Background In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.
Background
In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.Results Using the protozoan  Trypanosoma brucei  as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 ( TbPFR2 ), whose product is essential for flagellar motility. We demonstrate the complementation of  TbPFR2 , silenced  via  dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant  TbPFR2  encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of  TbPFR2 , silenced  via  dsRNA targeting its coding sequence, through heterologous expression of the  TbPFR2  orthologue gene from  Trypanosoma cruzi : the flagellum regained its motility.
Results
Using the protozoan  Trypanosoma brucei  as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 ( TbPFR2 ), whose product is essential for flagellar motility. We demonstrate the complementation of  TbPFR2 , silenced  via  dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant  TbPFR2  encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of  TbPFR2 , silenced  via  dsRNA targeting its coding sequence, through heterologous expression of the  TbPFR2  orthologue gene from  Trypanosoma cruzi : the flagellum regained its motility.Trypanosoma bruceiTbPFR2TbPFR2viaTbPFR2TbPFR2viaTbPFR2Trypanosoma cruziConclusions This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.
Conclusions
This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.

Background RNA interference (RNAi) can be triggered by introduction of long double-stranded RNA molecules (dsRNAs) in cells [ 1 ], and proceeds in a number of sequential steps, starting with the cleavage of long dsRNAs into shorter ≈ 21–23 nucleotide-long dsRNAs called short interfering RNAs (siRNAs; these were initially discovered in plants [ 2 ]). The enzyme responsible for this chopping (DICER; [ 3 , 4 ]) displays RNase III activity, producing characteristic siRNAs with a phosphorylated 5' end and a two nucleotide-overhanging 3'OH end. These siRNAs enter an RNA-induced silencing complex, or RISC [ 5 , 6 ]. A helicase activity unwinds the two strands of the siRNA, and RISC scans the mRNAs in the cytoplasm and cleaves the molecules that are found complementary to the RISC-contained siRNA [ 5 ]. RNA-silencing processes have been described in a variety of organisms: post-transcriptional gene silencing in plants [ 7 , 8 ], quelling in fungi [ 9 ], homology-dependent gene silencing in ciliates [ 10 ], or RNA interference in worms [ 1 ], flies [ 11 , 12 ], trypanosomes [ 13 , 14 ] and mammals [ 15 , 16 ]. It is thought that this machinery has evolved to protect cells against undesirable RNAs, like RNA viruses in plants [ 17 , 18 ], or to limit the mobility of transposable elements in animals [ 19 - 21 ]. While RNAi and associated phenomena constitute exceptional recent basic science findings, they also provided a basis for the elaboration of powerful research tools. RNAi methodologies have been set up to perform reverse-genetics studies in a number of organisms. RNAi potency and flexibility have allowed to perform high-throughput genetic screens in several organisms [ 22 - 26 ]. In mammalian cells, the presence of long dsRNA ( > 50 base pairs) triggers the activation of sequence-unspecific interferon-related pathways [ 27 - 29 ]. To circumvent this difficulty, researchers resorted to the transfection of small interfering RNAs [ 16 ] or  in vivo  synthesis of small hairpin RNAs, which were demonstrated to produce gene-specific silencing [ 27 , 30 , 31 ]; reviewed in [ 32 , 33 ]. However, an siRNA might trigger a number of potential unspecific events such as the degradation of partially complementary mRNA due to cross-hybridization, leading to unspecific RNAi, or the translational arrest due to a micro RNA-like effect where an siRNA hybridizes to a mRNA with one or few mismatches. It is thus of paramount importance to ensure that the phenotypic effects observed as a result of siRNA presence in cells are due to silencing of the target gene only. Two large-scale studies show that siRNA-induced gene silencing of transiently- or stably-expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genome-wide expression profiling [ 34 , 35 ]. In contrast, other works provided evidence that siRNAs can be target-unspecific, with the observation of silencing of genes that had limited sequence homology with the siRNA [ 36 , 37 ]. These reports should prompt scientists to assess the specificity of RNAi-silencing in any experiment. A solution to that problem, that we devised in trypanosomes and which is described in this report, is based on the rescueing of the RNAi-mediated loss-of-function phenotype by expressing an RNAi-resistant version of the target gene. Trypanosomes are protozoan parasites belonging to the Kinetoplastida order. These unicellular flagellated organisms diverged very early in eukaryotic evolution, and exhibit a number of original features [ 38 - 40 ]. Trypanosomes were amongst the first organisms where RNAi was discovered [ 13 , 14 ], and a number of strategies have been devised to either transiently or permanently induce gene-specific RNAi-silencing in these cells [ 14 , 41 - 43 ]. Examples of successful RNAi in trypanosomes used flagellar genes as targets which yielded easily monitored phenotypes [ 44 ]. From a structural point of view, the most conserved morphological feature of eukaryotic flagella is the axoneme, which is made of nine doublets of outer microtubules plus 2 central microtubules (so-called 9+2 axonemal structure). In trypanosomes, the flagellum not only has that axone-mal structure, but it also has a lattice-like structure called the paraflagellar rod (PFR) that is positioned along the axoneme. The two main components of the PFR are TbPFR2 and TbPFR1, that share 60% primary sequence identity [ 45 , 46 ].  TbPFR2  silencing leads to flagellar paralysis and trypanosomes do not swim anymore [ 13 , 47 ]. During the cell cycle, the cell first replicates its mitochondrial DNA (kinetoplast) and starts to grow a new flagellum whilst maintaining the old flagellum in place. Hence, a trypanosome which has two kinetoplasts and two nuclei will be close to completion of its cell cycle and will possess an old and a new flagellum [ 48 ]. This aspect is an interesting feature for RNAi-based studies of flagellar morphogenesis, because bi-flagellated cells have an "internal control" flagellum (the old one), while the new one has a phenotype corresponding to the RNAi-based gene knock-down. The presence of both the old and the new flagella in the same cell gives an indication of the time course of events when RNAi is induced in trypanosomes, leading to the appearance of a visible phenotype in the new flagellum while the older one is unchanged because it is not affected by gene silencing. We previously established the degree of identity between the gene sequences capable of leading to cross-RNAi [ 47 , 49 ]. However, as mentioned earlier, each time a phenotype is observed in RNAi experiments, it is necessary to ensure that it is indeed due to the specific silencing of the targeted gene(s). Inspired by the procedure with which gene knock-out is usually performed (the control experiment is done by re-introducing the knocked-out gene to ensure that the lost function gets complemented), we devised a functional complementation strategy aimed at assessing that RNAi indeed targets the intended gene. This strategy, elaborated using the  TbPFR2  gene as a model system, involved the silencing of the  TbPFR2  target  via  its UTRs and the expression of a RNAi-resistant copy of the targetted gene. The RNAi-resistant gene was either a copy of  TbPFR2  with different UTRs or its  Trypanosoma cruzi  orthologue:  TcPFR2 . We found that inter-species complementation experiments were straight forward. This strategy opens a venue for functional gene dissection experiments where modified gene sequences can be tested for their ability to encode functional protein that can complement the RNAi-based loss-of-function phenotype.
Background
RNA interference (RNAi) can be triggered by introduction of long double-stranded RNA molecules (dsRNAs) in cells [ 1 ], and proceeds in a number of sequential steps, starting with the cleavage of long dsRNAs into shorter ≈ 21–23 nucleotide-long dsRNAs called short interfering RNAs (siRNAs; these were initially discovered in plants [ 2 ]). The enzyme responsible for this chopping (DICER; [ 3 , 4 ]) displays RNase III activity, producing characteristic siRNAs with a phosphorylated 5' end and a two nucleotide-overhanging 3'OH end. These siRNAs enter an RNA-induced silencing complex, or RISC [ 5 , 6 ]. A helicase activity unwinds the two strands of the siRNA, and RISC scans the mRNAs in the cytoplasm and cleaves the molecules that are found complementary to the RISC-contained siRNA [ 5 ].RNA-silencing processes have been described in a variety of organisms: post-transcriptional gene silencing in plants [ 7 , 8 ], quelling in fungi [ 9 ], homology-dependent gene silencing in ciliates [ 10 ], or RNA interference in worms [ 1 ], flies [ 11 , 12 ], trypanosomes [ 13 , 14 ] and mammals [ 15 , 16 ]. It is thought that this machinery has evolved to protect cells against undesirable RNAs, like RNA viruses in plants [ 17 , 18 ], or to limit the mobility of transposable elements in animals [ 19 - 21 ].While RNAi and associated phenomena constitute exceptional recent basic science findings, they also provided a basis for the elaboration of powerful research tools. RNAi methodologies have been set up to perform reverse-genetics studies in a number of organisms. RNAi potency and flexibility have allowed to perform high-throughput genetic screens in several organisms [ 22 - 26 ]. In mammalian cells, the presence of long dsRNA ( > 50 base pairs) triggers the activation of sequence-unspecific interferon-related pathways [ 27 - 29 ]. To circumvent this difficulty, researchers resorted to the transfection of small interfering RNAs [ 16 ] or  in vivo  synthesis of small hairpin RNAs, which were demonstrated to produce gene-specific silencing [ 27 , 30 , 31 ]; reviewed in [ 32 , 33 ].