Res. Microbiol. 152 (2001) 123–129  2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923-2508(01)01182-2/REV

Mini-review

Genetic interference in protozoa Philippe Bastina,1 , Angélique Galvanib , Linda Sperlingb∗ a University of Manchester, School of Biological Sciences, Oxford Road, Manchester M13 9PT, UK b Centre de génétique moléculaire, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France

Received 24 October 2000; accepted 13 November 2000

Abstract – RNA interference first described in Caenorhabditis elegans and transgene-induced post-transcriptional gene silencing first described in plants and fungi now appear as different means of activating a conserved and ancient mechanism that can protect genomes against viruses and transposons and perhaps also control expression of endogenous genes. We present here similar genetic interference phenomena in highly divergent protozoa, Trypanosoma and Paramecium, and look ahead to what contribution these microorganisms could bring to this fast-moving area.  2001 Éditions scientifiques et médicales Elsevier SAS gene silencing / RNA, double-stranded / Paramecium / Trypanosoma

1. Introduction The discovery of post-transcriptional genetic interference phenomena is one of the most exciting advances in molecular genetics of recent years (many reviews are available, e.g. [7]). Introduction of transgenes in plants (post-transcriptional gene silencing, or PTGS) and fungi (quelling) or of double-stranded RNA in animals (RNA interference, or RNAi) can affect expression of homologous genes in a sequencespecific manner. The effect is maintained through mitosis but not usually meiosis and can, at least in plants and Caenorhabditis elegans, spread throughout the organism. Growing evidence that conserved molecular machinery is involved in PTGS in plants, quelling in Neurospora and RNAi in animals [11, 13, 14, 17, 24, 26, 27, 32, 41, 49, 51, 58] suggests an ancient evolutionary origin for RNA-mediated genetic interference. That this may indeed be the case is underscored by the recent discovery that highly divergent eukaryotic microorganisms (representatives of the kinetoplastid parasites and of the free-living ciliates) also

∗ Correspondence and reprints.

E-mail address: [email protected] (L. Sperling). 1 Present address: Muséum national d’histoire naturelle, Labo-

ratoire de biophysique, 43, rue Cuvier, 75231 Paris cedex 05, France.

present genetic interference phenomena. These protists, which are separated by greater evolutionary distance than yeast and man, have not only adopted very different life styles, but also have radically opposed strategies for gene expression. Genes in kinetoplastid parasites are transcribed in a polycistronic fashion and most or all regulation is post-transcriptional [52]. In contrast, ciliates regulate essentially all of their gene expression at the transcriptional level [50]. In this mini-review we will describe genetic interference in these microorganisms and discuss the biological implications. 2. RNAi in trypanosomes Trypanosomes and Leishmania, in addition to being protozoan parasites of medical and veterinary importance, have been extensively studied for their fascinating cellular and molecular biology [12, 15, 25, 35]. Over the last 10 years, spectacular progress in the understanding of these organisms has been achieved thanks to the rapid development of reverse genetics. Recently, three intriguing experiments have produced stunning phenotypes. First, transformation of Leishmania donovani with a circular plasmid expressing antisense RNA of the A2 gene led to molecular ablation of both A2 RNA and A2 protein ([59]; figure 1A). As a result, the mutant cells showed severe reduction in their ability to infect mice. This appeared

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Figure 1. Plasmids used to generate phenotypes by posttranscriptional gene silencing in Trypanosomatids. Large boxes represent gene coding sequences, small boxes represent processing signals, small boxes with arrow on top represent promoters. The white arrows indicate the orientation of coding sequences or of normal transcription for processing signals. Constructs used to silence (A) A2 expression in L. donovani [59]; (B) PFRA expression in T. brucei [5] and (C) α-tubulin expression in T. rhodesiense [42]. The thin lines represent the bacterial plasmid sequence.

to be the first successful “antisense” experiment in protozoan parasites. The A2 gene codes for proteins mostly composed of repetitions of a 10 aa motif and is expressed exclusively in the amastigote stage of the parasite [59]. Second, stable transformation of Trypanosoma brucei with a plasmid aimed at expressing antisense RNA of the PFRA gene produced the snl-1 mutant, with hardly any PFRA RNA or PFRA protein, that exhibits a dramatic paralysis phenotype ([4, 5]; figure 1B). The PFRA gene encodes a major protein of the paraflagellar rod, a large latticelike structure present inside the flagellum [3, 47]. Finally, transient transfection of trypanosomes with a plasmid called pGFPFAT (initially constructed for unrelated reasons) reduced α-tubulin expression and led to severe defects in cytokinesis, producing abnormally large cells termed FAT ([42]; figure 1C). The link between these apparently unrelated experiments is the fact that dsRNA can be expressed in all three situations (figures 1 and 2).

