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1.  Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi 
PLoS ONE  2010;5(6):e11407.
Background
Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility.
Methodology/Principal Findings
Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea.
Conclusions/Significance
Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating.
doi:10.1371/journal.pone.0011407
PMCID: PMC2894934  PMID: 20613980
2.  A single-cloning-step procedure for the generation of RNAi plasmids producing long stem-loop RNA 
RNA interference (RNAi), used as a tool, has revolutionized the studies of gene function. Long stem-loop dsRNA has been proven the most effective trigger for down-regulating target transcripts in RNAi-positive trypanosomatid parasites. Here we describe a protocol for constructing plasmids that produce long stem-loops by using a single cloning step. Inverted repeats are first obtained by self-ligation of PCR products that contain a randomized segment at one of their ends and then inserted in a plasmid vector. The random sequences create the loop (or “stuffer”) of the hairpin. This methodology was tested in Leishmania (Viannia) braziliensis to constitutively knock down the mRNAs for the well-studied paraflagellar rod protein 1 and 2 (PFR1 and PFR2) genes and revealed that mRNA cleavage products are unusually stable in these parasites. The protocol is suitable for any plasmid (for constitutive or inducible expression) and for any organism in which long stem-loops can be used to elicit RNAi.
doi:10.1016/j.molbiopara.2012.04.003
PMCID: PMC3358476  PMID: 22542486
RNAi; hairpin RNA; inverted repeat cloning; Leishmania braziliensis
3.  Cryptic Paraflagellar Rod in Endosymbiont-Containing Kinetoplastid Protozoa 
Eukaryotic Cell  2005;4(3):516-525.
Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.
doi:10.1128/EC.4.3.516-525.2005
PMCID: PMC1087800  PMID: 15755914
4.  Biogenesis of the Trypanosome Endo-Exocytotic Organelle Is Cytoskeleton Mediated 
PLoS Biology  2008;6(5):e105.
Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.
Author Summary
Trypanosomes are ubiquitous unicellular parasites that infect humans, animals, insects, and plants. African, Asian, and some South American trypanosomes have evolved the amazing ability to change their surface coat proteins, an essential strategy for their survival. The surface coat proteins are recycled and targeted to the surface of the parasite via an endocytic and exocytotic organelle called the flagellar pocket, which is sequestered in the trypanosome cell's cytoplasm. The flagellar pocket is also used to remove host-derived antibodies that are bound to the surface of the parasite, making this organelle critical for the parasite's evasion of the host immune system. We describe a novel protein, “BILBO1,” which was identified from the insect-form parasite of the African trypanosome Trypanosoma brucei. We show that BILBO1 is part of a ring or horseshoe-like cytoskeletal structure that is located in a region of the flagellar pocket called the collar. When BILBO1 transcripts were knocked down with inducible RNA interference, trypanosome cells became arrested in a post-mitotic cell-cycle stage. Induced cells lost the normal flagellum-to-cell-body attachment, were unable to regulate endocytosis and exocytosis, and most importantly, were unable to construct a new flagellar pocket. These results provide molecular evidence for the idea that flagellar pocket biogenesis is cytoskeletally mediated.
RNAi of the parasite protein BILBO1 prevents the biogenesis of the endocytic and exocytotic organelle in Trypanosoma brucei, kills the parasite, and reveals novel insights into how this pathogen organizes and uses one of its distinctive organelles.
doi:10.1371/journal.pbio.0060105
PMCID: PMC2365980  PMID: 18462016
5.  RNA interference: advances and questions. 
In animals and protozoa gene-specific double-stranded RNA triggers the degradation of homologous cellular RNAs, the phenomenon of RNA interference (RNAi). RNAi has been shown to represent a novel paradigm in eukaryotic biology and a powerful method for studying gene function. Here we discuss RNAi in terms of its mechanism, its relationship to other post-transcriptional gene silencing phenomena in plants and fungi, its connection to retroposon silencing and possibly to translation, and its biological role. Among the organisms where RNAi has been demonstrated the protozoan parasite Trypanosoma brucei represents the most ancient branch of the eukaryotic lineage. We provide a synopsis of what is currently known about RNAi in T. brucei and outline the recent advances that make RNAi the method of choice to disrupt gene function in these organisms.
doi:10.1098/rstb.2001.0952
PMCID: PMC1692925  PMID: 11839183
6.  Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum*S⃞ 
The Journal of Biological Chemistry  2009;284(9):5610-5619.
Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.
doi:10.1074/jbc.M808859200
PMCID: PMC2645819  PMID: 19074134
7.  TbAGO1, an Argonaute protein required for RNA interference, is involved in mitosis and chromosome segregation in Trypanosoma brucei 
BMC Biology  2003;1:2.
Background
RNA silencing processes are widespread in almost all eukaryotic organisms. They have various functions including genome protection, and the control of gene expression, development and heterochromatin formation. RNA interference (RNAi) is the post-transcriptional destruction of RNA, which is mediated by a ribonucleoprotein complex that contains, among several components, RNA helicases and Argonaute proteins. RNAi is functional in trypanosomes, protozoan parasites that separated very early from the main eukaryotic lineage and exhibit several intriguing features in terms of the control of gene expression. In this report, we investigated the functions of RNAi in Trypanosoma brucei.
Results
By searching through genome databases, novel Argonaute-like proteins were identified in several protozoa that belong to the kinetoplastid order, a group of organisms that diverged early from the main eukaryotic lineage. T. brucei possesses two Argonaute-like genes termed TbAGO1 and TbPWI1. Dual transient transfection assays suggest that TbAGO1, but not TbPWI1, is involved in RNAi. The entire coding region of TbAGO1 was deleted by double gene knockout. TbAGO1-/- cells turned out to be completely resistant to RNAi generated either by transfected double-stranded RNA or by expression of an inverted repeat. TbAGO1-/- cells were viable but showed a dramatically reduced growth rate. This was probably due to defects in mitosis and abnormal chromosome segregation as revealed by in situ analysis. The RNAi and growth phenotypes were complemented by the inducible expression of a GFP::TbAGO1 fusion protein that revealed the cytoplasmic location of the protein.
Conclusions
The requirement of TbAGO1 for RNAi in trypanosomes demonstrates the evolutionary ancient involvement of Argonaute proteins in RNAi silencing processes. RNAi-deficient TbAGO1-/- cells showed numerous defects in chromosome segregation and mitotic spindle assembly. We propose a working hypothesis in which RNAi would be involved in heterochromatin formation at the centromere and therefore in chromosome segregation.
doi:10.1186/1741-7007-1-2
PMCID: PMC317389  PMID: 14670085
8.  RNA Interference in Infectious Tropical Diseases 
Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.
doi:10.3347/kjp.2008.46.1.1
PMCID: PMC2526294  PMID: 18344671
RNAi; Parasitic protozoa; Helminth; Insect vector
9.  The Diverse Roles of RNA Helicases in RNAi 
Cell cycle (Georgetown, Tex.)  2009;8(21):3500-3505.
RNA interference (RNAi) is a regulatory gene silencing system found in nearly all eukaryotic organisms that employs small RNAs, typically 20–25 nucleotides long, to target complementary sequences found in mRNAs. RNA helicases use ATP to unwind double-stranded RNA (dsRNA), and are known to participate at nearly every level of RNA metabolism. A multitude of RNA helicases have been isolated from screens for essential RNAi factors, and even the earliest models of the RNAi pathway have presumed an RNA helicase to function at the level of small RNA duplex unwinding. However, while many components that function in RNAi have been uncovered and characterized, the exact placement in the pathway and ascription of a specific biochemical function of an RNA helicase in RNAi remains elusive. Recent studies have delved deeper into the precise role of some RNA helicases. Surprisingly, these studies have revealed nontraditional roles, which may not even require the helicase activity. Such findings suggest that RNA helicases regulate gene silencing at nearly every level of the RNAi pathways.
PMCID: PMC3016640  PMID: 19823018
gene silencing; RNA helicase; RNAi; small RNAs; unwind
10.  Retention and Loss of RNA Interference Pathways in Trypanosomatid Protozoans 
PLoS Pathogens  2010;6(10):e1001161.
RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.
