Christian Tschudi

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.

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

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 alpha-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.

Retention and loss of RNA interference pathways in trypanosomatid protozoans

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.

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

ABSTRACT 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.

B104, a new dispersed repeated gene family in Drosophila melanogaster and its analogies with retroviruses☆

Abstract The properties of B104, a new dispersed repeated gene family of Drosophila melanogaster are described. B104 was first discovered in a screen for genes differentially expressed at early embryonic development. The typical B104 element is 8.7 × 103 base-pairs in size and flanked by direct terminal repeats 429 base-pairs (bp) long. It is present in about 80 and 95 copies per haploid genome in Oregon R embryonic DNA and in Kcl cell line DNA, respectively, and accounts for about 0.4% of the D. melanogaster genome. While most of the B104 copies are closely conserved, a variant containing a 1.0 × 103 bp deletion as well as one containing a 2.4 × 103bp insertion have been found. B104 sequences are found in one large (about 8 × 103bp) and several short (0.8 to 1.2 × 103bp) transcripts. The DNA sequence of both terminal repeats in one element and of one terminal repeat in a second element was determined. In addition, two B104 complementary DNA clones originating from transcripts initiated and terminated, respectively, in the terminal repeat were also sequenced. The sequence data indicate: (1) that the B104 element is flanked by a 5 bp direct repeat; (2) that the sequence just internal to the left repeat shares homology with the transfer RNA primer binding site of retroviruses; and (3) that the B104 terminal repeat is redundantly transcribed in a manner typical of proviruses of vertebrate retroviruses.

Destruction of U2, U4, or U6 small nuclear RNA blocks Trans splicing in trypanosome cells

We have used permeable cells of Trypanosoma brucei to analyze the role of snRNAs in the trans splicing process. Degradation of U2, U4, or U6 snRNA by site-directed cleavage with complementary deoxyoligonucleotides and RNAase H inhibits trans splicing of the spliced leader (SL) RNA and newly synthesized alpha-tubulin pre-mRNAs. Cleavage of U snRNAs abolishes the appearance of putative trans splicing reaction intermediates and products, namely, linear branched molecules consisting of the SL intron joined to high molecular weight RNA (Y structures) and free SL intron. This indicates that U snRNAs are required for an early step in trans splicing. alpha-tubulin transcripts synthesized in the absence of trans splicing are unstable, suggesting that the addition of the SL sequence stabilizes pre-mRNAs against degradation. Our results provide direct evidence for the participation of U2 and U4/U6 snRNPs in trans splicing.

RNA Interference in Protozoan Parasites: Achievements and Challenges

ABSTRACT Protozoan parasites that profoundly affect mankind represent an exceptionally diverse group of organisms, including Plasmodium, Toxoplasma, Entamoeba, Giardia, trypanosomes, and Leishmania. Despite the overwhelming impact of these parasites, there remain many aspects to be discovered about mechanisms of pathogenesis and how these organisms survive in the host. Combined with the ever-increasing availability of sequenced genomes, RNA interference (RNAi), discovered a mere 13 years ago, has enormously facilitated the analysis of gene function, especially in organisms that are not amenable to classical genetic approaches. Here we review the current status of RNAi in studies of parasitic protozoa, with special emphasis on its use as a postgenomic tool.

Developmental Progression to Infectivity in Trypanosoma brucei Triggered by an RNA-Binding Protein

Awakening a Life Cycle Sleeping sickness continues to afflict populations in sub-Saharan Africa, but for the past 100 years, research on the Trypanosoma brucei protozoan parasite has been hampered by an inaccessibility of the insect vector stages to modern research tools. Kolev et al. (p. 1352) have identified an RNA-binding protein (RBP6) as a master regulator that drives the entire developmental program of the trypanosome through all its life-cycle stages. The ability to perform this transformation in vitro will allow study of the many biochemical and morphological changes as parasites change from dividing noninfectious forms to infectious nondividing antigenically variable forms. The developmental stages of the sleeping sickness parasite can now be observed without the tsetse fly. Unraveling the intricate interactions between Trypanosoma brucei, the protozoan parasite causing African trypanosomiasis, and the tsetse (Glossina) vector remains a challenge. Metacyclic trypanosomes, which inhabit the tsetse salivary glands, transmit the disease and are produced through a complex differentiation and unknown program. By overexpressing a single RNA-binding protein, TbRBP6, in cultured noninfectious trypanosomes, we recapitulated the developmental stages that have been observed in tsetse, including the generation of infective metacyclic forms expressing the variant surface glycoprotein. Thus, events leading to acquisition of infectivity in the insect vector are now accessible to laboratory investigation, providing an opening for new intervention strategies.

UPSTREAM TRNA GENES ARE ESSENTIAL FOR EXPRESSION OF SMALL NUCLEAR AND CYTOPLASMIC RNA GENES IN TRYPANOSOMES

An interesting feature of trypanosome genome organization involves genes transcribed by RNA polymerase III. The U6 small nuclear RNA (snRNA), U-snRNA B (the U3 snRNA homolog), and 7SL RNA genes are closely linked with different, divergently oriented tRNA genes. To test the hypothesis that this association is of functional significance, we generated deletion and block substitution mutants of all three small RNA genes and monitored their effects by transient expression in cultured insect-form cells of Trypanosoma brucei. In each case, two extragenic regulatory elements were mapped to the A and B boxes of the respective companion tRNA gene. In addition, the tRNA(Thr) gene, which is upstream of the U6 snRNA gene, was shown by two different tests to be expressed in T. brucei cells, thus confirming its identity as a gene. This association between tRNA and small RNA genes appears to be a general phenomenon in the family Trypanosomatidae, since it is also observed at the U6 snRNA loci in Leishmania pifanoi and Crithidia fasciculata and at the 7SL RNA locus in L. pifanoi. We propose that the A- and B-box elements of small RNA-associated tRNA genes serve a dual role as intragenic promoter elements for the respective tRNA genes and as extragenic regulatory elements for the linked small RNA genes. The possible role of tRNA genes in regulating small RNA gene transcription is discussed.

Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei

Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of siRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5′-monophosphate and a blocked 3′ terminus, suggesting that 3′ end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. brucei nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35- to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen.