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1.  RNAi Effector Diversity in Nematodes 
While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.
Author Summary
Many organisms regulate gene expression through an RNA interference (RNAi) pathway, first characterized in the nematode Caenorhabditis elegans. This pathway can be triggered experimentally using double-stranded (ds)RNA to selected gene targets, thereby allowing researchers to ‘silence’ individual genes and so investigate their function. It is hoped that this technology will facilitate gene silencing in important parasitic nematodes that impose a considerable health and economic burden on mankind. Unfortunately, differences in RNAi susceptibility have been observed between species. Here we investigated the possibility that differences in the complement of effector proteins involved in the RNAi pathway are responsible for these differences in susceptibility. Our data revealed that most facets of the RNAi pathway are well represented across parasitic nematodes, although there were fewer pathway proteins in other nematodes compared to C. elegans. In contrast, the proteins responsible for uptake and spread of dsRNA are not well represented in parasitic nematodes. However, the importance of these differences is undermined by our observation that the protein complements in all the parasites were qualitatively similar, regardless of RNAi-susceptibility. Clearly, differences in the RNAi pathway of parasitic nematodes do not explain the variations in susceptibility to experimental RNAi.
doi:10.1371/journal.pntd.0001176
PMCID: PMC3110158  PMID: 21666793
2.  An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans 
PLoS Pathogens  2009;5(2):e1000286.
Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense.
Author Summary
The genome of Caenorhabditis elegans encodes three Dicer-related helicases (DRHs) highly homologous to the DExD/H box helicase domain found in two distinct families of virus sensors, Dicer ribonucleases and RIG-I-like helicases (RLRs). Dicer initiates the specific, RNAi-mediated viral immunity in plants, fungi and invertebrates by producing virus-derived small interfering RNAs (siRNAs). By contrast, mammalian RLRs trigger interferon production and broad-spectrum viral immunity, although one of the three RLRs may act as both a negative and positive regulator of viral immunity. In this study we developed a transgenic C. elegans strain for high-throughput genetic screens and identified 35 genes including drh-1 that are required for RNAi-mediated viral immunity. Genetic epistatic analyses demonstrate that drh-1 mediates RNAi immunity downstream of the production of viral siRNAs. Notably, we found that drh-2 functions as a negative regulator of the viral immunity. Thus, both nematode DRHs and mammalian RLRs participate in antiviral immune responses. Unlike mammalian RLRs, however, nematode DRH-1 employs an RNAi effector mechanism and is unlikely to be involved in direct virus sensing.
doi:10.1371/journal.ppat.1000286
PMCID: PMC2629121  PMID: 19197349
3.  Systemic RNAi mediated gene silencing in the anhydrobiotic nematode Panagrolaimus superbus 
Background
Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. Although RNAi was first described in Caenorhabditis elegans, several nematode species are unable to mount an RNAi response when exposed to exogenous double stranded RNA (dsRNA). These include the satellite model organisms Pristionchus pacificus and Oscheius tipulae. Available data also suggest that the RNAi pathway targeting exogenous dsRNA may not be fully functional in some animal parasitic nematodes. The genus Panagrolaimus contains bacterial feeding nematodes which occupy a diversity of niches ranging from polar, temperate and semi-arid soils to terrestrial mosses. Thus many Panagrolaimus species are adapted to tolerate freezing and desiccation and are excellent systems to study the molecular basis of environmental stress tolerance. We investigated whether Panagrolaimus is susceptible to RNAi to determine whether this nematode could be used in large scale RNAi studies in functional genomics.
Results
We studied two species: Panagrolaimus sp. PS1159 and Panagrolaimus superbus. Both nematode species displayed embryonic lethal RNAi phenotypes following ingestion of Escherichia coli expressing dsRNA for the C. elegans embryonic lethal genes Ce-lmn-1 and Ce-ran-4. Embryonic lethal RNAi phenotypes were also obtained in both species upon ingestion of dsRNA for the Panagrolaimus genes ef1b and rps-2. Single nematode RT-PCR showed that a significant reduction in mRNA transcript levels occurred for the target ef1b and rps-2 genes in RNAi treated Panagrolaimus sp. 1159 nematodes. Visible RNAi phenotypes were also observed when P. superbus was exposed to dsRNA for structural genes encoding contractile proteins. All RNAi phenotypes were highly penetrant, particularly in P. superbus.
Conclusion
This demonstration that Panagrolaimus is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for P. superbus. This greatly enhances the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis and cryobiosis and as a possible satellite model nematode for comparative and functional genomics. Our data also identify another nematode infraorder which is amenable to RNAi and provide additional information on the diversity of RNAi phenotypes in nematodes.
doi:10.1186/1471-2199-9-58
PMCID: PMC2453295  PMID: 18565215
4.  Development of Functional Genomic Tools in Trematodes: RNA Interference and Luciferase Reporter Gene Activity in Fasciola hepatica 
The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis.
Author Summary
Reverse genetics tools allow assessing the function of unknown genes. Their application for the study of neglected infectious diseases could lead eventually to the identification of relevant gene products to be used in diagnosis, or as drug targets or immunization candidates. Being technically more simple and less demanding than other reverse genetics tools such as transgenesis or knockouts, the suppression of gene activity mediated by double-stranded RNA has emerged as a powerful tool for the analysis of gene function. RNAi appeared as an obvious alternative to apply in complex biological systems where information is still scarce, a situation common to several infectious and parasitic diseases. However, several technical or practical difficulties have hampered the development of this technique in parasites to the expectations originally generated. We developed a simple method to test the presence of a viable RNAi pathway by silencing an exogenous reporter gene. The method was tested in F. hepatica, describing the conditions for transfection and confirming the existence of a viable RNAi pathway in this parasite. The experimental design created can be useful as a first approach in organisms where genetic analysis is still unavailable, providing a tool to unravel gene function and probably advancing new candidates relevant in pathobiology, prevention or treatment.
doi:10.1371/journal.pntd.0000260
PMCID: PMC2440534  PMID: 18612418
5.  Clustering phenotype populations by genome-wide RNAi and multiparametric imaging 
How to predict gene function from phenotypic cues is a longstanding question in biology.Using quantitative multiparametric imaging, RNAi-mediated cell phenotypes were measured on a genome-wide scale.On the basis of phenotypic ‘neighbourhoods', we identified previously uncharacterized human genes as mediators of the DNA damage response pathway and the maintenance of genomic integrity.The phenotypic map is provided as an online resource at http://www.cellmorph.org for discovering further functional relationships for a broad spectrum of biological module
Genetic screens for phenotypic similarity have made key contributions for associating genes with biological processes. Aggregating genes by similarity of their loss-of-function phenotype has provided insights into signalling pathways that have a conserved function from Drosophila to human (Nusslein-Volhard and Wieschaus, 1980; Bier, 2005). Complex visual phenotypes, such as defects in pattern formation during development, greatly facilitated the classification of genes into pathways, and phenotypic similarities in many cases predicted molecular relationships. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cultured cells has become feasible in many organisms whose genome have been sequenced (Boutros and Ahringer, 2008). One of the current challenges is the computational categorization of visual phenotypes and the prediction of gene function and associated biological processes. With large parts of the genome still being in unchartered territory, deriving functional information from large-scale phenotype analysis promises to uncover novel gene–gene relationships and to generate functional maps to explore cellular processes.
In this study, we developed an automated approach using RNAi-mediated cell phenotypes, multiparametric imaging and computational modelling to obtain functional information on previously uncharacterized genes. To generate broad, computer-readable phenotypic signatures, we measured the effect of RNAi-mediated knockdowns on changes of cell morphology in human cells on a genome-wide scale. First, the several million cells were stained for nuclear and cytoskeletal markers and then imaged using automated microscopy. On the basis of fluorescent markers, we established an automated image analysis to classify individual cells (Figure 1A). After cell segmentation for determining nuclei and cell boundaries (Figure 1C), we computed 51 cell descriptors that quantified intensities, shape characteristics and texture (Figure 1F). Individual cells were categorized into 1 of 10 classes, which included cells showing protrusion/elongation, cells in metaphase, large cells, condensed cells, cells with lamellipodia and cellular debris (Figure 1D and E). Each siRNA knockdown was summarized by a phenotypic profile and differences between RNAi knockdowns were quantified by the similarity between phenotypic profiles. We termed the vector of scores a phenoprint (Figure 3C) and defined the phenotypic distance between a pair of perturbations as the distance between their corresponding phenoprints.
To visualize the distribution of all phenoprints, we plotted them in a genome-wide map as a two-dimensional representation of the phenotypic similarity relationships (Figure 3A). The complete data set and an interactive version of the phenotypic map are available at http://www.cellmorph.org. The map identified phenotypic ‘neighbourhoods', which are characterized by cells with lamellipodia (WNK3, ANXA4), cells with prominent actin fibres (ODF2, SOD3), abundance of large cells (CA14), many elongated cells (SH2B2, ELMO2), decrease in cell number (TPX2, COPB1, COPA), increase in number of cells in metaphase (BLR1, CIB2) and combinations of phenotypes such as presence of large cells with protrusions and bright nuclei (PTPRZ1, RRM1; Figure 3B).
To test whether phenotypic similarity might serve as a predictor of gene function, we focused our further analysis on two clusters that contained genes associated with the DNA damage response (DDR) and genomic integrity (Figure 3A and C). The first phenotypic cluster included proteins with kinetochore-associated functions such as NUF2 (Figure 3B) and SGOL1. It also contained the centrosomal protein CEP164 that has been described as an important mediator of the DNA damage-activated signalling cascade (Sivasubramaniam et al, 2008) and the largely uncharacterized genes DONSON and SON. A second phenotypically distinct cluster included previously described components of the DDR pathway such as RRM1 (Figure 3A–C), CLSPN, PRIM2 and SETD8. Furthermore, this cluster contained the poorly characterized genes CADM1 and CD3EAP.
Cells activate a signalling cascade in response to DNA damage induced by exogenous and endogenous factors. Central are the kinases ATM and ATR as they serve as sensors of DNA damage and activators of further downstream kinases (Harper and Elledge, 2007; Cimprich and Cortez, 2008). To investigate whether DONSON, SON, CADM1 and CD3EAP, which were found in phenotypic ‘neighbourhoods' to known DDR components, have a role in the DNA damage signalling pathway, we tested the effect of their depletion on the DDR on γ irradiation. As indicated by reduced CHEK1 phosphorylation, siRNA knock down of DONSON, SON, CD3EAP or CADM1 resulted in impaired DDR signalling on γ irradiation. Furthermore, knock down of DONSON or SON reduced phosphorylation of downstream effectors such as NBS1, CHEK1 and the histone variant H2AX on UVC irradiation. DONSON depletion also impaired recruitment of RPA2 onto chromatin and SON knockdown reduced RPA2 phosphorylation indicating that DONSON and SON presumably act downstream of the activation of ATM. In agreement to their phenotypic profile, these results suggest that DONSON, SON, CADM1 and CD3EAP are important mediators of the DDR. Further experiments demonstrated that they are also required for the maintenance of genomic integrity.
In summary, we show that genes with similar phenotypic profiles tend to share similar functions. The power of our computational and experimental approach is demonstrated by the identification of novel signalling regulators whose phenotypic profiles were found in proximity to known biological modules. Therefore, we believe that such phenotypic maps can serve as a resource for functional discovery and characterization of unknown genes. Furthermore, such approaches are also applicable for other perturbation reagents, such as small molecules in drug discovery and development. One could also envision combined maps that contain both siRNAs and small molecules to predict target–small molecule relationships and potential side effects.
Genetic screens for phenotypic similarity have made key contributions to associating genes with biological processes. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible. One of the current challenges however is the computational categorization of visual phenotypes and the prediction of biological function and processes. In this study, we describe a combined computational and experimental approach to discover novel gene functions and explore functional relationships. We performed a genome-wide RNAi screen in human cells and used quantitative descriptors derived from high-throughput imaging to generate multiparametric phenotypic profiles. We show that profiles predicted functions of genes by phenotypic similarity. Specifically, we examined several candidates including the largely uncharacterized gene DONSON, which shared phenotype similarity with known factors of DNA damage response (DDR) and genomic integrity. Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity. Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.
doi:10.1038/msb.2010.25
PMCID: PMC2913390  PMID: 20531400
DNA damage response signalling; massively parallel phenotyping; phenotype networks; RNAi screening
6.  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
7.  Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditis elegans 
PLoS Genetics  2010;6(7):e1001044.
Heme is a cofactor in proteins that function in almost all sub-cellular compartments and in many diverse biological processes. Heme is produced by a conserved biosynthetic pathway that is highly regulated to prevent the accumulation of heme—a cytotoxic, hydrophobic tetrapyrrole. Caenorhabditis elegans and related parasitic nematodes do not synthesize heme, but instead require environmental heme to grow and develop. Heme homeostasis in these auxotrophs is, therefore, regulated in accordance with available dietary heme. We have capitalized on this auxotrophy in C. elegans to study gene expression changes associated with precisely controlled dietary heme concentrations. RNA was isolated from cultures containing 4, 20, or 500 µM heme; derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. We identified 288 heme-responsive genes (hrgs) that were differentially expressed under these conditions. Of these genes, 42% had putative homologs in humans, while genomes of medically relevant heme auxotrophs revealed homologs for 12% in both Trypanosoma and Leishmania and 24% in parasitic nematodes. Depletion of each of the 288 hrgs by RNA–mediated interference (RNAi) in a transgenic heme-sensor worm strain identified six genes that regulated heme homeostasis. In addition, seven membrane-spanning transporters involved in heme uptake were identified by RNAi knockdown studies using a toxic heme analog. Comparison of genes that were positive in both of the RNAi screens resulted in the identification of three genes in common that were vital for organismal heme homeostasis in C. elegans. Collectively, our results provide a catalog of genes that are essential for metazoan heme homeostasis and demonstrate the power of C. elegans as a genetic animal model to dissect the regulatory circuits which mediate heme trafficking in both vertebrate hosts and their parasites, which depend on environmental heme for survival.
Author Summary
Heme is an iron-containing cofactor for proteins involved in many critical cellular processes. However, free heme is toxic to cells, suggesting that heme synthesis, acquisition, and transport is highly regulated. Efforts to understand heme trafficking in multicellular organisms have failed primarily due to the inability to separate the processes of endogenous heme synthesis from heme uptake and transport. Caenorhabditis elegans is unique among model organisms because it cannot synthesize heme but instead eats environmental heme to grow and develop normally. Thus, worms are an ideal genetic animal model to study heme homeostasis. This work identifies a novel list of 288 heme-responsive genes (hrgs) in C. elegans and a number of related genes in humans and medically relevant parasites. Knocking down the function of each of these hrgs reveals roles for several in heme uptake, transport, and detection within the organism. Our study provides insights into metazoan regulation of organismal heme homeostasis. The identification of parasite-specific hrg homologs may permit the selective design and screening of drugs that specifically target heme uptake pathways in parasites without affecting the host. Thus, this work has therapeutic implications for the treatment of human iron deficiency, one of the top ten mortality factors world-wide.
doi:10.1371/journal.pgen.1001044
PMCID: PMC2912396  PMID: 20686661
8.  Dissecting Systemic RNA Interference in the Red Flour Beetle Tribolium castaneum: Parameters Affecting the Efficiency of RNAi 
PLoS ONE  2012;7(10):e47431.
The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.
doi:10.1371/journal.pone.0047431
PMCID: PMC3484993  PMID: 23133513
9.  RNAi Dynamics in Juvenile Fasciola spp. Liver Flukes Reveals the Persistence of Gene Silencing In Vitro 
Background
Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).
Methodology/Principal Findings
Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.
Conclusions/Significance
In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.
Author Summary
RNA interference (RNAi) is a method for selectively silencing (or reducing expression of) mRNA transcripts, an approach which can be used to interrogate the function of genes and proteins, and enables the validation of potential targets for anthelmintic drugs or vaccines, by investigating the impact of silencing a particular gene on parasite survival or behaviour. This study focuses on liver fluke parasites, which cause serious disease in both humans and animals. We have only a handful of drugs with which to treat these infections, to which flukes are developing resistance, and no anti-fluke vaccines have yet been developed. New options for treatment and control of liver fluke parasites are sorely needed, and RNAi is a powerful tool in the development of such treatments. This study developed a set of simple methods for triggering RNAi in juvenile liver fluke, which show that although robust transcriptional suppression can be readily achieved across all targets tested, protein suppression occurs only after a target-specific lag period (likely related to protein half-life), which may require >25 days under current in vitro maintenance conditions. These findings are important for researchers aiming to employ RNAi in investigations of liver fluke biology and target validation.
doi:10.1371/journal.pntd.0003185
PMCID: PMC4177864  PMID: 25254508
10.  Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to Nodaviruses 
PLoS Biology  2011;9(1):e1000586.
Novel viruses have been discovered in wild Caenorahbditis nematode isolates and can now be used to explore host antiviral pathways, nematode ecology, and host-pathogen co-evolution.
An ideal model system to study antiviral immunity and host-pathogen co-evolution would combine a genetically tractable small animal with a virus capable of naturally infecting the host organism. The use of C. elegans as a model to define host-viral interactions has been limited by the lack of viruses known to infect nematodes. From wild isolates of C. elegans and C. briggsae with unusual morphological phenotypes in intestinal cells, we identified two novel RNA viruses distantly related to known nodaviruses, one infecting specifically C. elegans (Orsay virus), the other C. briggsae (Santeuil virus). Bleaching of embryos cured infected cultures demonstrating that the viruses are neither stably integrated in the host genome nor transmitted vertically. 0.2 µm filtrates of the infected cultures could infect cured animals. Infected animals continuously maintained viral infection for 6 mo (∼50 generations), demonstrating that natural cycles of horizontal virus transmission were faithfully recapitulated in laboratory culture. In addition to infecting the natural C. elegans isolate, Orsay virus readily infected laboratory C. elegans mutants defective in RNAi and yielded higher levels of viral RNA and infection symptoms as compared to infection of the corresponding wild-type N2 strain. These results demonstrated a clear role for RNAi in the defense against this virus. Furthermore, different wild C. elegans isolates displayed differential susceptibility to infection by Orsay virus, thereby affording genetic approaches to defining antiviral loci. This discovery establishes a bona fide viral infection system to explore the natural ecology of nematodes, host-pathogen co-evolution, the evolution of small RNA responses, and innate antiviral mechanisms.
Author Summary
The nematode C. elegans is a robust model organism that is broadly used in biology. It also has great potential for the study of host-microbe interactions, as it is possible to systematically knockout almost every gene in high-throughput fashion to examine the potential role of each gene in infection. While C. elegans has been successfully applied to the study of bacterial infections, only limited studies of antiviral responses have been possible since no virus capable of infecting any Caenorhabditis nematode in laboratory culture has previously been described. Here we report the discovery of natural viruses infecting wild isolates of C. elegans and its relative C. briggsae. These novel viruses are most closely related to the ssRNA nodaviruses, but have larger genomes than other described nodaviruses and clearly represent a new taxon of virus. We were able to use these viruses to infect a variety of laboratory nematode strains. We show that mutant worms defective in the RNA interference pathway, an antiviral system known to operate in a number of organisms, accumulate more viral RNA than wild type strains. The discovery of these viruses will enable further studies of host-virus interactions in C. elegans and the identification of other host mechanisms that counter viral infection.
doi:10.1371/journal.pbio.1000586
PMCID: PMC3026760  PMID: 21283608
11.  Repression of Germline RNAi Pathways in Somatic Cells by Retinoblastoma Pathway Chromatin Complexes 
PLoS Genetics  2012;8(3):e1002542.
The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.
Author Summary
In metazoans, soma and germline have specialized functions that require differential tissue-specific gene expression. In C. elegans, explicit chromatin marks deposited by the MES-4 histone methyltransferase and the MRG-1 chromodomain protein allow germline expression of particular suites of target genes. Conversely, the expression of germline-specific genes is repressed in somatic cells by other chromatin regulatory factors, including the retinoblastoma pathway genes. We characterized the distinct profiles of somatic misexpression of normally germline-specific genes in these mutants and mapped out three chromatin complexes that prevent misexpression. We demonstrate that one of the complexes closely counteracts the activity of MES-4 and MRG-1, whereas another complex interacts with additional regulators that are yet to be identified. We show that these intersecting chromatin complexes prevent the upregulation of a suite of germline-specific as well as ubiquitous small RNA pathway genes, which contributes to the enhanced RNAi response in retinoblastoma pathway mutant worms. We suggest that this function of the retinoblastoma pathway chromatin factors to prevent germline-associated gene expression programs in the soma and the upregulation of small RNA pathways may also underlie their role as tumor suppressors.
doi:10.1371/journal.pgen.1002542
PMCID: PMC3297578  PMID: 22412383
12.  Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference 
Genome Biology  2006;7(1):R4.
Mutations in lin-35, the worm ortholog of a mammalian tumor suppressor gene, and other synMuv B genes result in an increased sensitivity to RNAi and enhanced somatic transgene silencing.
Background
Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.
Results
We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.
Conclusion
A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.
doi:10.1186/gb-2006-7-1-r4
PMCID: PMC1431716  PMID: 16507136
13.  Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi 
Parasites & Vectors  2012;5:16.
Background
RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans.
Results
We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week.
Conclusions
We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.
doi:10.1186/1756-3305-5-16
PMCID: PMC3292814  PMID: 22243803
RNAi; nematode; immunostaining; Brugia; filaria
14.  GenomeRNAi: a database for cell-based RNAi phenotypes 
Nucleic Acids Research  2006;35(Database issue):D492-D497.
RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at
doi:10.1093/nar/gkl906
PMCID: PMC1747177  PMID: 17135194
15.  Neuron-Specific Feeding RNAi in C. elegans and Its Use in a Screen for Essential Genes Required for GABA Neuron Function 
PLoS Genetics  2013;9(11):e1003921.
Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.
Author Summary
Living organisms often reuse the same genes multiple times for different purposes. If one function of a gene is essential, death or arrest of the mutant masks other functions. Understanding the functions of essential genes is particularly critical in the nervous system, which must maintain plasticity and fend off disease long after development is complete. However, current strategies for generating conditional knockouts rely on making a new transgenic animal for each gene and thus are not useful for forward genetic screens or for other experiments involving a large number of genes. We have developed a technique in C. elegans for generating gene knockdown in selected neuron sub-types in response to feeding RNAi. Using this technique, we performed a screen aimed at identifying essential genes that are required for the function of mature GABAergic neurons. By knocking these genes down in only GABAergic neurons, we can circumvent the muddying effects of pleiotropy and find essential genes that function cell intrinsically to promote GABA neuron function. The genes we identified using this method provide a more complete understanding of the complex genetic requirements of post-developmental neurons.
doi:10.1371/journal.pgen.1003921
PMCID: PMC3820814  PMID: 24244189
16.  Relationship Between Mitochondrial Electron Transport Chain Dysfunction, Development, and Life Extension in Caenorhabditis elegans  
PLoS Biology  2007;5(10):e259.
Prior studies have shown that disruption of mitochondrial electron transport chain (ETC) function in the nematode Caenorhabditis elegans can result in life extension. Counter to these findings, many mutations that disrupt ETC function in humans are known to be pathologically life-shortening. In this study, we have undertaken the first formal investigation of the role of partial mitochondrial ETC inhibition and its contribution to the life-extension phenotype of C. elegans. We have developed a novel RNA interference (RNAi) dilution strategy to incrementally reduce the expression level of five genes encoding mitochondrial proteins in C. elegans: atp-3, nuo-2, isp-1, cco-1, and frataxin (frh-1). We observed that each RNAi treatment led to marked alterations in multiple ETC components. Using this dilution technique, we observed a consistent, three-phase lifespan response to increasingly greater inhibition by RNAi: at low levels of inhibition, there was no response, then as inhibition increased, lifespan responded by monotonically lengthening. Finally, at the highest levels of RNAi inhibition, lifespan began to shorten. Indirect measurements of whole-animal oxidative stress showed no correlation with life extension. Instead, larval development, fertility, and adult size all became coordinately affected at the same point at which lifespan began to increase. We show that a specific signal, initiated during the L3/L4 larval stage of development, is sufficient for initiating mitochondrial dysfunction–dependent life extension in C. elegans. This stage of development is characterized by the last somatic cell divisions normally undertaken by C. elegans and also by massive mitochondrial DNA expansion. The coordinate effects of mitochondrial dysfunction on several cell cycle–dependent phenotypes, coupled with recent findings directly linking cell cycle progression with mitochondrial activity in C. elegans, lead us to propose that cell cycle checkpoint control plays a key role in specifying longevity of mitochondrial mutants.
Author Summary
The worm Caenorhabditis elegans has afforded major advances in our understanding of aging, in part because a limited number of genetic pathways appear to govern aging in this organism. In this study, we explore one class of long-lived C. elegans, the Mit mutants, which are characterized by defective mitochondrial electron transport chain activity and, hence, ATP production. How disruption of mitochondrial function could lead to life extension has remained a mystery, especially because some of the same genes that cause life extension in worms (including nuo-2 and frh-1), result in pathology in people. Here, we resolve this paradox by showing that life extension of the Mit mutants is limited to a discrete window of electron transport chain dysfunction. We show that the onset of life extension correlates with the disruption of several cell cycle–related phenomena, including larval development, adult size, and fertility and fecundity. We find no overt correlation between levels of oxidative stress and longevity. We propose that life extension in the Mit mutants is intimately connected to DNA checkpoint signaling and that the Mit mutants provide a powerful model for studying human mitochondrial disorders and aging.
Previous publications showed that the inhibition of the mitochondrial electron transport in C. elegans both extended and decreased lifespan. Here this paradox is resolved by showing that the fate depends on the degree of inhibition.
doi:10.1371/journal.pbio.0050259
PMCID: PMC1994989  PMID: 17914900
17.  MicroRNAs that interfere with RNAi 
Worm  2013;2(1):e21835.
A recent study by Massirer et al. in the nematode C. elegans has shown that a family of microRNAs (miRNAs), miR-35-41, regulates the efficiency of RNA interference (RNAi), revealing a new connection between these small RNA pathways. In this commentary, we discuss the potential mechanisms for cross regulation in the miRNA and RNAi pathways and the implications for gene expression. While miRNAs are genetically encoded, the small interfering RNAs (siRNAs) that function in RNAi can originate from processing of exogenous dsRNA (exo-RNAi) or from the production of siRNAs from endogenous transcripts (endo-RNAi). These small RNA pathways involve Dicer and Argonaute proteins and typically use antisense base pairing to target mRNAs for downregulated expression. The discovery that loss of miR-35–41 results in enhanced exo-RNAi sensitivity and reduced endo-RNAi effectiveness suggests that these miRNAs normally help balance the RNAi pathways. The effect of mir-35–41 on RNAi is largely through lin-35, the C. elegans homolog of the tumor suppressor Retinoblastoma (Rb) gene. lin-35/Rb previously has been shown to regulate RNAi sensitivity through unclear mechanisms and the new finding that accumulation of LIN-35/Rb protein is dependent on miR-35–41 adds another layer of complexity to this process. The utilization of miRNAs to control the responsiveness of RNAi exemplifies the cross-regulation embedded in small RNA-directed pathways.
doi:10.4161/worm.21835
PMCID: PMC3670461  PMID: 24058860
C. elegans; RNAi; lin-35; miR-35-41; miRNA; retinoblastoma (Rb)
18.  Diverse Chromatin Remodeling Genes Antagonize the Rb-Involved SynMuv Pathways in C. elegans 
PLoS Genetics  2006;2(5):e74.
In Caenorhabditis elegans, vulval cell-fate specification involves the activities of multiple signal transduction and regulatory pathways that include a receptor tyrosine kinase/Ras/mitogen-activated protein kinase pathway and synthetic multivulva (SynMuv) pathways. Many genes in the SynMuv pathways encode transcription factors including the homologs of mammalian Rb, E2F, and components of the nucleosome-remodeling deacetylase complex. To further elucidate the functions of the SynMuv genes, we performed a genome-wide RNA interference (RNAi) screen to search for genes that antagonize the SynMuv gene activities. Among those that displayed a varying degree of suppression of the SynMuv phenotype, 32 genes are potentially involved in chromatin remodeling (called SynMuv suppressor genes herein). Genetic mutations of two representative genes (zfp-1 and mes-4) were used to further characterize their positive roles in vulval induction and relationships with Ras function. Our analysis revealed antagonistic roles of the SynMuv suppressor genes and the SynMuv B genes in germline-soma distinction, RNAi, somatic transgene silencing, and tissue specific expression of pgl-1 and the lag-2/Delta genes. The opposite roles of these SynMuv B and SynMuv suppressor genes on transcriptional regulation were confirmed in somatic transgene silencing. We also report the identifications of ten new genes in the RNAi pathway and six new genes in germline silencing. Among the ten new RNAi genes, three encode homologs of proteins involved in both protein degradation and chromatin remodeling. Our findings suggest that multiple chromatin remodeling complexes are involved in regulating the expression of specific genes that play critical roles in developmental decisions.