Ngô et al. [42] noticed that transcription of the pGFPFAT plasmid would produce dsRNA of the 5 untranslated region (UTR) of α-tubulin. Transient transfection of an in vitro transcribed and annealed dsRNA of the 5 UTR α-tubulin reproduced the FAT phenotype, whereas introduction of the singlestranded RNA (sense or antisense) had no apparent effect. The same phenotype was observed when trypanosomes were transfected with dsRNA derived from a 115-nt fragment of the α-tubulin coding region, or with dsRNA of the 5 UTR of β-tubulin (which is only 59 nt long). A few months before, Fire et al. [21] had convincingly demonstrated that microinjection of C. elegans worms with dsRNA of a particular gene leads to rapid, potent and specific degradation of the corresponding mRNA. This effect was termed RNA interference (RNAi), and was proposed as an explanation for various posttranscriptional gene silencing phenomena reported in plants and fungi [39]. A similar explanation was provided by Bastin et al. [2], who noticed that the antisense plasmid had to be inserted in the PFRA gene cluster to elicit the paralysis phenotype (trypanosomes possess 4 nearly identical copies of the PFRA gene that are transcribed in a polycistronic fashion; [18]). On the contrary, direct expression of PFRA antisense RNA had little effect on PFRA expression or cell motility. Integration of the antisense plasmid in the PFRA gene cluster means that transcription can now occur on both strands and therefore PFRA sense and antisense RNAs can be produced in situ (figure 2A). Moreover, the hybrid PFRA genes generated by the homologous recombination event are not flanked by valid processing signals and so are likely to accumulate at the transcription site [16], hence facilitating the formation of dsRNA. This interpretation was confirmed by stable transformation of trypanosomes with a plasmid able to express linked sense and antisense copies of the PFRA gene (figure 2B). Expression of this PFRA dsRNA was controlled by a tetracyclineinducible promoter [56]. In the absence of induction, the transformed trypanosomes displayed normal amounts of PFRA and behaved normally, but after induction, PFRA RNA rapidly disappeared and production of new PFRA protein was halted. As a result, the cells stopped swimming and looked paralysed. Interestingly, the interference effect proved to be reversible once the tetracycline inducer was removed

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initiation of transcription takes place. It is possible that both strands of circular plasmids transformed in Leishmania are transcribed, making the interpretation of the above results rather delicate. Direct transformation with A2 dsRNA should help resolve this issue. 3. Homology-dependent gene silencing in Paramecium

Figure 2. Production of PFRA dsRNA is responsible for PFRA ablation in the T. brucei snl -1 mutant. (A) Insertion of the PFRA ‘antisense’ plasmid within the PFRA gene cluster (snl -1 genotype) leads to overlapping production of sense and antisense PFRA RNA. As a result of the insertion, several PFRA RNAs do not possess valid 5 or 3 processing signals (indicated by stars, above the gene for the sense orientation, below it for the antisense orientation). Trypanosomes possess four PFRA genes in a cluster and are diploid. Insertion occurred in only one allele, the other one, shown on top, is not modified. (B) The pαPFRA430 plasmid allows direct production of PFRA dsRNA under the control of an inducible promoter (snl -2 cell line, see text). None of the endogenous PFRA gene clusters (shown on top) are affected.

from the medium, arguing against permanent genetic modification at the DNA level. More unexpected results were reported by Zhang and Matlashewski [60], who dissected the original ‘antisense’ A2 plasmid (figure 1), in an attempt to understand what elements were requested for successful A2 ablation. Surprisingly, the expression of an RNA linking A2 sense and antisense from an episome did not produce A2 ablation, whereas expression of A2 antisense alone did [60]. However, two elements have to be taken into consideration. First, the transformed populations in these experiments have not been cloned and the variability within experiments has not been assessed. Second, one has to remember that true RNA polymerase promoters have not been identified in Leishmania and it is still not clear how