>in vivoHowever, an siRNA might trigger a number of potential unspecific events such as the degradation of partially complementary mRNA due to cross-hybridization, leading to unspecific RNAi, or the translational arrest due to a micro RNA-like effect where an siRNA hybridizes to a mRNA with one or few mismatches. It is thus of paramount importance to ensure that the phenotypic effects observed as a result of siRNA presence in cells are due to silencing of the target gene only. Two large-scale studies show that siRNA-induced gene silencing of transiently- or stably-expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genome-wide expression profiling [ 34 , 35 ]. In contrast, other works provided evidence that siRNAs can be target-unspecific, with the observation of silencing of genes that had limited sequence homology with the siRNA [ 36 , 37 ]. These reports should prompt scientists to assess the specificity of RNAi-silencing in any experiment. A solution to that problem, that we devised in trypanosomes and which is described in this report, is based on the rescueing of the RNAi-mediated loss-of-function phenotype by expressing an RNAi-resistant version of the target gene.Trypanosomes are protozoan parasites belonging to the Kinetoplastida order. These unicellular flagellated organisms diverged very early in eukaryotic evolution, and exhibit a number of original features [ 38 - 40 ]. Trypanosomes were amongst the first organisms where RNAi was discovered [ 13 , 14 ], and a number of strategies have been devised to either transiently or permanently induce gene-specific RNAi-silencing in these cells [ 14 , 41 - 43 ]. Examples of successful RNAi in trypanosomes used flagellar genes as targets which yielded easily monitored phenotypes [ 44 ]. From a structural point of view, the most conserved morphological feature of eukaryotic flagella is the axoneme, which is made of nine doublets of outer microtubules plus 2 central microtubules (so-called 9+2 axonemal structure). In trypanosomes, the flagellum not only has that axone-mal structure, but it also has a lattice-like structure called the paraflagellar rod (PFR) that is positioned along the axoneme. The two main components of the PFR are TbPFR2 and TbPFR1, that share 60% primary sequence identity [ 45 , 46 ].  TbPFR2  silencing leads to flagellar paralysis and trypanosomes do not swim anymore [ 13 , 47 ]. During the cell cycle, the cell first replicates its mitochondrial DNA (kinetoplast) and starts to grow a new flagellum whilst maintaining the old flagellum in place. Hence, a trypanosome which has two kinetoplasts and two nuclei will be close to completion of its cell cycle and will possess an old and a new flagellum [ 48 ]. This aspect is an interesting feature for RNAi-based studies of flagellar morphogenesis, because bi-flagellated cells have an "internal control" flagellum (the old one), while the new one has a phenotype corresponding to the RNAi-based gene knock-down. The presence of both the old and the new flagella in the same cell gives an indication of the time course of events when RNAi is induced in trypanosomes, leading to the appearance of a visible phenotype in the new flagellum while the older one is unchanged because it is not affected by gene silencing.TbPFR2We previously established the degree of identity between the gene sequences capable of leading to cross-RNAi [ 47 , 49 ]. However, as mentioned earlier, each time a phenotype is observed in RNAi experiments, it is necessary to ensure that it is indeed due to the specific silencing of the targeted gene(s). Inspired by the procedure with which gene knock-out is usually performed (the control experiment is done by re-introducing the knocked-out gene to ensure that the lost function gets complemented), we devised a functional complementation strategy aimed at assessing that RNAi indeed targets the intended gene. This strategy, elaborated using the  TbPFR2  gene as a model system, involved the silencing of the  TbPFR2  target  via  its UTRs and the expression of a RNAi-resistant copy of the targetted gene. The RNAi-resistant gene was either a copy of  TbPFR2  with different UTRs or its  Trypanosoma cruzi  orthologue:  TcPFR2 . We found that inter-species complementation experiments were straight forward. This strategy opens a venue for functional gene dissection experiments where modified gene sequences can be tested for their ability to encode functional protein that can complement the RNAi-based loss-of-function phenotype.TbPFR2TbPFR2viaTbPFR2Trypanosoma cruziTcPFR2Results and discussion Multiple RNAi on trypanosomes We wanted to establish if the co-transfection of two distinct dsRNAs, targeting two different genes, could trigger their simultaneous silencing. The genes selected were  TbPFR2  and  FLA1 ;  TbPFR2  encodes one of the two major components of the paraflagellar rod and is necessary for flagellum motility [ 13 ];  FLA1  encodes a protein required for flagellum attachment to the cell body [ 50 ]. These dsRNAs were transfected simultaneously in wild-type trypanosomes. As a control experiment, we used  GFP  dsRNA. Cells were monitored for their acquired phenotype 15 h and 22 h after transfection (Table  1 ). The extinction of TbPFR2 was followed by immunofluorescence microscopy using the L8C4 anti-TbPFR2 monoclonal antibody.  FLA1  gene silencing was analyzed by differential interference contrast microscopy, as it results in the visible detachement of the flagellum from the cell body (Figure  2 ). The transfection of  TbPFR2  dsRNA yielded potent silencing, as more than 60 % of the cells showed no staining for L8C4 15 h later. Since old flagella pre-exist in cells which were affected by RNAi at the beginning of cell replication, the real percentage of silenced cells is probably higher than 60 %, which is confirmed by the fact that it built up to more than 74 % at time point 22 h. The transfection of  FLA1  dsRNA produced a phenotype in which the flagellum was detached from the cell body in more than 50 % of the cells. When both dsRNAs were co-transfected, both phenotypes were indeed observed, with similar frequencies to experiments where only one dsRNA was transfected. All the transfected cell populations did show a comparable growth rate (data not shown). The trypanosomes shown in Figure  2  had been transfected with both dsRNAs and the cell on the right is starting cytokinesis. The old flagellum of that cell is detached, while the new flagellum is attached along the cell body. The new flagellum exhibits a dilation of its distal tip, probably corresponding to the accumulation of TbPFR1, that is not assembled but still transported to the distal tip of the flagellum in the absence of TbPFR2 [ 13 ]. This observation demonstrates the usefulness of double-transfection experiments also for kinetics analysis. In our case, 22 h after dsRNAs transient transfection, the phenotype due to the  FLA1  silencing is no longer visible in the new flagellum while that same flagellum still exhibits the phenotype due to the  TbPFR2  silencing, clearly indicating different turn-over for TbPFR2 and FLA1 proteins. The RNAi machinery could cope with two different dsRNA populations, without – in our conditions – any visible saturation effect. These results show the feasibility of experiments involving the use of multiple dsRNAs, thus allowing studies on complex processes in the cell physiology. However, such complex experiments can only be envisaged after ensuring that the phenotypes resulting from RNAi are specifically due to silencing of the target gene. In order to address that specific problem, we elaborated a method that involves RNAi experiments on trypanosomes that were engineered to possess an extra RNAi-resistant copy of the targeted gene, leading to functional complementation. Gene silencing by dsRNA targeting UTRs As a model for this study, we chose the  TbPFR2  gene, which is present in four copies in the WT trypanosome genome (Figure  1A ), all transcribed as a single long polycistronic mRNA. All these gene copies are separated by three identical intergenic UTRs (igUTR), while the first copy has a unique 5' UTR and the last copy has a unique 3'UTR. Three types of dsRNA populations were used in our experiments, and termed as follows. dsRNA homologous to the  GFP  sequence was labelled "GFP dsRNA"; dsRNA homologous to coding sequence of the  TbPFR2  gene was labelled "CDS dsRNA"; finally, the mixture of three dsRNAs homologous to the 5' UTR, igUTR and 3'UTR of the  TbPFR2  gene was termed "UTRs MIX dsRNAs" (Figure  1A ). These dsRNAs were transfected into three cell lines: WT,  TbPFR2tag  and  TbPFR2tag-ΔHLA  (see Methods). For each experiment, the presence or absence of TbPFR2 in the new flagellum of bi-nucleated/bi-flagellated cells was monitored by immunofluorescence 14 h after the transfection (Table  2 ). Reports in [ 14 , 24 ] showed that RNAi silencing of a gene can be accomplished by targeting transcribed non-coding sequences. Here, we wanted to make sure that this kind of experiment was still feasible with a more complex system such as the  TbPFR2  multigene locus, where multiple and distinct UTRs regulate the expression of four  TbPFR2  isogenes. We first transfected WT trypanosomes with GFP dsRNA as a negative control and did not detect any  TbPFR2  silencing (Figure  3A ). Second, WT trypanosomes were transfected with the CDS dsRNA: 88 % of cells showed typical  TbPFR2  silencing with an anti-TbPFR2 immunofluorescence showing that the protein was missing from the new flagellum (Figure  3B ). Finally, the WT trypanosomes were transfected with the UTRs MIX dsRNAs, yielding the same phenotype as for the CDS dsRNA, although the silencing appeared less pronounced (Figure  3C  and Table  2 ). Overall, these results demonstrate that RNAi could efficiently silence all of the  TbPFR2  gene copies by targeting non-coding sequences present at the mRNA level. When WT trypanosomes were transfected with  TbPFR2  dsRNA complementary to only one UTR, the cells did not display any specific phenotype (data not shown). This observation is probably explained by the organization of the  TbPFR2  locus: the polycistronic transcript is rapidly spliced into three different types of mRNA, each encoding one of the four copies of  TbPFR2  [ 51 - 53 ]. Thus, even if one type of  TbPFR2  RNA is destroyed, the three remaining ones would likely provide enough RNA to synthesize TbPFR2 levels compatible with normal PFR formation. To demonstrate that the silencing observed upon transfection of WT trypanosomes with the UTRs MIX dsRNAs was due to the actual targeting of  TbPFR2 , we used two cell lines expressing a supplementary tagged  TbPFR2  gene copy. The  TbPFR2tag  cell line expresses the TbPFR2-TAG protein which correctly localizes to the flagellum. The  TbPFR2tag-ΔHLA  cell line expresses TbPFR2-TAG-ΔHLA, lacking the HLA tripeptide, which prevents its localization to the flagellum. To determine both the cellular localization of the tagged TbPFR2 proteins (TbPFR2-TAG and TbPFR2-TAG-ΔHLA) and the completeness of the PFR assembly, immunofluorescence experiments were carried out with the BB2 and ROD-1 antibodies; the former recognizes the Ty-1 epitope tag present on the two tagged TbPFR2 proteins [ 54 ], while the latter is a marker for full PFR assembly [ 55 , 56 ]. GFP dsRNA was transfected as a negative control in each cell line. As expected, this did not yield any  TbPFR2  silencing: TbPFR2 was decorated in both the old and new flagella by the anti-TbPFR2 antibody, and the PFR could be assembled fully, as evidenced by its staining with the ROD-1 antibody (data not shown, Table  2 ). In  TbPFR2tag  cells, TbPFR2-TAG was able to localize to the PFR, as evidenced by the PFR decoration with BB2 (red color, Figure  4A ). In contrast, TbPFR2-TAG-ΔHLA failed to do so in  TbPFR2tag-ΔHLA  cells, and the BB2 signal was detected in the cytoplasm, as expected (red color, Figure  4D ). We next compared  TbPFR2tag  trypanosomes after transfection with either CDS dsRNA or UTRs MIX dsRNAs.  TbPFR2tag  cells transfected with CDS dsRNA had a flagellum not (or faintly) decorated with the anti-TbPFR2 antibody, demonstrating that both the WT and the recombinant  TbPFR2  gene copies were effciently silenced (Table  2 ). That result was confirmed with anti-TAG immunofluorescence that showed no staining of the flagellum, demonstrating that TbPFR2-TAG was absent (no red color, Figure  4B ). This lack of both TbPFR2 and TbPFR2-TAG led to an incomplete assembly of the PFR, which was therefore not decorated with the ROD-1 antibody (no yellow color, Figure  4B ). In contrast, cells transfected with the UTRs MIX dsRNAs exhibited a WT phenotype, with only 2 % of the cells displaying  TbPFR2  silencing in the flagellum (Table  2 ). In this case, the tagged protein was expressed, leading to complete assembly of the PFR (yellow color, Figure  4C ) because the protein is functional and localized to the flagellum (red color, Figure  4C ; [ 56 ]). This remarkable result indicates a complementation phenomenon that is explained by the fact that the tagged  TbPFR2  gene was not silenced, as it was expressed from a coding sequence flanked by UTRs from the expression vector: from the 5' UTR of the procyclin gene and from the 3'UTR of the aldolase gene (Figure  1B ; [ 57 ]). To definitely demonstrate that the complementation described above is indeed due to the expression of functional TbPFR2-TAG, we transfected the same dsRNA into  TbPFR2tag-ΔHLA  trypanosomes expressing a modified TbPFR2 protein missing three amino acids (that is nine nucleotides out of 1800). TbPFR2tag-ΔHLA does not access the flagellar compartment and thus cannot be functional [ 58 ]. Transfecting either CDS dsRNA or UTRs MIX dsRNAs produced cells in which the new flagellum was not (or faintly) decorated by the anti-TbPFR2 antibody (Table  2 ). TbPFR2-TAG-ΔHLA was not decorated by the anti-TAG antibody when cells were transfected with CDS dsRNA (red color, Figure  4E ), indicating that both the WT and the tagged  TbPFR2  copies were silenced, thus leading to an incomplete PFR edification (yellow color, Figure  4E ). In contrast, transfection of UTRs MIX dsRNAs did not prevent the expression of the recombinant TbPFR2-TAG-ΔHLA protein, as it appeared stained by the anti-TAG antibody (red color, Figure  4F ). However, that non-functional protein could not participate in the construction of the PFR, as shown by the absence of ROD-1 signal in the new flagellum, since it cannot access the flagellum (yellow color, Figure  4F ). RNA-directed RNA polymerase activity (RdRP) has been implicated as one possible step in the formation of siRNA in fungi [ 59 ], plants [ 17 , 18 ], and worms [ 60 ]. The fact that we could specifically silence WT  TbPFR2  by targeting its UTRs, without interfering with the tagged  TbPFR2  genes, suggests that spreading of silencing beyond the initial targeted sequence does not occur in trypanosomes [ 61 - 64 ]. Functional complementation with orthologue genes We next asked if an RNAi-mediated loss of function could be complemented by the expression of a gene orthologue to the silenced one. The system used to answer that question involved the  TbPFR2i  cell line – that expresses  TbPFR2  dsRNA under the control of a tetracycline-inducible promoter [ 47 ] – into which constitutive expression of  Trypanosoma cruzi TbPFR2  orthologue ( TcPFR2 ) was established using stable transfection procedures. TbPFR2 and TcPFR2 proteins share 90 % identity (both of them are recognized by the anti-TbPFR2 L8C4 antibody), but their gene sequences have diverged enough for us to envisage that the RNAi-silencing of  TbPFR2  would not affect significantly the introduced  TcPFR2  gene (83 % overall nucleotide identity). We thus created two new cell lines based on the previously described  TbPFR2i  cells [ 47 ] (see Methods). TbPFR2 expression and cell motility were analyzed. Our first experiment showed that the  PCGFP  cells constitutively expressed GFP, as detected by microscope observation of living cells (data not shown). Both the  PCGFP  and  PCTcPFR2  cell lines were induced to express  TbPFR2  dsRNA for 48 hours. Immunofluorescence revealed that non-induced  PCGFP  cells exhibit a WT-like phenotype (Figure  5A ,  - TET). When these cells were induced with tetracycline, expected  TbPFR2  silencing occurred (Figure  5A , +TET). Non-induced  PCTcPFR2  cells displayed an intense anti-TbPFR2 antibody decoration with bright dots in the cytoplasm, indicative of TcPFR2 overexpression (such overexpression by the EP procyclin promoter is frequent; Figure  5B ,  - TET). When these same cells were tetracycline-induced, the flagellar staining was still perfectly visible, at a level comparable to the one previously observed in the non-induced  PCGFP  control cells (Figure  5B , +TET). Bright dots previously observed had disappeared, probably as a result of  TbPFR2  silencing. The fact that the paraflagellar rod was still neatly decorated by the anti-TbPFR2 antibody demonstrated that the  structural  inter-species complementation had indeed taken place in these cells, with TcPFR2 being effectively located at the flagellum. Did these structurally-complemented cells show a  functional  complementation, i.e. a normal flagellum motility (hence a normal cellular mobility)? To address this question, we performed a sedimentation assay [ 56 ] on non-induced and tetracycline-induced  PCGFP  and  PCTcPFR2  trypanosomes (Figure  6 ). Non-induced  PCGFP  cells showed a little tendency to sediment due to the fact that expression of  TbPFR2  dsRNA in  TbPFR2i  cells is partially leaky, producing low amounts of  TbPFR2  dsRNA even in the absence of tetracycline ([ 43 ]; Durand-Dubief and Bastin, unpublished data). When expression of  TbPFR2  dsRNA was fully induced, motility stopped leading to increased sedimentation (Figure  6 , left panel). In contrast, expression of  TbPFR2  dsRNA in  PCTcPFR2  cells did not reduce motility (Figure  6 , right panel). That result definitely demonstrates that the ortholog protein TcPFR2 fully complemented the loss of function resulting from  TbPFR2  silencing. The complementation described above shows the robustness of our strategy, because TbPFR2 and TcPFR2 are highly similar (they share 82 % identity at the nucleotide level [ 65 ]) and are nonetheless correctly differenciated by the RNAi machinery. However, our complementation strategy might be more diffcult to implement when the gene studied is too similar to the  T. brucei  counterpart. While this unfavorable case might happen with extremely evolutionarily-related organisms, studies have shown that the overall genetic sequence identity between  Trypanosoma brucei  and  Trypanosoma cruzi , for example (the closest evolutionarily-related organisms envisaged for these studies), is roughly 80 % ([ 65 ] and [ 66 ]). [ 47 ] showed that this identity percentage is still compatible with an RNAi-based complementation strategy. It goes without saying that when the organisms are evolutionarily-distant, gene sequences diverge more rapidly than the protein sequences, thus laying off a field where our strategy can be implemented with good confidence that complementation will occur.
Results and discussion
Multiple RNAi on trypanosomes We wanted to establish if the co-transfection of two distinct dsRNAs, targeting two different genes, could trigger their simultaneous silencing. The genes selected were  TbPFR2  and  FLA1 ;  TbPFR2  encodes one of the two major components of the paraflagellar rod and is necessary for flagellum motility [ 13 ];  FLA1  encodes a protein required for flagellum attachment to the cell body [ 50 ]. These dsRNAs were transfected simultaneously in wild-type trypanosomes. As a control experiment, we used  GFP  dsRNA. Cells were monitored for their acquired phenotype 15 h and 22 h after transfection (Table  1 ). The extinction of TbPFR2 was followed by immunofluorescence microscopy using the L8C4 anti-TbPFR2 monoclonal antibody.  FLA1  gene silencing was analyzed by differential interference contrast microscopy, as it results in the visible detachement of the flagellum from the cell body (Figure  2 ). The transfection of  TbPFR2  dsRNA yielded potent silencing, as more than 60 % of the cells showed no staining for L8C4 15 h later. Since old flagella pre-exist in cells which were affected by RNAi at the beginning of cell replication, the real percentage of silenced cells is probably higher than 60 %, which is confirmed by the fact that it built up to more than 74 % at time point 22 h. The transfection of  FLA1  dsRNA produced a phenotype in which the flagellum was detached from the cell body in more than 50 % of the cells. When both dsRNAs were co-transfected, both phenotypes were indeed observed, with similar frequencies to experiments where only one dsRNA was transfected. All the transfected cell populations did show a comparable growth rate (data not shown). The trypanosomes shown in Figure  2  had been transfected with both dsRNAs and the cell on the right is starting cytokinesis. The old flagellum of that cell is detached, while the new flagellum is attached along the cell body. The new flagellum exhibits a dilation of its distal tip, probably corresponding to the accumulation of TbPFR1, that is not assembled but still transported to the distal tip of the flagellum in the absence of TbPFR2 [ 13 ]. This observation demonstrates the usefulness of double-transfection experiments also for kinetics analysis. In our case, 22 h after dsRNAs transient transfection, the phenotype due to the  FLA1  silencing is no longer visible in the new flagellum while that same flagellum still exhibits the phenotype due to the  TbPFR2  silencing, clearly indicating different turn-over for TbPFR2 and FLA1 proteins. The RNAi machinery could cope with two different dsRNA populations, without – in our conditions – any visible saturation effect. These results show the feasibility of experiments involving the use of multiple dsRNAs, thus allowing studies on complex processes in the cell physiology. However, such complex experiments can only be envisaged after ensuring that the phenotypes resulting from RNAi are specifically due to silencing of the target gene. In order to address that specific problem, we elaborated a method that involves RNAi experiments on trypanosomes that were engineered to possess an extra RNAi-resistant copy of the targeted gene, leading to functional complementation.
Multiple RNAi on trypanosomes
We wanted to establish if the co-transfection of two distinct dsRNAs, targeting two different genes, could trigger their simultaneous silencing. The genes selected were  TbPFR2  and  FLA1 ;  TbPFR2  encodes one of the two major components of the paraflagellar rod and is necessary for flagellum motility [ 13 ];  FLA1  encodes a protein required for flagellum attachment to the cell body [ 50 ]. These dsRNAs were transfected simultaneously in wild-type trypanosomes. As a control experiment, we used  GFP  dsRNA.TbPFR2FLA1TbPFR2FLA1GFPCells were monitored for their acquired phenotype 15 h and 22 h after transfection (Table  1 ). The extinction of TbPFR2 was followed by immunofluorescence microscopy using the L8C4 anti-TbPFR2 monoclonal antibody.  FLA1  gene silencing was analyzed by differential interference contrast microscopy, as it results in the visible detachement of the flagellum from the cell body (Figure  2 ).FLA1The transfection of  TbPFR2  dsRNA yielded potent silencing, as more than 60 % of the cells showed no staining for L8C4 15 h later. Since old flagella pre-exist in cells which were affected by RNAi at the beginning of cell replication, the real percentage of silenced cells is probably higher than 60 %, which is confirmed by the fact that it built up to more than 74 % at time point 22 h. The transfection of  FLA1  dsRNA produced a phenotype in which the flagellum was detached from the cell body in more than 50 % of the cells. When both dsRNAs were co-transfected, both phenotypes were indeed observed, with similar frequencies to experiments where only one dsRNA was transfected. All the transfected cell populations did show a comparable growth rate (data not shown). The trypanosomes shown in Figure  2  had been transfected with both dsRNAs and the cell on the right is starting cytokinesis. The old flagellum of that cell is detached, while the new flagellum is attached along the cell body. The new flagellum exhibits a dilation of its distal tip, probably corresponding to the accumulation of TbPFR1, that is not assembled but still transported to the distal tip of the flagellum in the absence of TbPFR2 [ 13 ]. This observation demonstrates the usefulness of double-transfection experiments also for kinetics analysis. In our case, 22 h after dsRNAs transient transfection, the phenotype due to the  FLA1  silencing is no longer visible in the new flagellum while that same flagellum still exhibits the phenotype due to the  TbPFR2  silencing, clearly indicating different turn-over for TbPFR2 and FLA1 proteins.TbPFR2FLA1FLA1TbPFR2The RNAi machinery could cope with two different dsRNA populations, without – in our conditions – any visible saturation effect. These results show the feasibility of experiments involving the use of multiple dsRNAs, thus allowing studies on complex processes in the cell physiology. However, such complex experiments can only be envisaged after ensuring that the phenotypes resulting from RNAi are specifically due to silencing of the target gene. In order to address that specific problem, we elaborated a method that involves RNAi experiments on trypanosomes that were engineered to possess an extra RNAi-resistant copy of the targeted gene, leading to functional complementation.Gene silencing by dsRNA targeting UTRs As a model for this study, we chose the  TbPFR2  gene, which is present in four copies in the WT trypanosome genome (Figure  1A ), all transcribed as a single long polycistronic mRNA. All these gene copies are separated by three identical intergenic UTRs (igUTR), while the first copy has a unique 5' UTR and the last copy has a unique 3'UTR. Three types of dsRNA populations were used in our experiments, and termed as follows. dsRNA homologous to the  GFP  sequence was labelled "GFP dsRNA"; dsRNA homologous to coding sequence of the  TbPFR2  gene was labelled "CDS dsRNA"; finally, the mixture of three dsRNAs homologous to the 5' UTR, igUTR and 3'UTR of the  TbPFR2  gene was termed "UTRs MIX dsRNAs" (Figure  1A ). These dsRNAs were transfected into three cell lines: WT,  TbPFR2tag  and  TbPFR2tag-ΔHLA  (see Methods). For each experiment, the presence or absence of TbPFR2 in the new flagellum of bi-nucleated/bi-flagellated cells was monitored by immunofluorescence 14 h after the transfection (Table  2 ). Reports in [ 14 , 24 ] showed that RNAi silencing of a gene can be accomplished by targeting transcribed non-coding sequences. Here, we wanted to make sure that this kind of experiment was still feasible with a more complex system such as the  TbPFR2  multigene locus, where multiple and distinct UTRs regulate the expression of four  TbPFR2  isogenes. We first transfected WT trypanosomes with GFP dsRNA as a negative control and did not detect any  TbPFR2  silencing (Figure  3A ). Second, WT trypanosomes were transfected with the CDS dsRNA: 88 % of cells showed typical  TbPFR2  silencing with an anti-TbPFR2 immunofluorescence showing that the protein was missing from the new flagellum (Figure  3B ). Finally, the WT trypanosomes were transfected with the UTRs MIX dsRNAs, yielding the same phenotype as for the CDS dsRNA, although the silencing appeared less pronounced (Figure  3C  and Table  2 ). Overall, these results demonstrate that RNAi could efficiently silence all of the  TbPFR2  gene copies by targeting non-coding sequences present at the mRNA level. When WT trypanosomes were transfected with  TbPFR2  dsRNA complementary to only one UTR, the cells did not display any specific phenotype (data not shown). This observation is probably explained by the organization of the  TbPFR2  locus: the polycistronic transcript is rapidly spliced into three different types of mRNA, each encoding one of the four copies of  TbPFR2  [ 51 - 53 ]. Thus, even if one type of  TbPFR2  RNA is destroyed, the three remaining ones would likely provide enough RNA to synthesize TbPFR2 levels compatible with normal PFR formation. To demonstrate that the silencing observed upon transfection of WT trypanosomes with the UTRs MIX dsRNAs was due to the actual targeting of  TbPFR2 , we used two cell lines expressing a supplementary tagged  TbPFR2  gene copy. The  TbPFR2tag  cell line expresses the TbPFR2-TAG protein which correctly localizes to the flagellum. The  TbPFR2tag-ΔHLA  cell line expresses TbPFR2-TAG-ΔHLA, lacking the HLA tripeptide, which prevents its localization to the flagellum. To determine both the cellular localization of the tagged TbPFR2 proteins (TbPFR2-TAG and TbPFR2-TAG-ΔHLA) and the completeness of the PFR assembly, immunofluorescence experiments were carried out with the BB2 and ROD-1 antibodies; the former recognizes the Ty-1 epitope tag present on the two tagged TbPFR2 proteins [ 54 ], while the latter is a marker for full PFR assembly [ 55 , 56 ]. GFP dsRNA was transfected as a negative control in each cell line. As expected, this did not yield any  TbPFR2  silencing: TbPFR2 was decorated in both the old and new flagella by the anti-TbPFR2 antibody, and the PFR could be assembled fully, as evidenced by its staining with the ROD-1 antibody (data not shown, Table  2 ). In  TbPFR2tag  cells, TbPFR2-TAG was able to localize to the PFR, as evidenced by the PFR decoration with BB2 (red color, Figure  4A ). In contrast, TbPFR2-TAG-ΔHLA failed to do so in  TbPFR2tag-ΔHLA  cells, and the BB2 signal was detected in the cytoplasm, as expected (red color, Figure  4D ). We next compared  TbPFR2tag  trypanosomes after transfection with either CDS dsRNA or UTRs MIX dsRNAs.  TbPFR2tag  cells transfected with CDS dsRNA had a flagellum not (or faintly) decorated with the anti-TbPFR2 antibody, demonstrating that both the WT and the recombinant  TbPFR2  gene copies were effciently silenced (Table  2 ). That result was confirmed with anti-TAG immunofluorescence that showed no staining of the flagellum, demonstrating that TbPFR2-TAG was absent (no red color, Figure  4B ). This lack of both TbPFR2 and TbPFR2-TAG led to an incomplete assembly of the PFR, which was therefore not decorated with the ROD-1 antibody (no yellow color, Figure  4B ). In contrast, cells transfected with the UTRs MIX dsRNAs exhibited a WT phenotype, with only 2 % of the cells displaying  TbPFR2  silencing in the flagellum (Table  2 ). In this case, the tagged protein was expressed, leading to complete assembly of the PFR (yellow color, Figure  4C ) because the protein is functional and localized to the flagellum (red color, Figure  4C ; [ 56 ]). This remarkable result indicates a complementation phenomenon that is explained by the fact that the tagged  TbPFR2  gene was not silenced, as it was expressed from a coding sequence flanked by UTRs from the expression vector: from the 5' UTR of the procyclin gene and from the 3'UTR of the aldolase gene (Figure  1B ; [ 57 ]). To definitely demonstrate that the complementation described above is indeed due to the expression of functional TbPFR2-TAG, we transfected the same dsRNA into  TbPFR2tag-ΔHLA  trypanosomes expressing a modified TbPFR2 protein missing three amino acids (that is nine nucleotides out of 1800). TbPFR2tag-ΔHLA does not access the flagellar compartment and thus cannot be functional [ 58 ]. Transfecting either CDS dsRNA or UTRs MIX dsRNAs produced cells in which the new flagellum was not (or faintly) decorated by the anti-TbPFR2 antibody (Table  2 ). TbPFR2-TAG-ΔHLA was not decorated by the anti-TAG antibody when cells were transfected with CDS dsRNA (red color, Figure  4E ), indicating that both the WT and the tagged  TbPFR2  copies were silenced, thus leading to an incomplete PFR edification (yellow color, Figure  4E ). In contrast, transfection of UTRs MIX dsRNAs did not prevent the expression of the recombinant TbPFR2-TAG-ΔHLA protein, as it appeared stained by the anti-TAG antibody (red color, Figure  4F ). However, that non-functional protein could not participate in the construction of the PFR, as shown by the absence of ROD-1 signal in the new flagellum, since it cannot access the flagellum (yellow color, Figure  4F ). RNA-directed RNA polymerase activity (RdRP) has been implicated as one possible step in the formation of siRNA in fungi [ 59 ], plants [ 17 , 18 ], and worms [ 60 ]. The fact that we could specifically silence WT  TbPFR2  by targeting its UTRs, without interfering with the tagged  TbPFR2  genes, suggests that spreading of silencing beyond the initial targeted sequence does not occur in trypanosomes [ 61 - 64 ].
Gene silencing by dsRNA targeting UTRs
As a model for this study, we chose the  TbPFR2  gene, which is present in four copies in the WT trypanosome genome (Figure  1A ), all transcribed as a single long polycistronic mRNA. All these gene copies are separated by three identical intergenic UTRs (igUTR), while the first copy has a unique 5' UTR and the last copy has a unique 3'UTR. Three types of dsRNA populations were used in our experiments, and termed as follows. dsRNA homologous to the  GFP  sequence was labelled "GFP dsRNA"; dsRNA homologous to coding sequence of the  TbPFR2  gene was labelled "CDS dsRNA"; finally, the mixture of three dsRNAs homologous to the 5' UTR, igUTR and 3'UTR of the  TbPFR2  gene was termed "UTRs MIX dsRNAs" (Figure  1A ). These dsRNAs were transfected into three cell lines: WT,  TbPFR2tag  and  TbPFR2tag-ΔHLA  (see Methods). For each experiment, the presence or absence of TbPFR2 in the new flagellum of bi-nucleated/bi-flagellated cells was monitored by immunofluorescence 14 h after the transfection (Table  2 ).TbPFR2GFPTbPFR2TbPFR2TbPFR2tagTbPFR2tag-ΔHLAReports in [ 14 , 24 ] showed that RNAi silencing of a gene can be accomplished by targeting transcribed non-coding sequences. Here, we wanted to make sure that this kind of experiment was still feasible with a more complex system such as the  TbPFR2  multigene locus, where multiple and distinct UTRs regulate the expression of four  TbPFR2  isogenes. We first transfected WT trypanosomes with GFP dsRNA as a negative control and did not detect any  TbPFR2  silencing (Figure  3A ). Second, WT trypanosomes were transfected with the CDS dsRNA: 88 % of cells showed typical  TbPFR2  silencing with an anti-TbPFR2 immunofluorescence showing that the protein was missing from the new flagellum (Figure  3B ). Finally, the WT trypanosomes were transfected with the UTRs MIX dsRNAs, yielding the same phenotype as for the CDS dsRNA, although the silencing appeared less pronounced (Figure  3C  and Table  2 ). Overall, these results demonstrate that RNAi could efficiently silence all of the  TbPFR2  gene copies by targeting non-coding sequences present at the mRNA level.TbPFR2TbPFR2TbPFR2TbPFR2TbPFR2When WT trypanosomes were transfected with  TbPFR2  dsRNA complementary to only one UTR, the cells did not display any specific phenotype (data not shown). This observation is probably explained by the organization of the  TbPFR2  locus: the polycistronic transcript is rapidly spliced into three different types of mRNA, each encoding one of the four copies of  TbPFR2  [ 51 - 53 ]. Thus, even if one type of  TbPFR2  RNA is destroyed, the three remaining ones would likely provide enough RNA to synthesize TbPFR2 levels compatible with normal PFR formation.TbPFR2TbPFR2TbPFR2TbPFR2To demonstrate that the silencing observed upon transfection of WT trypanosomes with the UTRs MIX dsRNAs was due to the actual targeting of  TbPFR2 , we used two cell lines expressing a supplementary tagged  TbPFR2  gene copy. The  TbPFR2tag  cell line expresses the TbPFR2-TAG protein which correctly localizes to the flagellum.TbPFR2TbPFR2TbPFR2tagThe  TbPFR2tag-ΔHLA  cell line expresses TbPFR2-TAG-ΔHLA, lacking the HLA tripeptide, which prevents its localization to the flagellum. To determine both the cellular localization of the tagged TbPFR2 proteins (TbPFR2-TAG and TbPFR2-TAG-ΔHLA) and the completeness of the PFR assembly, immunofluorescence experiments were carried out with the BB2 and ROD-1 antibodies; the former recognizes the Ty-1 epitope tag present on the two tagged TbPFR2 proteins [ 54 ], while the latter is a marker for full PFR assembly [ 55 , 56 ].TbPFR2tag-ΔHLAGFP dsRNA was transfected as a negative control in each cell line. As expected, this did not yield any  TbPFR2  silencing: TbPFR2 was decorated in both the old and new flagella by the anti-TbPFR2 antibody, and the PFR could be assembled fully, as evidenced by its staining with the ROD-1 antibody (data not shown, Table  2 ). In  TbPFR2tag  cells, TbPFR2-TAG was able to localize to the PFR, as evidenced by the PFR decoration with BB2 (red color, Figure  4A ). In contrast, TbPFR2-TAG-ΔHLA failed to do so in  TbPFR2tag-ΔHLA  cells, and the BB2 signal was detected in the cytoplasm, as expected (red color, Figure  4D ).TbPFR2TbPFR2tagTbPFR2tag-ΔHLAWe next compared  TbPFR2tag  trypanosomes after transfection with either CDS dsRNA or UTRs MIX dsRNAs.  TbPFR2tag  cells transfected with CDS dsRNA had a flagellum not (or faintly) decorated with the anti-TbPFR2 antibody, demonstrating that both the WT and the recombinant  TbPFR2  gene copies were effciently silenced (Table  2 ). That result was confirmed with anti-TAG immunofluorescence that showed no staining of the flagellum, demonstrating that TbPFR2-TAG was absent (no red color, Figure  4B ). This lack of both TbPFR2 and TbPFR2-TAG led to an incomplete assembly of the PFR, which was therefore not decorated with the ROD-1 antibody (no yellow color, Figure  4B ). In contrast, cells transfected with the UTRs MIX dsRNAs exhibited a WT phenotype, with only 2 % of the cells displaying  TbPFR2  silencing in the flagellum (Table  2 ). In this case, the tagged protein was expressed, leading to complete assembly of the PFR (yellow color, Figure  4C ) because the protein is functional and localized to the flagellum (red color, Figure  4C ; [ 56 ]). This remarkable result indicates a complementation phenomenon that is explained by the fact that the tagged  TbPFR2  gene was not silenced, as it was expressed from a coding sequence flanked by UTRs from the expression vector: from the 5' UTR of the procyclin gene and from the 3'UTR of the aldolase gene (Figure  1B ; [ 57 ]).TbPFR2tagTbPFR2tagTbPFR2TbPFR2TbPFR2To definitely demonstrate that the complementation described above is indeed due to the expression of functional TbPFR2-TAG, we transfected the same dsRNA into  TbPFR2tag-ΔHLA  trypanosomes expressing a modified TbPFR2 protein missing three amino acids (that is nine nucleotides out of 1800). TbPFR2tag-ΔHLA does not access the flagellar compartment and thus cannot be functional [ 58 ]. Transfecting either CDS dsRNA or UTRs MIX dsRNAs produced cells in which the new flagellum was not (or faintly) decorated by the anti-TbPFR2 antibody (Table  2 ). TbPFR2-TAG-ΔHLA was not decorated by the anti-TAG antibody when cells were transfected with CDS dsRNA (red color, Figure  4E ), indicating that both the WT and the tagged  TbPFR2  copies were silenced, thus leading to an incomplete PFR edification (yellow color, Figure  4E ). In contrast, transfection of UTRs MIX dsRNAs did not prevent the expression of the recombinant TbPFR2-TAG-ΔHLA protein, as it appeared stained by the anti-TAG antibody (red color, Figure  4F ). However, that non-functional protein could not participate in the construction of the PFR, as shown by the absence of ROD-1 signal in the new flagellum, since it cannot access the flagellum (yellow color, Figure  4F ).TbPFR2tag-ΔHLATbPFR2RNA-directed RNA polymerase activity (RdRP) has been implicated as one possible step in the formation of siRNA in fungi [ 59 ], plants [ 17 , 18 ], and worms [ 60 ]. The fact that we could specifically silence WT  TbPFR2  by targeting its UTRs, without interfering with the tagged  TbPFR2  genes, suggests that spreading of silencing beyond the initial targeted sequence does not occur in trypanosomes [ 61 - 64 ].TbPFR2TbPFR2Functional complementation with orthologue genes We next asked if an RNAi-mediated loss of function could be complemented by the expression of a gene orthologue to the silenced one. The system used to answer that question involved the  TbPFR2i  cell line – that expresses  TbPFR2  dsRNA under the control of a tetracycline-inducible promoter [ 47 ] – into which constitutive expression of  Trypanosoma cruzi TbPFR2  orthologue ( TcPFR2 ) was established using stable transfection procedures. TbPFR2 and TcPFR2 proteins share 90 % identity (both of them are recognized by the anti-TbPFR2 L8C4 antibody), but their gene sequences have diverged enough for us to envisage that the RNAi-silencing of  TbPFR2  would not affect significantly the introduced  TcPFR2  gene (83 % overall nucleotide identity). We thus created two new cell lines based on the previously described  TbPFR2i  cells [ 47 ] (see Methods). TbPFR2 expression and cell motility were analyzed. Our first experiment showed that the  PCGFP  cells constitutively expressed GFP, as detected by microscope observation of living cells (data not shown). Both the  PCGFP  and  PCTcPFR2  cell lines were induced to express  TbPFR2  dsRNA for 48 hours. Immunofluorescence revealed that non-induced  PCGFP  cells exhibit a WT-like phenotype (Figure  5A ,  - TET). When these cells were induced with tetracycline, expected  TbPFR2  silencing occurred (Figure  5A , +TET). Non-induced  PCTcPFR2  cells displayed an intense anti-TbPFR2 antibody decoration with bright dots in the cytoplasm, indicative of TcPFR2 overexpression (such overexpression by the EP procyclin promoter is frequent; Figure  5B ,  - TET). When these same cells were tetracycline-induced, the flagellar staining was still perfectly visible, at a level comparable to the one previously observed in the non-induced  PCGFP  control cells (Figure  5B , +TET). Bright dots previously observed had disappeared, probably as a result of  TbPFR2  silencing. The fact that the paraflagellar rod was still neatly decorated by the anti-TbPFR2 antibody demonstrated that the  structural  inter-species complementation had indeed taken place in these cells, with TcPFR2 being effectively located at the flagellum. Did these structurally-complemented cells show a  functional  complementation, i.e. a normal flagellum motility (hence a normal cellular mobility)? To address this question, we performed a sedimentation assay [ 56 ] on non-induced and tetracycline-induced  PCGFP  and  PCTcPFR2  trypanosomes (Figure  6 ). Non-induced  PCGFP  cells showed a little tendency to sediment due to the fact that expression of  TbPFR2  dsRNA in  TbPFR2i  cells is partially leaky, producing low amounts of  TbPFR2  dsRNA even in the absence of tetracycline ([ 43 ]; Durand-Dubief and Bastin, unpublished data). When expression of  TbPFR2  dsRNA was fully induced, motility stopped leading to increased sedimentation (Figure  6 , left panel). In contrast, expression of  TbPFR2  dsRNA in  PCTcPFR2  cells did not reduce motility (Figure  6 , right panel). That result definitely demonstrates that the ortholog protein TcPFR2 fully complemented the loss of function resulting from  TbPFR2  silencing. The complementation described above shows the robustness of our strategy, because TbPFR2 and TcPFR2 are highly similar (they share 82 % identity at the nucleotide level [ 65 ]) and are nonetheless correctly differenciated by the RNAi machinery. However, our complementation strategy might be more diffcult to implement when the gene studied is too similar to the  T. brucei  counterpart. While this unfavorable case might happen with extremely evolutionarily-related organisms, studies have shown that the overall genetic sequence identity between  Trypanosoma brucei  and  Trypanosoma cruzi , for example (the closest evolutionarily-related organisms envisaged for these studies), is roughly 80 % ([ 65 ] and [ 66 ]). [ 47 ] showed that this identity percentage is still compatible with an RNAi-based complementation strategy. It goes without saying that when the organisms are evolutionarily-distant, gene sequences diverge more rapidly than the protein sequences, thus laying off a field where our strategy can be implemented with good confidence that complementation will occur.
Functional complementation with orthologue genes
We next asked if an RNAi-mediated loss of function could be complemented by the expression of a gene orthologue to the silenced one. The system used to answer that question involved the  TbPFR2i  cell line – that expresses  TbPFR2  dsRNA under the control of a tetracycline-inducible promoter [ 47 ] – into which constitutive expression of  Trypanosoma cruzi TbPFR2  orthologue ( TcPFR2 ) was established using stable transfection procedures. TbPFR2 and TcPFR2 proteins share 90 % identity (both of them are recognized by the anti-TbPFR2 L8C4 antibody), but their gene sequences have diverged enough for us to envisage that the RNAi-silencing of  TbPFR2  would not affect significantly the introduced  TcPFR2  gene (83 % overall nucleotide identity). We thus created two new cell lines based on the previously described  TbPFR2i  cells [ 47 ] (see Methods). TbPFR2 expression and cell motility were analyzed.TbPFR2iTbPFR2Trypanosoma cruzi TbPFR2TcPFR2TbPFR2TcPFR2TbPFR2iOur first experiment showed that the  PCGFP  cells constitutively expressed GFP, as detected by microscope observation of living cells (data not shown). Both the  PCGFP  and  PCTcPFR2  cell lines were induced to express  TbPFR2  dsRNA for 48 hours. Immunofluorescence revealed that non-induced  PCGFP  cells exhibit a WT-like phenotype (Figure  5A ,  - TET). When these cells were induced with tetracycline, expected  TbPFR2  silencing occurred (Figure  5A , +TET). Non-induced  PCTcPFR2  cells displayed an intense anti-TbPFR2 antibody decoration with bright dots in the cytoplasm, indicative of TcPFR2 overexpression (such overexpression by the EP procyclin promoter is frequent; Figure  5B ,  - TET). When these same cells were tetracycline-induced, the flagellar staining was still perfectly visible, at a level comparable to the one previously observed in the non-induced  PCGFP  control cells (Figure  5B , +TET). Bright dots previously observed had disappeared, probably as a result of  TbPFR2  silencing. The fact that the paraflagellar rod was still neatly decorated by the anti-TbPFR2 antibody demonstrated that the  structural  inter-species complementation had indeed taken place in these cells, with TcPFR2 being effectively located at the flagellum.PCGFPPCGFPPCTcPFR2TbPFR2PCGFP-TbPFR2PCTcPFR2-PCGFPTbPFR2structuralDid these structurally-complemented cells show a  functional  complementation, i.e. a normal flagellum motility (hence a normal cellular mobility)? To address this question, we performed a sedimentation assay [ 56 ] on non-induced and tetracycline-induced  PCGFP  and  PCTcPFR2  trypanosomes (Figure  6 ). Non-induced  PCGFP  cells showed a little tendency to sediment due to the fact that expression of  TbPFR2  dsRNA in  TbPFR2i  cells is partially leaky, producing low amounts of  TbPFR2  dsRNA even in the absence of tetracycline ([ 43 ]; Durand-Dubief and Bastin, unpublished data). When expression of  TbPFR2  dsRNA was fully induced, motility stopped leading to increased sedimentation (Figure  6 , left panel). In contrast, expression of  TbPFR2  dsRNA in  PCTcPFR2  cells did not reduce motility (Figure  6 , right panel). That result definitely demonstrates that the ortholog protein TcPFR2 fully complemented the loss of function resulting from  TbPFR2  silencing.functionalPCGFPPCTcPFR2PCGFPTbPFR2TbPFR2iTbPFR2TbPFR2TbPFR2PCTcPFR2TbPFR2The complementation described above shows the robustness of our strategy, because TbPFR2 and TcPFR2 are highly similar (they share 82 % identity at the nucleotide level [ 65 ]) and are nonetheless correctly differenciated by the RNAi machinery. However, our complementation strategy might be more diffcult to implement when the gene studied is too similar to the  T. brucei  counterpart. While this unfavorable case might happen with extremely evolutionarily-related organisms, studies have shown that the overall genetic sequence identity between  Trypanosoma brucei  and  Trypanosoma cruzi , for example (the closest evolutionarily-related organisms envisaged for these studies), is roughly 80 % ([ 65 ] and [ 66 ]). [ 47 ] showed that this identity percentage is still compatible with an RNAi-based complementation strategy. It goes without saying that when the organisms are evolutionarily-distant, gene sequences diverge more rapidly than the protein sequences, thus laying off a field where our strategy can be implemented with good confidence that complementation will occur.T. bruceiTrypanosoma bruceiTrypanosoma cruziConclusions In this report, we demonstrated that RNAi-mediated silencing of a gene by targeting its UTRs is useful in studies where the loss of function resulting from this silencing must be complemented with the expression of an RNAi-resistant copy of the silenced gene, in order to demonstrate that the phenotype is indeed due to silencing of that gene, and not to inactivation of another one. The results obtained in this work are of particular interest when reverse-genetics studies cannot be easily achieved in organisms not amenable to RNAi, like  Leishmania  [ 67 ] or  Trypanosoma cruzi  [ 68 ], or where genetics experiments are hardly set up, like mammals. When genes from these organisms are to be studied, a complementation experiment can be set up as a three-step procedure whereby: 1) the ortholog gene in  Trypanosoma brucei  is RNAi-silenced and the loss-of-function phenotype is established; 2)  T. brucei  cells are engineered to ensure constitutive heterologous expression of the gene of interest, still allowing RNAi-mediated silencing of the  T. brucei  gene; 3) function of the investigated gene is assessed by checking if the loss-of-function phenotype observed in the first place gets complemented. Additionally, one application of the strategy described herein is genetic functional dissection, which is of interest when protein domains are to be characterized with respect to their function (e.g. the HLA tripeptide sequence in TbPFR2 that localizes the protein to the flagellum). Complementation had previously been demonstrated following transformation of mammalian cells with EGFP siRNA and expression of a codon-modified, but functional, EGFP version [ 69 ]. Our strategies are increasing flexibility for complementation studies after RNAi as unmodified genes can be used for rescue.
Conclusions
In this report, we demonstrated that RNAi-mediated silencing of a gene by targeting its UTRs is useful in studies where the loss of function resulting from this silencing must be complemented with the expression of an RNAi-resistant copy of the silenced gene, in order to demonstrate that the phenotype is indeed due to silencing of that gene, and not to inactivation of another one. The results obtained in this work are of particular interest when reverse-genetics studies cannot be easily achieved in organisms not amenable to RNAi, like  Leishmania  [ 67 ] or  Trypanosoma cruzi  [ 68 ], or where genetics experiments are hardly set up, like mammals. When genes from these organisms are to be studied, a complementation experiment can be set up as a three-step procedure whereby: 1) the ortholog gene in  Trypanosoma brucei  is RNAi-silenced and the loss-of-function phenotype is established; 2)  T. brucei  cells are engineered to ensure constitutive heterologous expression of the gene of interest, still allowing RNAi-mediated silencing of the  T. brucei  gene; 3) function of the investigated gene is assessed by checking if the loss-of-function phenotype observed in the first place gets complemented. Additionally, one application of the strategy described herein is genetic functional dissection, which is of interest when protein domains are to be characterized with respect to their function (e.g. the HLA tripeptide sequence in TbPFR2 that localizes the protein to the flagellum).LeishmaniaTrypanosoma cruziTrypanosoma bruceiT. bruceiT. bruceiComplementation had previously been demonstrated following transformation of mammalian cells with EGFP siRNA and expression of a codon-modified, but functional, EGFP version [ 69 ]. Our strategies are increasing flexibility for complementation studies after RNAi as unmodified genes can be used for rescue.Methods Trypanosomes The procyclic  T. brucei brucei  strain 427 (or its derivatives) was used throughout this work. Cells were cultured at 27°C in semi-defined medium 79 (SDM 79) containing 10% foetal calf serum.  PFRAi  cells were described in [ 47 ]. The  TbPFR2i  trypanosomes can be tetracycline-induced to express  TbPFR2  dsRNA, thus eliciting an RNAi response against that gene. Note that this cell line is referred to as  TbPFR2i  in this article because of a change in the gene nomenclature [ 70 ]. RNAi assays by transient transfection RNA was synthesized  in vitro  with T3 and Sp6 polymerases using PCR products as templates [ 71 ]. The following primers (incorporating T3 or Sp6 promoters) were used: for  GFP  (from the nucleotide coding sequence 476–691 of the  EGFPN2  gene; Clontech),  AATTAACCCTCACTAAAGGGAGAAG  AACGGCATCAAGGTGAAC (T3 promoter italicized) and  ATTTAGGTGACACTATAGAAG  AGTGATCCCGGCGGCGGTCACG (Sp6 promoter italicized); for  FLA1 ,  AATTAACCCTCACTAAAGGGAGA  CCAAACCGTGGGCACCAAGG (T3 promoter italicized) and  ATTTAGGTGAACTATAGAAGAG  GTGGGATGATTAAAACGAGC (Sp6 promoter italicized); for the  TbPFR2  5' untranslated region (5' UTR; nucleotide sequence [-545→-1] upstream of  TbPFR2  ATG start codon),  AATTAACCCTCACTAAAGGGAGA  (T3 promoter) and  ATTTAGGTGACACT-ATAGAAGAG  (Sp6 promoter); for the  TbPFR2  intergenic untranslated region (igUTR),  AATTAACCCTCACTAAAGGGAGA  CGCTGCGCTTAAATGTCTT (T3 promoter italicized) and  ATTTAGGTGACACTATAGAAGA  GTGATGCTTTATTGCTTTCT (Sp6 promoter italicized); for the  TbPFR2  3'untranslated region (3'UTR; nucleotide sequence [1→533] downstream of the  TbPFR2  TAG stop codon),  AATTAACCCTCACTAAAGGGAGA  (universal T3 promoter) and  ATTTAGGTGACACTATAGAAGAG  (universal Sp6 promoter); for the  TbPFR2  coding sequence (CDS; nucleotide coding sequence [1084→1358]),  ATTTAGGTGACACTATAGA  GAGGTGAAGCGCCGTATTGAGGA (Sp6 promoter italicized) and  AATTAACCCTCACTAAAGGGAGA  GTTTTGTACAGGCGACGGAA (T3 promoter italicized); Figure  1A  shows the  TbPFR2  locus and the position of the two dsRNA populations that were used, and their homology to either the coding sequence (labelled "CDS dsRNA") or the different 5'UTR, igUTR and 3'UTR all together (labelled "UTRs MIX dsRNAs"). A third dsRNA, homologous to the  GFP  gene is labelled "GFP dsRNA" throughout this work and was used as a control dsRNA. dsRNA was introduced into trypanosomes by electroporation, as described [ 14 ]. Plasmids Plasmid pPC was generated from plasmid pSk1-GFP [ 50 ] as follows: pSk1-GFP was digested with  Hin d III and  Eco  RI to remove the  GFP  gene. Oligonucleotides  AGCT  GTCTAGCGATATCGGATCCG (forward) and  AATT  CGGATCCGATATCGCTAGCA (reverse) were annealed (protruding ends italicized) and the resulting double-strand oligonucleotide was ligated into the pSk1-GFP plasmid, resulting in the insertion of a poly-linker containing restriction sites  Cla  I,  Hin d III,  Nhe  I,  Eco  RV,  Bam  HI and  Eco  RI (Branche and Bastin; unpublished data). Plas-mid pPCTcPFR2 was generated as follows: amplification of the  TcPFR2  gene was performed using  Trypanosoma cruzi  genomic DNA (kind gift of Cécile Gallet and Philippe Grellier, MNHN) and the two primers TcPFR2H (GAGTCTAAGCTTATGAGCTACAAGGAGGCATC) and TcPFR2ER (GCGTGGAATTCTTACTGTGTGATCTGCTGCAC). Both the amplified DNA fragment and the pPC plasmid were digested with  Eco  RI and  Hin d III. The fragment was ligated into pPC so as to yield the plasmid pPCTcPFR2 (Figure  1B ). Cell lines The different constructs used to transform trypanosomes are shown on Figure  1B . The cell lines were established as follows. WT-derived trypanosomes constitutively expressing TbPFR2-TAG proteins The  TbPFR2tag  cell line was derived from the WT cell line into which the pTbPFR2TAG430 plasmid [ 72 ] was transfected. The recombinant cells constitutively expressed the TbPFR2-TAG protein, that is localized in the flagellum (Fig  4D ). Tagged TbPFR2 is known to be functional [ 56 , 72 ]. In contrast, transformation of WT cells with the pTbPFR2TAGΔHLA430 plasmid lead to the expression of slightly modified TbPFR2 protein, missing only three amino acids, that failed to enter the flagellum compartment and hence was found in the cell body cytoplasm [ 58 ] (Fig  4G ). This cell line was called  TbPFR2tag-ΔHLA . After electroporation [ 73 ], cells were grown overnight and then distributed in 24-well plates in the presence of phleomycin (2  μ g/mL) for selection. TbPFR2i-derived trypanosomes constitutively expressing GFP and TcPFR2 TbPFR2i  cells [ 47 ] constituted the genetic background into which we established the  PCGFP  and  PCTcPFR2  new cell lines. The  PCGFP  cell line was established by transfecting  TbPFR2i  cells with plasmid pPCGFP after linearization with  BstX  I. For establishing the  PCTcPFR2  cell line, the pPCTcPFR2 plasmid was linearized with  BstX  I and transfected into  TbPFR2i  cells. Recombinant cells were selected by addition of puromycin (1  μ g/mL), phleomycin (2  μ g/mL), G418 (15  μ g/mL) and hygromycin (20  μ g/mL) to the culture medium. Immunofluorescence and microscopy Three different monoclonal antibodies were used as hybridoma supernatants: L8C4, IgG recognizing  T. brucei  TbPFR2 and cross-reacting with  T. cruzi  orthologue TcPFR2 [ 74 ]; BB2, IgG recognizing the Ty-1 tag of the TbPFR2-TAG and TbPFR2-TAG-ΔHLA recombinant proteins [ 54 ]; and ROD-1, IgM recognizing a doublet of minor PFR proteins [ 55 ]. For immunofluorescence, trypanosomes were spread onto poly-L-lysine-coated slides, fixed in cold methanol and processed as described [ 75 ]. Experiments involving the use of L8C4 only were performed with an FITC-conjugated anti-mouse IgG secondary antibody. Double-staining experiments using BB2 and ROD-1 were performed with a TRITC-conjugated specific anti-mouse IgG secondary antibody and an FITC-conjugated specific anti-mouse IgM secondary antibody. DNA was systematically stained with 4',6-diamidino-2-phenylindole (DAPI). Slides were examined with a Leica DMR microscope, images were captured using a cooled CCD camera (Cool Snap HQ, Roper Scientific) and processed with the GNU image manipulation program version 2 [ 76 ]. Cell sedimentation assay The trypanosome sedimentation assay was performed as described in [ 56 ]. Briefly: trypanosomes were grown at ≈ 5.10 6  cells/mL in normal culture medium, with or without 48 hour tetracycline induction. 1 mL of these cultures was dispensed to 5 plastic spectrophotometry cuvettes, for time points 0, 2, 4, 6, 8 hours, and left still. At each time point, the optical density at 600 nm was measured twice: first without mixing (O.D. no mix ) and second after mixing the cuvette (O.D. mix ). Data were plotted as a function of time.
Methods
Trypanosomes The procyclic  T. brucei brucei  strain 427 (or its derivatives) was used throughout this work. Cells were cultured at 27°C in semi-defined medium 79 (SDM 79) containing 10% foetal calf serum.  PFRAi  cells were described in [ 47 ]. The  TbPFR2i  trypanosomes can be tetracycline-induced to express  TbPFR2  dsRNA, thus eliciting an RNAi response against that gene. Note that this cell line is referred to as  TbPFR2i  in this article because of a change in the gene nomenclature [ 70 ].
Trypanosomes
The procyclic  T. brucei brucei  strain 427 (or its derivatives) was used throughout this work. Cells were cultured at 27°C in semi-defined medium 79 (SDM 79) containing 10% foetal calf serum.  PFRAi  cells were described in [ 47 ]. The  TbPFR2i  trypanosomes can be tetracycline-induced to express  TbPFR2  dsRNA, thus eliciting an RNAi response against that gene. Note that this cell line is referred to as  TbPFR2i  in this article because of a change in the gene nomenclature [ 70 ].T. brucei bruceiPFRAiTbPFR2iTbPFR2TbPFR2iRNAi assays by transient transfection RNA was synthesized  in vitro  with T3 and Sp6 polymerases using PCR products as templates [ 71 ]. The following primers (incorporating T3 or Sp6 promoters) were used: for  GFP  (from the nucleotide coding sequence 476–691 of the  EGFPN2  gene; Clontech),  AATTAACCCTCACTAAAGGGAGAAG  AACGGCATCAAGGTGAAC (T3 promoter italicized) and  ATTTAGGTGACACTATAGAAG  AGTGATCCCGGCGGCGGTCACG (Sp6 promoter italicized); for  FLA1 ,  AATTAACCCTCACTAAAGGGAGA  CCAAACCGTGGGCACCAAGG (T3 promoter italicized) and  ATTTAGGTGAACTATAGAAGAG  GTGGGATGATTAAAACGAGC (Sp6 promoter italicized); for the  TbPFR2  5' untranslated region (5' UTR; nucleotide sequence [-545→-1] upstream of  TbPFR2  ATG start codon),  AATTAACCCTCACTAAAGGGAGA  (T3 promoter) and  ATTTAGGTGACACT-ATAGAAGAG  (Sp6 promoter); for the  TbPFR2  intergenic untranslated region (igUTR),  AATTAACCCTCACTAAAGGGAGA  CGCTGCGCTTAAATGTCTT (T3 promoter italicized) and  ATTTAGGTGACACTATAGAAGA  GTGATGCTTTATTGCTTTCT (Sp6 promoter italicized); for the  TbPFR2  3'untranslated region (3'UTR; nucleotide sequence [1→533] downstream of the  TbPFR2  TAG stop codon),  AATTAACCCTCACTAAAGGGAGA  (universal T3 promoter) and  ATTTAGGTGACACTATAGAAGAG  (universal Sp6 promoter); for the  TbPFR2  coding sequence (CDS; nucleotide coding sequence [1084→1358]),  ATTTAGGTGACACTATAGA  GAGGTGAAGCGCCGTATTGAGGA (Sp6 promoter italicized) and  AATTAACCCTCACTAAAGGGAGA  GTTTTGTACAGGCGACGGAA (T3 promoter italicized); Figure  1A  shows the  TbPFR2  locus and the position of the two dsRNA populations that were used, and their homology to either the coding sequence (labelled "CDS dsRNA") or the different 5'UTR, igUTR and 3'UTR all together (labelled "UTRs MIX dsRNAs"). A third dsRNA, homologous to the  GFP  gene is labelled "GFP dsRNA" throughout this work and was used as a control dsRNA. dsRNA was introduced into trypanosomes by electroporation, as described [ 14 ].
RNAi assays by transient transfection
RNA was synthesized  in vitro  with T3 and Sp6 polymerases using PCR products as templates [ 71 ]. The following primers (incorporating T3 or Sp6 promoters) were used:in vitrofor  GFP  (from the nucleotide coding sequence 476–691 of the  EGFPN2  gene; Clontech),  AATTAACCCTCACTAAAGGGAGAAG  AACGGCATCAAGGTGAAC (T3 promoter italicized) and  ATTTAGGTGACACTATAGAAG  AGTGATCCCGGCGGCGGTCACG (Sp6 promoter italicized);GFPEGFPN2AATTAACCCTCACTAAAGGGAGAAGATTTAGGTGACACTATAGAAGfor  FLA1 ,  AATTAACCCTCACTAAAGGGAGA  CCAAACCGTGGGCACCAAGG (T3 promoter italicized) and  ATTTAGGTGAACTATAGAAGAG  GTGGGATGATTAAAACGAGC (Sp6 promoter italicized);FLA1AATTAACCCTCACTAAAGGGAGAATTTAGGTGAACTATAGAAGAGfor the  TbPFR2  5' untranslated region (5' UTR; nucleotide sequence [-545→-1] upstream of  TbPFR2  ATG start codon),  AATTAACCCTCACTAAAGGGAGA  (T3 promoter) and  ATTTAGGTGACACT-ATAGAAGAG  (Sp6 promoter);TbPFR2TbPFR2AATTAACCCTCACTAAAGGGAGAATTTAGGTGACACT-ATAGAAGAGfor the  TbPFR2  intergenic untranslated region (igUTR),  AATTAACCCTCACTAAAGGGAGA  CGCTGCGCTTAAATGTCTT (T3 promoter italicized) and  ATTTAGGTGACACTATAGAAGA  GTGATGCTTTATTGCTTTCT (Sp6 promoter italicized);TbPFR2AATTAACCCTCACTAAAGGGAGAATTTAGGTGACACTATAGAAGAfor the  TbPFR2  3'untranslated region (3'UTR; nucleotide sequence [1→533] downstream of the  TbPFR2  TAG stop codon),  AATTAACCCTCACTAAAGGGAGA  (universal T3 promoter) and  ATTTAGGTGACACTATAGAAGAG  (universal Sp6 promoter);TbPFR2TbPFR2AATTAACCCTCACTAAAGGGAGAATTTAGGTGACACTATAGAAGAGfor the  TbPFR2  coding sequence (CDS; nucleotide coding sequence [1084→1358]),  ATTTAGGTGACACTATAGA  GAGGTGAAGCGCCGTATTGAGGA (Sp6 promoter italicized) and  AATTAACCCTCACTAAAGGGAGA  GTTTTGTACAGGCGACGGAA (T3 promoter italicized);TbPFR2ATTTAGGTGACACTATAGAAATTAACCCTCACTAAAGGGAGAFigure  1A  shows the  TbPFR2  locus and the position of the two dsRNA populations that were used, and their homology to either the coding sequence (labelled "CDS dsRNA") or the different 5'UTR, igUTR and 3'UTR all together (labelled "UTRs MIX dsRNAs"). A third dsRNA, homologous to the  GFP  gene is labelled "GFP dsRNA" throughout this work and was used as a control dsRNA. dsRNA was introduced into trypanosomes by electroporation, as described [ 14 ].TbPFR2GFPPlasmids Plasmid pPC was generated from plasmid pSk1-GFP [ 50 ] as follows: pSk1-GFP was digested with  Hin d III and  Eco  RI to remove the  GFP  gene. Oligonucleotides  AGCT  GTCTAGCGATATCGGATCCG (forward) and  AATT  CGGATCCGATATCGCTAGCA (reverse) were annealed (protruding ends italicized) and the resulting double-strand oligonucleotide was ligated into the pSk1-GFP plasmid, resulting in the insertion of a poly-linker containing restriction sites  Cla  I,  Hin d III,  Nhe  I,  Eco  RV,  Bam  HI and  Eco  RI (Branche and Bastin; unpublished data). Plas-mid pPCTcPFR2 was generated as follows: amplification of the  TcPFR2  gene was performed using  Trypanosoma cruzi  genomic DNA (kind gift of Cécile Gallet and Philippe Grellier, MNHN) and the two primers TcPFR2H (GAGTCTAAGCTTATGAGCTACAAGGAGGCATC) and TcPFR2ER (GCGTGGAATTCTTACTGTGTGATCTGCTGCAC). Both the amplified DNA fragment and the pPC plasmid were digested with  Eco  RI and  Hin d III. The fragment was ligated into pPC so as to yield the plasmid pPCTcPFR2 (Figure  1B ).
