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Christian Tschudi

Publications and source records attributed to Christian Tschudi.

At least 19 recordsLinked to original sources
PubMed2006

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei.

RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi. However, the identity of Dicer has not been resolved. Here, we report the identification and functional characterization of a T. brucei Dicer-like enzyme (TbDcl1). Using genetic and biochemical approaches, we provide evidence that TbDcl1 is required for the generation of siRNA-size molecules and for RNAi. Whereas Dicer and Dicer-like proteins are endowed with two adjacent RNase III domains at the carboxyl terminus (RNase IIIa and RNase IIIb), the arrangement of these two domains is unusual in TbDcl1. RNase IIIa is close to the amino terminus, and RNase IIIb is located approximately in the center of the molecule. This domain organization is specific to trypanosomatids and further illustrates the variable structures of protozoan Dicer-like proteins as compared to fungal and metazoan Dicer.

Amino Acid Sequence↗
PubMed2006

Functional replacement of Trypanosoma brucei Argonaute by the human slicer Argonaute2.

RNA interference (RNAi) is widespread throughout the eukaryotic lineage, from protozoa to man. Central to all RNAi phenomena is a member of the Argonaute protein family, and, in the case of dsRNA-triggered mRNA cleavage, the Ago protein functions as the RNAi endonuclease or slicer. However, at present there is no definite experimental evidence that slicer Argonautes can be interchanged between distantly related organisms. Here, we show that the human slicer Argonaute2 (HsAgo2), but not HsAgo1, functions in RNAi in the early divergent protozoan Trypanosoma brucei, thus mimicking the situation in mammalian cells. This finding indicates that the basic features of the RNAi mechanism are conserved from T. brucei to man.

Animals↗
PubMed2005

A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei.

The spliced-leader (SL) RNA plays a key role in the biogenesis of mRNA in trypanosomes by providing the m(7)G-capped SL sequence to the 5' end of every mRNA. The cap structure of the SL RNA is unique in eukaryotes with 4 nucleotides after the cap carrying a total of seven methyl groups and by convention is referred to as "cap 4". Although the enzymatic machinery for cap addition has been characterized in several organisms, including Trypanosoma brucei, the identification of methyltransferases dedicated to the generation of higher order cap structures has lagged behind, except in viruses. Here we describe T. brucei MT57 (TbMT57), a primarily nuclear polypeptide with structural and functional similarities to vaccinia virus VP39, a bifunctional protein acting at the mRNA 5' end as a cap-specific 2'-O-methyltransferase. Down-regulation by RNAi or genetic ablation of TbMT57 resulted in the accumulation of SL RNA missing 2'-O-methyl groups at positions +3 and +4 and thus bearing a cap 2 rather than a cap 4. Furthermore, competitive binding studies indicated that modifications at the +3 and +4 positions are important for binding to the nuclear cap-binding complex. Genetic ablation of MT57 resulted in viable cells with no apparent defect in SL RNA trans-splicing, suggesting that MT57 is not essential or that trypanosomes have developed alternate mechanisms to counteract the absence of this protein. Interestingly, MT57 homologs are only found in trypanosomatid protozoa that have a cap 4 structure and in poxviruses, of which vaccinia virus is a prototype.

Amino Acid Sequence↗
PubMed2005

Analysis of spliceosomal complexes in Trypanosoma brucei and silencing of two splicing factors Prp31 and Prp43.

In trypanosomatids all mRNAs undergo trans-splicing, whereas cis-splicing is restricted to a few transcripts. Trans-splicing is mechanistically similar to cis-splicing, however, little is known about the trans-splicing machinery and its underlying mechanism. In this study, we examined the involvement of splicing factors in cis- and trans-splicing by RNA interference (RNAi). Two factors (Prp31 and Prp43) were found to be essential for both pathways, suggesting that splicing factors are shared by these two reactions. We identified a 45S complex carrying pre-mRNA and all the U-snRNAs, including U1 and the SL RNA, suggesting that a single spliceosomal complex may potentially conduct both trans- and cis-splicing.