Author Summary
RNAi interference pathways play fundamental roles in eukaryotes and provide important methods for the analysis of gene function. Occasionally RNAi has been lost, precluding its use as a tool, as well as raising the question of what forces could lead to loss of such a key pathway. Genomic and functional studies previously showed that within trypanosomatids protozoans RNAi was absent in both Leishmania major and Trypanosoma cruzi. The genome of L. braziliensis, a member of the early diverging Leishmania subgenus Viannia, retained key genes required for RNAi such as an Argonaute. We demonstrated that in fact L. braziliensis shows strong RNAi activity with reporter and endogenous genes affecting flagellar function. These data suggest that RNAi may be productively applied for functional genomic studies in L. braziliensis. We mapped the evolutionary point at which RNAi was lost in lineage leading to Leishmania and Crithidia, and establish that RNAi must have been lost at least twice in the trypanosomatids, once on the lineage leading to T. cruzi and independently following the divergence of the Viannia subgenus from other Leishmania species. Lastly, we discuss hypotheses concerning the forces leading to the loss of RNAi in Leishmania evolution, including viral invasion, increased genome plasticity, and altered virulence.
doi:10.1371/journal.ppat.1001161
PMCID: PMC2965760  PMID: 21060810
11.  Three-Dimensional Structure of the Trypanosome Flagellum Suggests that the Paraflagellar Rod Functions as a Biomechanical Spring 
PLoS ONE  2012;7(1):e25700.
Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.
doi:10.1371/journal.pone.0025700
PMCID: PMC3250385  PMID: 22235240
12.  A computational study of off-target effects of RNA interference 
Nucleic Acids Research  2005;33(6):1834-1847.
RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silencing that is frequently used to study gene function. RNAi is initiated by short interfering RNA (siRNA) of ∼21 nt in length, either generated from the double-stranded RNA (dsRNA) by using the enzyme Dicer or introduced experimentally. Following association with an RNAi silencing complex, siRNA targets mRNA transcripts that have sequence identity for destruction. A phenotype resulting from this knockdown of expression may inform about the function of the targeted gene. However, ‘off-target effects’ compromise the specificity of RNAi if sequence identity between siRNA and random mRNA transcripts causes RNAi to knockdown expression of non-targeted genes. The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. This computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100–400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. Remarkably, high-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5′ termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C.elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of ∼10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.
doi:10.1093/nar/gki324
PMCID: PMC1072799  PMID: 15800213
13.  The miR-35-41 Family of MicroRNAs Regulates RNAi Sensitivity in Caenorhabditis elegans 
PLoS Genetics  2012;8(3):e1002536.
RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) to direct silencing of specific genes through transcriptional and post-transcriptional mechanisms. The siRNA guides can originate from exogenous (exo–RNAi) or natural endogenous (endo–RNAi) sources of double-stranded RNA (dsRNA). In Caenorhabditis elegans, inactivation of genes that function in the endo–RNAi pathway can result in enhanced silencing of genes targeted by siRNAs from exogenous sources, indicating cross-regulation between the pathways. Here we show that members of another small RNA pathway, the mir-35-41 cluster of microRNAs (miRNAs) can regulate RNAi. In worms lacking miR-35-41, there is reduced expression of lin-35/Rb, the C. elegans homolog of the tumor suppressor Retinoblastoma gene, previously shown to regulate RNAi responsiveness. Genome-wide microarray analyses show that targets of endo–siRNAs are up-regulated in mir-35-41 mutants, a phenotype also displayed by lin-35/Rb mutants. Furthermore, overexpression of lin-35/Rb specifically rescues the RNAi hypersensitivity of mir-35-41 mutants. Although the mir-35-41 miRNAs appear to be exclusively expressed in germline and embryos, their effect on RNAi sensitivity is transmitted to multiple tissues and stages of development. Additionally, we demonstrate that maternal contribution of miR-35-41 or lin-35/Rb is sufficient to reduce RNAi effectiveness in progeny worms. Our results reveal that miRNAs can broadly regulate other small RNA pathways and, thus, have far reaching effects on gene expression beyond directly targeting specific mRNAs.