Paramecium is a free-living ciliate which has been used for over 50 years for genetic analysis of a variety of cellular processes such as membrane excitability, regulated secretion, surface antigen variation, cellular morphogenesis and programmed genome rearrangements [10, 30, 34, 37, 46, 54]. One of the most fascinating aspects of Paramecium biology is nuclear dimorphism, a characteristic of ciliates. Each individual has two types of nuclei, a diploid germline nucleus (micronucleus) that intervenes in sexual processes and a polyploid somatic nucleus (macronucleus) that assures transcriptional activity during vegetative growth. At each sexual generation the macronucleus is destroyed and a new one is formed from the zygotic micronucleus, by programmed rearrangements of the entire genome (review: [43]). Analysis of gene function has recently become possible in Paramecium, thanks to the discovery of a homology-dependent epigenetic phenomenon that shares many of the characteristics of post-transcriptional gene silencing. The introduction of the coding region of a gene into the somatic macronucleus at high copy number leads to reduced expression of the cellular homologues of the gene and striking phenotypes [45]. Promoter sequences are not required to obtain silencing. Silenced cells are characterized by the specific reduction of mRNA and the appearance of aberrantly sized RNA molecules both larger and smaller than the mRNA, arguing that the phenomenon is post-transcriptional. The silencing effect is stable through vegetative growth but is lost at sexual processes when the transformed macronucleus is degraded. Does the mechanism involve dsRNA? For the moment, evidence to the affirmative is indirect. To appreciate the indirect evidence, we need to consider the mechanics of transformation in Paramecium. Transformation of the Paramecium somatic macronucleus is achieved by microinjection of DNA. Transformation is remarkably efficient because the injected DNA

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Figure 3. Gene silencing in Paramecium by transformation with gene coding regions. The ND7 gene is required for exocytotic membrane fusion, easily evaluated on single cells by use of the fixative picric acid (inset on left). Microinjection of wild-type cells with the coding region of the ND7 gene (top) yields cells that have lost their exocytotic capacity (exo− phenotype) while microinjection of an equivalent number of copies of the complete ND7 gene with flanking sequences sufficient for expression does not affect exocytotic capacity (exo+ phenotype). Data from [45].

is processed and maintained as a pseudo macronuclear chromosome rather than integrated into the host genome [8, 23]. Microinjected DNA is linearized if circular, the linear molecules are joined end-to-end to produce a series of multimers and the ends of these multimers receive telomeric repeats. These linear episomes are replicated along with the other macronuclear chromosomes and are thus maintained for the vegetative life of the clone. If DNA containing complete genes with their 5 and 3 flanking sequences is microinjected into the macronucleus, the genes are expressed. In this way, mutant phenotypes can be complemented by injection of the corresponding wild-type gene, allowing genes to be cloned by functional complementation [28, 48]. Cells transformed with complete genes produce the corresponding mRNA and no aberrant RNA. Transformation with only the coding region of a gene, in the absence of a promoter and downstream signals for transcription termination, gives a radically different result, i.e. reduced expression of the homologous cellular genes (figure 3). Silenced cells contain reduced amounts of the corresponding mRNA along with large quantities of aberrantly sized RNA molecules homologous to the transforming DNA. These aberrantly sized RNA molecules hybridize on Northern blots with both sense and antisense probes (Galvani, unpublished data) and with plasmid sequences present in the transforming DNA [45]. The aberrant RNA is thus produced by transcription of the transforming DNA, from cryptic initiation sites situated

on either strand. The aberrant RNA very likely contains at least some double-stranded RNA molecules since both sense and corresponding antisense RNA are detected on northern blots. It thus seems highly probable that in Paramecium as in trypanosomes, double-stranded RNA is instrumental in obtaining genetic interference effects. Experiments with constructs designed to express sense, antisense or doublestranded RNA under the control of defined regulatory sequences should clarify this point. 4. Biological implications and perspectives The discovery of genetic interference phenomena is perfectly timed with the post-genomic era. In trypanosomes, the combination of RNAi and the tetracycline-inducible expression system offers a fantastic tool to study gene function. We can now study over time how a particular gene product disappears from the cell, but also how it reappears, features that have already allowed interesting observations on cytoskeletal structure assembly in trypanosomes [2]. In Paramecium, homology-dependent gene silencing already provides a powerful tool for functional analysis, rapidly yielding spectacular phenotypes for most of the genes that have been studied, for example genes involved in cytoskeleton organization [45], basal body duplication [44] and regulated secretion [22, 45]. Moreover, the function of essential genes can be assessed with far less ambiguity than in knockout experiments since it is now possible to

