A zinc finger protein, TbZC3H20, stabilizes two developmentally regulated mRNAs in trypanosomes.

CCCH zinc finger proteins (ZC3Hs) are a novel class of RNA-binding protein involved in post-transcriptional mechanisms controlling gene expression. We show TbZC3H20 from Trypanosoma brucei, the causative agent of sleeping sickness and other diseases, stabilizes two developmentally regulated transcripts encoding a mitochondrial carrier protein (MCP12) and trans-sialidase (TS-like E). TbZC3H20 is shown to be an RNA-binding protein that is enriched in insect procyclic form T. brucei and is the first ZC3H discovered controlling gene expression through modulating mRNA abundance in trypanosomes. Previous studies have demonstrated that RNA recognition motif-containing and PUF family RNA-binding proteins can control gene expression by stabilizing specific target mRNA levels. This work is the first to describe a ZC3H stabilizing rather than destabilizing target mRNAs as a regulatory mechanism and the first report of a ZC3H regulating a gene encoding a mitochondrial protein. This suggests a broader role for ZC3Hs in post-transcriptional regulation of gene expression than previously thought.

Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point.

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.

Functional expression of the gene encoding cytidine triphosphate synthetase from Plasmodium falciparum which contains two novel sequences that are potential antimalarial targets.

CTP synthetase (E C 6.3.4.2 UTP: ammonia ligase (ADP-forming)) catalyses the formation of CTP from UTP and, in the human parasite Plasmodium falciparum, is the sole source of cytidine nucleotides. It is thus a potential chemotherapeutic target, especially as the gene sequence indicated that the encoded GAT-domain of the enzyme contains two extended peptide segments (42aa and 223aa as compared to the host enzyme). Here, we circumvent the codon usage problems associated with the high A/T content of the P. falciparum sequence, especially evident in sequences encoding the extra peptides, to successfully express active recombinant P. falciparum CTP synthetase using preferred E. coli codons. This partially synthetic gene produced recombinant enzyme, containing the additional segments, which was functionally assayed for activity in vitro. We also show the native enzyme contains the additional peptides using immunoblots with antibodies derived from the recombinant protein. Confocal microscopy, using antibodies to the recombinant protein, provided evidence that the enzyme is expressed in vivo. This establishes for the first time that P. falciparum contain active CTP synthetase and that this enzyme contains two novel insert sequences in the functional enzyme.

Disruption of the developmental programme of Trypanosoma brucei by genetic ablation of TbZFP1, a differentiation-enriched CCCH protein.

The regulation of differentiation is particularly important in microbial eukaryotes that inhabit multiple environments. The parasite Trypanosoma brucei is an extreme example of this, requiring exquisite gene regulation during transmission from mammals to the tsetse fly vector. Unusually, trypanosomes rely almost exclusively on post-transcriptional mechanisms for regulated gene expression. Hence, RNA binding proteins are potentially of great significance in controlling stage-regulated processes. We have previously identified TbZFP1 as a trypanosome molecule transiently enriched during differentiation to tsetse midgut procyclic forms. This small protein (101 amino acids) contains the unusual CCCH zinc finger, an RNA binding motif. Here, we show that genetic ablation of TbZFP1 compromises repositioning of the mitochondrial genome, a specific event in the strictly regulated differentiation programme. Despite this, other events that occur both before and after this remain intact. Significantly, this phenotype correlates with the TbZFP1 expression profile during differentiation. This is the first genetic disruption of a developmental regulator in T. brucei. It demonstrates that programmed events in parasite development can be uncoupled at the molecular level. It also further supports the importance of CCCH proteins in key aspects of trypanosome cell function.

tbCPSF30 depletion by RNA interference disrupts polycistronic RNA processing in Trypanosoma brucei.

Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs. In Trypanosoma brucei, mRNA processing is of particular importance, since most transcripts are derived from polycistronic transcription units. This organization dictates that regulated gene expression is promoter-independent and governed at the posttranscriptional level. We have identified tbCPSF30, a protein containing five CCCH zinc finger motifs, which is a homologue of the cleavage and polyadenylation specificity factor (CPSF) 30-kDa subunit, a component of the machinery required for 3'-end formation in yeast and mammals. Using gene silencing of tbCPSF30 by RNA interference, we demonstrate that this gene is essential in bloodstream and procyclic forms of T. brucei. Interestingly, tbCPSF30-specific RNA interference results in the accumulation of an aberrant tbCPSF30 mRNA species concomitant with depletion of tbCPSF30 protein. tbCPSF30 protein depletion is accompanied by the accumulation of unprocessed tubulin RNAs, implicating tbCPSF30 in polycistronic RNA processing. By genome data base mining, we also identify several other putative components of the T. brucei cleavage and polyadenylation machinery, indicating their conservation throughout eukaryotic evolution. This study is the first to identify and characterize a core component of the T. brucei CPSF and show its involvement in polycistronic RNA processing.