valuable tool for the study of trypanosome biochemistry, pathogenicity, and cell and molecular biology.

Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of ϳ57 kDa (band IV) and ϳ50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.In kinetoplastid protozoans, many mitochondrial transcripts undergo RNA editing, a specific insertion and deletion of U residues at multiple sites, directed by guide RNAs (reviewed in references 2, 13, 15, 38, and 39). Both U deletional and U insertional editing cycles have been reproduced in vitro and shown to involve three enzymatic steps ( Fig. 1A) (9,20,34,35; see also reference 7). First, the mRNA is cleaved by a guide RNA (gRNA)-directed endonuclease, U residues are then added to or removed from the 3Ј end of the upstream cleavage product by a terminal-U-transferase or 3Ј-U-exonuclease, and the mRNA is then rejoined by RNA ligase. A complex consisting of seven different polypeptides that contains all these activities and catalyzes both U-deletional and U-insertional editing rounds has been purified from Trypanosoma brucei mitochondria (10, 29). We have undertaken the cloning and characterization of the polypeptides that make up this complex, beginning with one identified as an RNA ligase.RNA ligases are used by many cells in tRNA splicing (e.g., see references 5, 14, 45, and 49) and by bacteriophage T4 in tRNA repair (reviewed in reference 41), and they are also present in trypanosome mitochondria (3,17,46). These enzymes join RNA 3Ј hydroxyl and 5Ј phosphate termini, evidently by a common mechanism (30, 31; Fig. 1B). First, the ligase autoadenylylates, using ATP to form a covalent protein-AMP intermediate while releasing pyrophosphate (PP i ). This reaction occurs in the absence of RNA and reverses with high concentrations of PP i . The AMP is then transferred to the 5Ј phosphate of a donor RNA, generating a 5Ј-5Ј linkage, and the 3Ј hydroxyl of the acceptor RNA finally displaces this 5Ј AMP, forming the new phosphodiester bond. T. brucei mitochondrial extract ...

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.

Inability of T7 RNA polymerase to processively transcribe higher eukaryotic chromatin is interpreted as a correlate of its reported inhibition by nucleosomes on reconstituted templates in vitro . We used chromosomally integrated reporter cassettes to examine features of T7 transcription in a lower eukaryotic system. Luciferase reporters were targeted to rDNA in transgenic Trypanosoma brucei stably expressing the phage polymerase. Because trypanosome mRNAs are capped by RNA splicing in trans , T7 transcription could be gauged by luciferase activity. In contrast to findings from higher eukaryotes, T7 transcription is vigorous and processive on chromatin templates in T.brucei , surpassing levels achieved with endogenous promoters, including those recruiting RNA polymerase I. This may be a reflection of intrinsic differences in chromatin structure between differently evolved eukaryotes or of an integration site that is exceptionally permissive for T7 transcription due to a local accessible chromatin conformation. T7 transcription could be manipulated to achieve different levels of constitutive expression, through the use of promoter mutations. Moreover, T7 initiation could be regulated by the prokaryotic Tet repressor and elongation halted by T7 terminator sequences. We have exploited these features to construct a robust inducible expression system, whose utility potentially extends to other trans -splicing organisms.