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visualise how a transformed cell dies, as documented in both organisms (L. Kohl, P.B. and K. Gull, unpublished data; [44]). In trypanosomes, the knowledge of the full sequence of a gene and its flanking sequences is not needed, since dsRNA corresponding to gene fragments is sufficient to confer the interference effect ([42]; L. Kohl, P. Bastin and K. Gull, unpublished data). This may also turn out to be the case in Paramecium. Aside from providing biologists with the ideal means of inactivating genes, what are the natural functions of genetic interference? Genetic analysis in Neurospora [11, 13, 14], Arabidopsis [17, 41] and C. elegans [24, 32, 49, 51] and in vitro experiments in Drosophila [26, 58] suggest a system of ‘genetic immunity’ that plays an important role in genome defense. Double-stranded RNA produced by replication of viruses or transcription of multi-copy foreign DNA such as transposons, induces assembly of ribonucleoprotein complexes that specifically degrade RNA homologous to the dsRNA molecules. These complexes contain 21–23-nt dsRNA molecules which confer the sequence-specificity [27, 58]. A seductive model [1] proposes that the small RNAs of the complex base pair with an invading mRNA strand in a reaction involving ATP-dependent helicase activity. An endonuclease activity then cleaves the invading mRNA thus achieving sequence-specific mRNA degradation and at the same time regenerating an active ribonucleoprotein particle. These small 21–23-nt RNAs have been detected in conjunction with PTGS and RNAi in plants [26] and Drosophila [27], respectively. Experimental evidence is available in plants that genetic interference can protect the genome against viruses [9, 31, 55]. In C. elegans, several genes necessary for RNAi are implicated in the control of transposition [24, 32]. Such a role for genetic interference is possible in Paramecium. There is good evidence that the genomes of ciliates once contained and may still harbour transposons. One class of germline-limited sequences, that are precisely removed during macronuclear development (internal eliminated sequences or IESs, present as ∼ 60 000 unique sequence elements in the germline), appear to be degenerate Tc1/mariner family transposons [6, 33, 34]. Intact transposons have not yet been found in Paramecium, but have been reported in the germline of other ciliates [29, 57]. Vegetative gene silencing would provide a means of inactivating transposons or

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other parasitic DNA not excised during macronuclear development. Another homology-dependent phenomenon occurs in Paramecium, during macronuclear development. Certain characters, such as mating type in Paramecium tetraurelia, display maternal heredity, i.e. they are determined by the somatic macronucleus at the previous sexual generation. Genetic and molecular analyses of maternal heredity indicate that it involves transfer of information from the old macronucleus to the new one. This information perpetuates the pattern of the developmental DNA rearrangements across sexual generations with no modification of the germline genome [20, 36 – 38]. As the transferred information is sequence-specific, it has been proposed that nucleic acid (most likely RNA) produced by the old macronucleus travels through the cytoplasm to the macronucleus being neoformed and acts through base pairing to guide the DNA rearrangements. It will be important to determine whether these two homologydependent phenomena (vegetative gene silencing and epigenetic control of developmental DNA rearrangements) which operate in alternative phases of the Paramecium life cycle, rely on the same cellular machinery. Genetic interference may fulfill functions other than genome defense, for example in C. elegans some genes required for RNAi are also involved in germline development [49] and a subset of the smg genes required for the nonsense-mediated RNA decay pathway also appear to be involved in RNAi [19]. Interestingly, in trypanosomatids, control of gene expression is mostly achieved at the post-transcriptional level, where the 3 untranslated region plays a central role [52]. In some cases, initiation of transcription takes place but mature RNA is not detected [53]. This can be paralleled with RNAi, that does not seem to affect transcription initiation [40, 42]. RNAi may reveal pathways of RNA metabolism that at least partially overlap with this particular mode of posttranscriptional regulation of gene expression. In the past, particular properties of protozoa have facilitated important discoveries in molecular biology, such as ribozymes, RNA editing, telomerase, histone acetylase and GPI anchors, which all turned out to be of major significance in metazoa. The pronounced and often original characteristics evolved by protozoa may also bring new insights into the biological significance of genetic interference mechanisms.

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