Author Summary
RNA interference (RNAi) has become a widely used approach for silencing genes of interest. This tool is possible because endogenous RNA silencing pathways exist broadly across organisms, including humans, worms, and plants. The general RNAi pathway utilizes small ∼21-nucleotide RNAs to target specific protein-coding genes through base-pairing interactions. Since RNAs from exogenous sources require some of the same factors as endogenous small RNAs to silence gene expression, there can be competition between the pathways. Thus, perturbations in the endogenous RNAi pathway can result in enhanced silencing efficiency by exogenous small RNAs. MicroRNAs (miRNAs) comprise another endogenous small RNA pathway, but their biogenesis and mechanism of gene silencing are distinct in many ways from RNAi pathways. Here we show that a family of miRNAs regulates the effectiveness of RNAi in Caenorhabditis elegans. Loss of mir-35-41 results in enhanced RNAi by exogenous RNAs and reduced silencing of endogenous RNAi targets. The embryonic miR-35-41 miRNAs regulate the sensitivity to RNAi through lin-35/Rb, a homolog of the human Retinoblastoma tumor suppressor gene previously shown to regulate RNAi effectiveness in C. elegans. Additionally, we show that this sensitivity can be passed on to the next generation of worms, demonstrating a far-reaching effect of the miR-35-41 miRNAs on gene regulation by other small RNA pathways.
doi:10.1371/journal.pgen.1002536
PMCID: PMC3297572  PMID: 22412382
14.  Targeting of Dicer-2 and RNA by a Viral RNA Silencing Suppressor in Drosophila Cells 
Journal of Virology  2012;86(10):5763-5773.
RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2–Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.
doi:10.1128/JVI.07229-11
PMCID: PMC3347268  PMID: 22438534
15.  Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei ▿ † 
Eukaryotic Cell  2011;10(7):884-894.
The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.
doi:10.1128/EC.00298-10
PMCID: PMC3147417  PMID: 21378260
16.  The major component of the paraflagellar rod of Trypanosoma brucei is a helical protein that is encoded by two identical, tandemly linked genes 
The Journal of Cell Biology  1989;109(4):1695-1709.
The flagellum of the parasitic hemoflagellate Trypanosoma brucei contains two major structures: (a) the microtubule axoneme, and (b) a highly ordered, filamentous array, the paraflagellar rod (PFR). This is a complex, three-dimensional structure, of yet unknown function, that extends along most of the axoneme and is closely linked to it. Its major structural component is a single protein of 600 amino acids. This PFR protein can assume two different conformations, resulting in two distinct bands of apparent molecular masses of 73 and 69 kD in SDS-gel electrophoresis. Secondary structure predictions indicate a very high helix content. Despite its biochemical similarity to the intermediate filament proteins (solubility properties, amino acid composition, and high degree of helicity), the PFR protein does not belong in this class of cytoskeletal proteins. The PFR protein is coded for by two tandemly linked genes of identical nucleotide sequence. Both genes are transcribed into stable mRNAs of very similar length that carry the mini-exon sequence at their 5' termini.
PMCID: PMC2115804  PMID: 2793936
17.  The Ebola Virus VP35 Protein Is a Suppressor of RNA Silencing 
PLoS Pathogens  2007;3(6):e86.
RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.
Author Summary
Cells have evolved mechanisms to protect themselves from virus infection. A well-known antiviral mechanism in mammals is the interferon (IFN) response of the innate immune system. In plants, insects, and worms, RNA silencing or RNA interference (RNAi) is a strong antiviral defence mechanism. It is still debated whether RNAi is also used as an antiviral mechanism in mammals. Many mammalian viruses encode essential factors that suppress the innate antiviral responses of the host. Such innate immunity suppressor proteins, or IFN antagonists, have recently been reported to also suppress RNAi in mammalian cells. We now demonstrate that the Ebola virus VP35 protein, a known IFN antagonist, suppresses RNAi in human cells. In addition, VP35 restores the production of an HIV-1 variant with a defective RNAi suppressor Tat protein. These results indicate that RNAi is part of the innate antiviral defence response in mammals and that viruses need to counteract this response in order to replicate. Whereas RNAi and INF act in concert to prevent the infection of mammalian cells, the invading viruses encode a protein that counteracts both defence mechanisms.
doi:10.1371/journal.ppat.0030086
PMCID: PMC1894824  PMID: 17590081
18.  Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes 
Molecular and Cellular Biology  1999;19(12):8191-8200.
The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.