Plasmids
Plasmid pPC was generated from plasmid pSk1-GFP [ 50 ] as follows: pSk1-GFP was digested with  Hin d III and  Eco  RI to remove the  GFP  gene. Oligonucleotides  AGCT  GTCTAGCGATATCGGATCCG (forward) and  AATT  CGGATCCGATATCGCTAGCA (reverse) were annealed (protruding ends italicized) and the resulting double-strand oligonucleotide was ligated into the pSk1-GFP plasmid, resulting in the insertion of a poly-linker containing restriction sites  Cla  I,  Hin d III,  Nhe  I,  Eco  RV,  Bam  HI and  Eco  RI (Branche and Bastin; unpublished data). Plas-mid pPCTcPFR2 was generated as follows: amplification of the  TcPFR2  gene was performed using  Trypanosoma cruzi  genomic DNA (kind gift of Cécile Gallet and Philippe Grellier, MNHN) and the two primers TcPFR2H (GAGTCTAAGCTTATGAGCTACAAGGAGGCATC) and TcPFR2ER (GCGTGGAATTCTTACTGTGTGATCTGCTGCAC). Both the amplified DNA fragment and the pPC plasmid were digested with  Eco  RI and  Hin d III. The fragment was ligated into pPC so as to yield the plasmid pPCTcPFR2 (Figure  1B ).HinEcoGFPAGCTAATTClaHinNheEcoBamEcoTcPFR2Trypanosoma cruziEcoHinCell lines The different constructs used to transform trypanosomes are shown on Figure  1B . The cell lines were established as follows. WT-derived trypanosomes constitutively expressing TbPFR2-TAG proteins The  TbPFR2tag  cell line was derived from the WT cell line into which the pTbPFR2TAG430 plasmid [ 72 ] was transfected. The recombinant cells constitutively expressed the TbPFR2-TAG protein, that is localized in the flagellum (Fig  4D ). Tagged TbPFR2 is known to be functional [ 56 , 72 ]. In contrast, transformation of WT cells with the pTbPFR2TAGΔHLA430 plasmid lead to the expression of slightly modified TbPFR2 protein, missing only three amino acids, that failed to enter the flagellum compartment and hence was found in the cell body cytoplasm [ 58 ] (Fig  4G ). This cell line was called  TbPFR2tag-ΔHLA . After electroporation [ 73 ], cells were grown overnight and then distributed in 24-well plates in the presence of phleomycin (2  μ g/mL) for selection. TbPFR2i-derived trypanosomes constitutively expressing GFP and TcPFR2 TbPFR2i  cells [ 47 ] constituted the genetic background into which we established the  PCGFP  and  PCTcPFR2  new cell lines. The  PCGFP  cell line was established by transfecting  TbPFR2i  cells with plasmid pPCGFP after linearization with  BstX  I. For establishing the  PCTcPFR2  cell line, the pPCTcPFR2 plasmid was linearized with  BstX  I and transfected into  TbPFR2i  cells. Recombinant cells were selected by addition of puromycin (1  μ g/mL), phleomycin (2  μ g/mL), G418 (15  μ g/mL) and hygromycin (20  μ g/mL) to the culture medium.
Cell lines
The different constructs used to transform trypanosomes are shown on Figure  1B . The cell lines were established as follows.WT-derived trypanosomes constitutively expressing TbPFR2-TAG proteins The  TbPFR2tag  cell line was derived from the WT cell line into which the pTbPFR2TAG430 plasmid [ 72 ] was transfected. The recombinant cells constitutively expressed the TbPFR2-TAG protein, that is localized in the flagellum (Fig  4D ). Tagged TbPFR2 is known to be functional [ 56 , 72 ]. In contrast, transformation of WT cells with the pTbPFR2TAGΔHLA430 plasmid lead to the expression of slightly modified TbPFR2 protein, missing only three amino acids, that failed to enter the flagellum compartment and hence was found in the cell body cytoplasm [ 58 ] (Fig  4G ). This cell line was called  TbPFR2tag-ΔHLA . After electroporation [ 73 ], cells were grown overnight and then distributed in 24-well plates in the presence of phleomycin (2  μ g/mL) for selection.
WT-derived trypanosomes constitutively expressing TbPFR2-TAG proteins
The  TbPFR2tag  cell line was derived from the WT cell line into which the pTbPFR2TAG430 plasmid [ 72 ] was transfected. The recombinant cells constitutively expressed the TbPFR2-TAG protein, that is localized in the flagellum (Fig  4D ). Tagged TbPFR2 is known to be functional [ 56 , 72 ]. In contrast, transformation of WT cells with the pTbPFR2TAGΔHLA430 plasmid lead to the expression of slightly modified TbPFR2 protein, missing only three amino acids, that failed to enter the flagellum compartment and hence was found in the cell body cytoplasm [ 58 ] (Fig  4G ). This cell line was called  TbPFR2tag-ΔHLA . After electroporation [ 73 ], cells were grown overnight and then distributed in 24-well plates in the presence of phleomycin (2  μ g/mL) for selection.TbPFR2tagTbPFR2tag-ΔHLAμTbPFR2i-derived trypanosomes constitutively expressing GFP and TcPFR2 TbPFR2i  cells [ 47 ] constituted the genetic background into which we established the  PCGFP  and  PCTcPFR2  new cell lines. The  PCGFP  cell line was established by transfecting  TbPFR2i  cells with plasmid pPCGFP after linearization with  BstX  I. For establishing the  PCTcPFR2  cell line, the pPCTcPFR2 plasmid was linearized with  BstX  I and transfected into  TbPFR2i  cells. Recombinant cells were selected by addition of puromycin (1  μ g/mL), phleomycin (2  μ g/mL), G418 (15  μ g/mL) and hygromycin (20  μ g/mL) to the culture medium.
TbPFR2i-derived trypanosomes constitutively expressing GFP and TcPFR2
TbPFR2i  cells [ 47 ] constituted the genetic background into which we established the  PCGFP  and  PCTcPFR2  new cell lines. The  PCGFP  cell line was established by transfecting  TbPFR2i  cells with plasmid pPCGFP after linearization with  BstX  I. For establishing the  PCTcPFR2  cell line, the pPCTcPFR2 plasmid was linearized with  BstX  I and transfected into  TbPFR2i  cells. Recombinant cells were selected by addition of puromycin (1  μ g/mL), phleomycin (2  μ g/mL), G418 (15  μ g/mL) and hygromycin (20  μ g/mL) to the culture medium.TbPFR2iPCGFPPCTcPFR2PCGFPTbPFR2iBstXPCTcPFR2BstXTbPFR2iμμμμImmunofluorescence and microscopy Three different monoclonal antibodies were used as hybridoma supernatants: L8C4, IgG recognizing  T. brucei  TbPFR2 and cross-reacting with  T. cruzi  orthologue TcPFR2 [ 74 ]; BB2, IgG recognizing the Ty-1 tag of the TbPFR2-TAG and TbPFR2-TAG-ΔHLA recombinant proteins [ 54 ]; and ROD-1, IgM recognizing a doublet of minor PFR proteins [ 55 ]. For immunofluorescence, trypanosomes were spread onto poly-L-lysine-coated slides, fixed in cold methanol and processed as described [ 75 ]. Experiments involving the use of L8C4 only were performed with an FITC-conjugated anti-mouse IgG secondary antibody. Double-staining experiments using BB2 and ROD-1 were performed with a TRITC-conjugated specific anti-mouse IgG secondary antibody and an FITC-conjugated specific anti-mouse IgM secondary antibody. DNA was systematically stained with 4',6-diamidino-2-phenylindole (DAPI). Slides were examined with a Leica DMR microscope, images were captured using a cooled CCD camera (Cool Snap HQ, Roper Scientific) and processed with the GNU image manipulation program version 2 [ 76 ].
Immunofluorescence and microscopy
Three different monoclonal antibodies were used as hybridoma supernatants: L8C4, IgG recognizing  T. brucei  TbPFR2 and cross-reacting with  T. cruzi  orthologue TcPFR2 [ 74 ]; BB2, IgG recognizing the Ty-1 tag of the TbPFR2-TAG and TbPFR2-TAG-ΔHLA recombinant proteins [ 54 ]; and ROD-1, IgM recognizing a doublet of minor PFR proteins [ 55 ]. For immunofluorescence, trypanosomes were spread onto poly-L-lysine-coated slides, fixed in cold methanol and processed as described [ 75 ]. Experiments involving the use of L8C4 only were performed with an FITC-conjugated anti-mouse IgG secondary antibody. Double-staining experiments using BB2 and ROD-1 were performed with a TRITC-conjugated specific anti-mouse IgG secondary antibody and an FITC-conjugated specific anti-mouse IgM secondary antibody. DNA was systematically stained with 4',6-diamidino-2-phenylindole (DAPI). Slides were examined with a Leica DMR microscope, images were captured using a cooled CCD camera (Cool Snap HQ, Roper Scientific) and processed with the GNU image manipulation program version 2 [ 76 ].T. bruceiT. cruziCell sedimentation assay The trypanosome sedimentation assay was performed as described in [ 56 ]. Briefly: trypanosomes were grown at ≈ 5.10 6  cells/mL in normal culture medium, with or without 48 hour tetracycline induction. 1 mL of these cultures was dispensed to 5 plastic spectrophotometry cuvettes, for time points 0, 2, 4, 6, 8 hours, and left still. At each time point, the optical density at 600 nm was measured twice: first without mixing (O.D. no mix ) and second after mixing the cuvette (O.D. mix ). Data were plotted as a function of time.
Cell sedimentation assay
The trypanosome sedimentation assay was performed as described in [ 56 ]. Briefly: trypanosomes were grown at ≈ 5.10 6  cells/mL in normal culture medium, with or without 48 hour tetracycline induction. 1 mL of these cultures was dispensed to 5 plastic spectrophotometry cuvettes, for time points 0, 2, 4, 6, 8 hours, and left still. At each time point, the optical density at 600 nm was measured twice: first without mixing (O.D. no mix ) and second after mixing the cuvette (O.D. mix ). Data were plotted as a function of time.Authors' contributions F.R. carried out most of the experiments reported and wrote the manuscript, M.D.-D. performed the double transfection reported at Table  1  & Figure  2  and P.B. conceived the study and participated in its design and coordination.
Authors' contributions
F.R. carried out most of the experiments reported and wrote the manuscript, M.D.-D. performed the double transfection reported at Table  1  & Figure  2  and P.B. conceived the study and participated in its design and coordination.