Animals↗
PubMed2005

Novel and essential subunits in the 300-kilodalton nuclear cap binding complex of Trypanosoma brucei.

One of the unique aspects of RNA processing in trypanosomatid protozoa is the presence of a cap 4 structure (m7Gpppm2(6)AmpAmpCmpm3Um) at the 5' end of all mRNAs. The cap 4 becomes part of the mRNA through trans-splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans-splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-alpha and three novel proteins that are only present in organisms featuring a cap 4 structure and trans-splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans-splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans-splicing.

Amino Acid Sequence↗
PubMed2004

Function of the Trypanosome Argonaute 1 protein in RNA interference requires the N-terminal RGG domain and arginine 735 in the Piwi domain.

Argonaute proteins are central components of RNA interference (RNAi) and related phenomena in a wide variety of eukaryotes, including the early diverging protozoan Trypanosoma brucei. The single T. brucei Argonaute protein (TbAGO1) is in a complex with small interfering RNAs (siRNAs), and a fraction of this ribonucleoprotein particle is associated with polyribosomes. In this study, we generated a panel of insertion, deletion, and single point mutants of TbAGO1 and assayed them in vivo for their function in RNAi. In addition to the signature domains of Argonaute proteins, PAZ and Piwi, TbAGO1 has an N-terminal domain with a high abundance of RGG repeats. Deletion of the N-terminal domain blocked association of AGO1 with polyribosomes and severely affected mRNA cleavage. Nevertheless, the mutant protein was in a complex with siRNAs. In contrast, deletion of the Piwi domain led to a loss of siRNAs but did not abolish polyribosome association. Site-directed mutagenesis of conserved amino acids in the Piwi domain identified arginine 735 as essential for RNAi. Although the R735A mutant bound siRNAs and associated with polyribosomes, it displayed a severe defect in the cleavage of target mRNA.

Animals↗
PubMed2004

Disruption of Ixodes scapularis anticoagulation by using RNA interference.

Ixodes scapularis ticks transmit many pathogens, including Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Vaccines directed against arthropod proteins injected into the host during tick engorgement could prevent numerous infectious diseases. Salp14, a salivary anticoagulant, poses a key target for such intervention. Salp14 is the prototypic member of a family of potential I. scapularis anticoagulants, expressed and secreted in tick saliva during tick feeding. RNA interference was used to assess the role of Salp14 in tick feeding. Salp14 and its paralogs were silenced, as demonstrated by the reduction of mRNA and protein specific for these antigens. Tick salivary glands lacking Salp14 had reduced anticoagulant activity, as revealed by a 60-80% reduction of anti-factor Xa activity. Silencing the expression of salp14 and its paralogs also reduced the ability of I. scapularis to feed, as demonstrated by a 50-70% decline in the engorgement weights. Because ticks have several anticoagulants, it is likely that the expression of multiple anticoagulants in I. scapularis saliva would have to be ablated simultaneously to abolish tick feeding. These studies demonstrate that RNA interference can silence I. scapularis genes and disrupt their physiologic function in vivo, and they identify vaccine candidates that can alter vector engorgement.

Actins↗
PubMed2004

RNA interference in protozoan parasites.

RNA interference or RNAi is defined as the mechanism through which gene-specific, double-stranded RNA (dsRNA) triggers degradation of homologous transcripts. Besides providing an invaluable tool to downregulate gene expression in a variety of organisms, it is now evident that RNAi extends its tentacles into both the nucleus and the cytoplasm and is involved in a variety of gene silencing phenomena. Here we review the current status of RNAi in protozoan parasites that cause diseases of considerable medical and veterinary importance throughout Africa, Asia and the Americas. RNAi was first discovered in Trypanosoma brucei, a species of the family Trypanosomatidae, and it rapidly became the method of choice to downregulate gene expression in these organisms. At the same time, mechanistic studies exposed a role for RNAi in the control of retroposon transcript abundance. Whereas RNAi is also present in T. congolense, other members of the same family of organisms, namely T. cruzi and Leishmania major, are RNAi-negative. In apicomplexan parasites, there is experimental evidence for RNAi in Plasmodium, but this is not supported by their genetic make up. In contrast, the genome of Toxoplasma gondii harbours gene candidates with convincing similarity to 'classical' RNAi genes. Thus, as previously shown in fungi, protozoan parasites are genetically heterogeneous as far as the RNAi pathway is concerned. Finally, database mining predicts that Entamoeba histolytica and Giardia intestinalis have an RNAi pathway and the presence of RNAi genes in Giardia supports the view that gene silencing by dsRNA appeared very early during evolution of the eukaryotic lineage.

Animals↗
PubMed2004

Role of a 300-kilodalton nuclear complex in the maturation of Trypanosoma brucei initiator methionyl-tRNA.

tRNAs are transcribed as precursors containing 5' leader and 3' extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNA(Met) in Trypanosoma brucei, an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of T. brucei that is required for trimming the 5' leader of initiator tRNA(Met) precursors. One of the subunits of the complex (T. brucei MT40 [TbMT40]) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNA(Met) containing 5' extensions but processed 3' ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNA(Met) molecules with 5' and 3' extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNA(Met). Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNA(Met), 3' trimming precedes 5' processing.

Amino Acid Sequence↗
PubMed2004

Selection and characterization of RNA interference-deficient trypanosomes impaired in target mRNA degradation.

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).

Alleles↗
PubMed2004

Argonaute protein in the early divergent eukaryote Trypanosoma brucei: control of small interfering RNA accumulation and retroposon transcript abundance.

Members of the Argonaute protein family have been linked through a combination of genetic and biochemical studies to RNA interference (RNAi) and related phenomena. Here, we describe the characterization of the first Argonaute protein (AGO1) in Trypanosoma brucei, the earliest divergent eukaryote where RNAi has been described so far. AGO1 is predominantly cytoplasmic and is found in a ribonucleoprotein particle with small interfering RNAs (siRNAs), and this particle is present in a soluble form, as well as associated with polyribosomes. A genetic knockout of AGO1 leads to a loss of RNAi, and concomitantly, endogenous retroposon-derived siRNAs as well as siRNAs derived from transgenic double-stranded RNA are reduced to almost undetectable levels. Furthermore, AGO1 deficiency leads to an increase in retroposon transcript abundance via mechanisms operating at the transcriptional level and at the RNA stability level. Our results suggest that AGO1 function is required for production and/or stabilization of siRNAs and provide the first evidence for an Argonaute protein being involved in the regulation of retroposon transcript levels.

Amino Acid Sequence↗
PubMed2004

Functional characterization of a Trypanosoma brucei TATA-binding protein-related factor points to a universal regulator of transcription in trypanosomes.

Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3' portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.

Amino Acid Sequence↗
PubMed2004

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. Currently, the method of choice to examine gene function in these organisms is RNA interference (RNAi). mRNA degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. In this chapter, we describe some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA and analysis of dsRNA, and target mRNA.

Animals↗
PubMed2004

A PCR-based method for gene deletion and protein tagging in Trypanosoma brucei.

Sequence information on the Trypanosoma brucei genome is rapidly accumulating. As a consequence, there is a need for techniques to analyze gene function systematically. Here, we describe a polymerase chain reaction (PCR)-based method for direct gene deletion and the generation of epitope-tagged fusion proteins. The approach is based on methodologies developed for Saccharomyces cerevisiae and involves PCR amplification of a reporter cassette using primers containing flanking sequences specific to the target gene. The PCR product is then transfected directly into procyclic T. brucei cells, and homologous recombinants that carry the deleted or tagged target gene are identified.

Animals↗