PMCID: PMC84903  PMID: 10567544
19.  The possible impact of persistent virus infection on the function of the RNAi machinery in insects: a hypothesis 
RNAi experiments in insects are characterized by great variability in efficiency; for instance beetles and locusts are very amenable to dsRNA-mediated gene silencing, while other insect groups, most notably lepidopterans, are more refractory to RNAi. Several factors can be forwarded that could affect the efficiency of RNAi, such as the composition and function of the intracellular RNAi machinery, the mechanism of dsRNA uptake, the presence of dsRNA- and siRNA-degrading enzymes and non-specific activation of the innate immune response. In this essay, we investigate the evidence whether persistent infection with RNA viruses could be a major factor that affects the response to exogenous dsRNA in insects. The occurrence of RNA viruses in different insect groups will be discussed, as well as several mechanisms by which viruses could interfere with the process of RNAi. Finally, the impact of RNA virus infection on the design of dsRNA-based insect control strategies will be considered.
doi:10.3389/fphys.2013.00319
PMCID: PMC3817476  PMID: 24204347
RNAi; persistent virus infection; RNA virus; insects; pest control; innate immune response
20.  Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells 
Molecular and Cellular Biology  2001;21(22):7807-7816.
Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.
doi:10.1128/MCB.21.22.7807-7816.2001
PMCID: PMC99950  PMID: 11604515
21.  Small nucleolar RNA interference in Trypanosoma brucei: mechanism and utilization for elucidating the function of snoRNAs 
Nucleic Acids Research  2010;38(20):7236-7247.
Expression of dsRNA complementary to small nucleolar RNAs (snoRNAs) in Trypanosoma brucei results in snoRNA silencing, termed snoRNAi. Here, we demonstrate that snoRNAi requires the nuclear TbDCL2 protein, but not TbDCL1, which is involved in RNA interference (RNAi) in the cytoplasm. snoRNAi depends on Argonaute1 (Slicer), and on TbDCL2, suggesting that snoRNA dicing and slicing takes place in the nucleus, and further suggesting that AGO1 is active in nuclear silencing. snoRNAi was next utilized to elucidate the function of an abundant snoRNA, TB11Cs2C2 (92 nt), present in a cluster together with the spliced leader associated RNA (SLA1) and snR30, which are both H/ACA RNAs with special nuclear functions. Using AMT-UV cross-linking and RNaseH cleavage, we provide evidence for the interaction of TB11Cs2C2 with the small rRNAs, srRNA-2 and srRNA-6, which are part of the large subunit (LSU) rRNA. snoRNAi of TB11Cs2C2 resulted in defects in generating srRNA-2 and LSUβ rRNA. This is the first snoRNA described so far to engage in trypanosome-specific processing events.
doi:10.1093/nar/gkq599
PMCID: PMC2978370  PMID: 20601683
22.  Stable and heritable gene silencing in the malaria vector Anopheles stephensi 
Nucleic Acids Research  2003;31(15):e85.
Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles. This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.
PMCID: PMC169974  PMID: 12888537
23.  Role of RNA Interference (RNAi) in the Moss Physcomitrella patens 
RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species.
doi:10.3390/ijms14011516
PMCID: PMC3565333  PMID: 23344055
RNAi; non-coding RNAs; miRNA; siRNA; gene silencing; Physcomitrella patens
24.  RNA Interference with Special Reference to Combating Viruses of Crustacea 
RNA interference has evolved from being a nuisance biological phenomenon to a valuable research tool to determine gene function and as a therapeutic agent. Since pioneering observations regarding RNA interference were first reported in the 1990s from the nematode worm, plants and Drosophila, the RNAi phenomenon has since been reported in all eukaryotic organisms investigated from protozoans, plants, arthropods, fish and mammals. The design of RNAi therapeutics has progressed rapidly to designing dsRNA that can specifically and effectively silence disease related genes. Such technology has demonstrated the effective use of short interfering as therapeutics. In the absence of a B cell lineage in arthropods, and hence no long term vaccination strategy being available, the introduction of using RNA interference in crustacea may serve as an effective control and preventative measure for viral diseases for application in aquaculture.
doi:10.1007/s13337-012-0084-1
PMCID: PMC3550745  PMID: 23997446
RNA interference; Crustacea; Viruses; WSSV; YHV;  TSV; PmergDNV; MrNV
25.  Generation of RNAi Libraries for High-Throughput Screens 
The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.
doi:10.1155/JBB/2006/45716
PMCID: PMC1559919  PMID: 17057364

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