Synopsis
In animal cells, DNA and genes are packed into a structure called chromatin. Chromatin-modifying protein complexes play a critical role in the regulation of gene expression. These complexes can alter the chemical and structural properties of the chromosome leading to either the repression or activation of gene expression. How these different complexes coordinate to regulate animal development remains to be explored. Several developmental processes in the nematode Caenorhabditis elegans present excellent model systems to study the functions of chromatin modifications. Using a genome-wide screen, the authors have identified 32 genes that encode potential chromatin-modifying proteins that antagonize the function of another set of transcription regulators including homologs of the mammalian Rb tumor suppressor and components of other chromatin-modifying complexes. The antagonistic roles of these two sets of genes have been observed in a variety of cellular and developmental processes, including organ development and expression of genes in particular tissues. This work indicates that multiple chromatin-modifying complexes are involved in maintaining proper expression of many genes that are critical for precise developmental decisions. Studies on these worm genes should shed light on the roles of the mammalian counterparts in development and related human diseases.
doi:10.1371/journal.pgen.0020074
PMCID: PMC1463046  PMID: 16710447
19.  The Caenorhabditis elegans HEN1 Ortholog, HENN-1, Methylates and Stabilizes Select Subclasses of Germline Small RNAs 
PLoS Genetics  2012;8(4):e1002617.
Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2′-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family.
Author Summary
Small RNAs serve as sentinels of the genome, policing activity of selfish genetic elements, modulating chromatin dynamics, and fine-tuning gene expression. Nowhere is this more important than in the germline, where endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) promote formation of functional gametes and ensure viable, fertile progeny. Small RNAs act primarily by associating with effector proteins called Argonautes to direct repression of complementary mRNAs. HEN1 methyltransferases, which methylate small RNAs, play a critical role in accumulation of these silencing signals. In this study, we report that the 26G RNAs, a class of C. elegans endo-siRNAs, are differentially methylated in male and female germlines. 26G RNAs derived from the two germlines are virtually indistinguishable, except that they associate with evolutionarily divergent Argonautes. Our data support a model wherein the methylation status and, consequently, stability of a small RNA are determined by the associated Argonaute. Therefore, selective expression of Argonautes that permit or prohibit methylation may represent a new mechanism for regulating small RNA turnover. As we observe this phenomenon in the germline, it may be particularly pertinent for directing inheritance of small RNAs, which can carry information not encoded in progeny DNA that is essential for continued transgenerational genome surveillance.
doi:10.1371/journal.pgen.1002617
PMCID: PMC3330095  PMID: 22548001
20.  An Integrated Strategy to Study Muscle Development and Myofilament Structure in Caenorhabditis elegans 
PLoS Genetics  2009;5(6):e1000537.
A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.
Author Summary
Muscular diseases affect many people worldwide. While we have learned much about the sarcomere, the basic building block of muscle cells, there are still numerous questions that remain to be answered. We must learn more about proteins expressed in muscle and how they interact so that better treatments for myopathies can be developed. The nematode Caenorhabditis elegans is a valuable model organism for the study of muscle due to similarities between worm body wall muscle and vertebrate muscle, along with its semi-transparent cuticle that allows for visualization of muscle structures in live animals. We have used transcriptional profiling methods to identify the majority of genes that are expressed in the embryonic body wall muscle cells of C. elegans. To gain insight into possible functions performed by these genes and their corresponding proteins, we examined animals and muscle cells for abnormalities after the targeted inactivation of about 3,300 genes. We identified 122 genes necessary for proper myofilament organization, 108 of which had no previously characterized role in muscle. This approach proved to be a rapid and sensitive means to identify genes that affect muscle differentiation and sarcomere assembly.
doi:10.1371/journal.pgen.1000537
PMCID: PMC2694363  PMID: 19557190
21.  Probing the microRNA pathway with small molecules 
Bioorganic & medicinal chemistry  2013;21(20):6119-6123.
MicroRNA (miRNA)/RNA interference (RNAi) is recognized as one of the most important mechanisms regulating gene expression at the posttranscriptional level in eukaryotic cells. The main components within the miRNA/RNAi pathway are now known and well characterized, but studies on the molecular mechanisms that regulate the activity of the miRNA/RNAi pathway are just beginning to emerge. High-throughput reporter assays have been developed to monitor the activity of the miRNA/RNAi pathway and applied in a proof-of-concept pilot screening, which has led to the identification of some inhibitors and activators that either generally or specifically regulate the activity of the miRNA/RNAi pathway. In addition, combined with multidisciplinary approaches like proteomics, biochemistry, and genetics, some protein co-factors were found to play important roles in the regulation of the miRNA/RNAi pathway. Herein we highlight the high-throughput reporter assays developed in recent years and the resulting discovery of miRNA/RNAi enhancers and inhibitors.
doi:10.1016/j.bmc.2013.05.030
PMCID: PMC3789859  PMID: 23791866
microRNA/RNAi pathway; reporter system; high-throughput screening; small molecule; chemical biology approach; enhancer; inhibitor
22.  MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans 
PLoS Genetics  2010;6(4):e1000903.
RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi–related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.
Author Summary
Due to its intrinsic characteristics, RNA interference (RNAi) has become one of the most widely used tools in cell biology and has revolutionized approaches to elucidate gene function. The process, also known as RNA silencing, is triggered by dsRNA molecules that are cleaved by Dicer proteins into small interfering RNAs (siRNAs). The rde-1 gene from Caenorhabditis elegans was one of the first genes found in association with this mechanism and encodes the only Argonaute protein in worms, which is by itself essential for the classical RNAi pathway triggered by exogenously introduced dsRNA. However, little is known about endogenous functions of RDE-1. Here we show that RDE-1 binds to many classes of small RNAs, including microRNAs. We show that miR-243 is efficiently bound by RDE-1 and triggers regular RNAi on an endogenous target, implying that many RNA species, including miRNAs, are constantly being screened against the transcriptome using the canonical exogenous RNAi pathway.
doi:10.1371/journal.pgen.1000903
PMCID: PMC2851571  PMID: 20386745
23.  Manipulation of Behavioral Decline in Caenorhabditis elegans with the Rag GTPase raga-1 
PLoS Genetics  2010;6(5):e1000972.
Normal aging leads to an inexorable decline in motor performance, contributing to medical morbidity and decreased quality of life. While much has been discovered about genetic determinants of lifespan, less is known about modifiers of age-related behavioral decline and whether new gene targets may be found which extend vigorous activity, with or without extending lifespan. Using Caenorhabditis elegans, we have developed a model of declining neuromuscular function and conducted a screen for increased behavioral activity in aged animals. In this model, behavioral function suffers from profound reductions in locomotory frequency, but coordination is strikingly preserved until very old age. By screening for enhancers of locomotion at advanced ages we identified the ras-related Rag GTPase raga-1 as a novel modifier of behavioral aging. raga-1 loss of function mutants showed vigorous swimming late in life. Genetic manipulations revealed that a gain of function raga-1 curtailed behavioral vitality and shortened lifespan, while a dominant negative raga-1 lengthened lifespan. Dietary restriction results indicated that a raga-1 mutant is relatively protected from the life-shortening effects of highly concentrated food, while RNAi experiments suggested that raga-1 acts in the highly conserved target of rapamycin (TOR) pathway in C. elegans. Rag GTPases were recently shown to mediate nutrient-dependent activation of TOR. This is the first demonstration of their dramatic effects on behavior and aging. This work indicates that novel modulators of behavioral function can be identified in screens, with implications for future study of the clinical amelioration of age-related decline.
Author Summary
As humans and animals age, there is an inevitable decrease in functional capacity. Elderly individuals can suffer from a decline in motor function, or the ability to move. Genetic studies in model organisms have led to the identification of genes that can prolong lifespan. Elongation of lifespan is less appealing, however, if there is not also an extension of vitality or enhanced functionality. Here, we have used a genetic model organism, the nematode worm Caenorhabditis elegans, to screen for mutations that result in enhanced vitality in older animals. We identified a new modifier of the aging of motor function, RAGA-1, a protein present in species from worms and fruit flies to humans. Animals with a raga-1 mutation move more vigorously at advanced ages and also live longer, on average, than wild-type. In contrast, animals engineered with an excessively active version of RAGA-1 show decreases in behavioral activity earlier in life than wild-type and a strikingly shortened lifespan. This offers the possibility that manipulating raga-1 could also produce beneficial effects, such as enhanced vitality, in aging humans.
doi:10.1371/journal.pgen.1000972
PMCID: PMC2877737  PMID: 20523893
24.  Genome wide screening of RNAi factors of Sf21 cells reveal several novel pathway associated proteins 
BMC Genomics  2014;15(1):775.
Background
RNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda, we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application.
Results
The genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera (Bombyx mori) or Coleoptera (Tribolium castaneum) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects.
Conclusion
The database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-775) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-775
PMCID: PMC4247154  PMID: 25199785
RNA interference; siRNA screening; Sf21 cells; Genome-wide screening; Insect RNAi; Spodoptera frugiperda
25.  A C. elegans Model for Mitochondrial Fatty Acid Synthase II: The Longevity-Associated Gene W09H1.5/mecr-1 Encodes a 2-trans-Enoyl-Thioester Reductase 
PLoS ONE  2009;4(11):e7791.
Our recognition of the mitochondria as being important sites of fatty acid biosynthesis is continuously unfolding, especially in light of new data becoming available on compromised fatty acid synthase type 2 (FASII) in mammals. For example, perturbed regulation of murine 17β-HSD8 encoding a component of the mitochondrial FASII enzyme 3-oxoacyl-thioester reductase is implicated in polycystic kidney disease. In addition, over-expression in mice of the Mecr gene coding for 2-trans-enoyl-thioester reductase, also of mitochondrial FASII, leads to impaired heart function. However, mouse knockouts for mitochondrial FASII have hitherto not been reported and, hence, there is a need to develop alternate metazoan models such as nematodes or fruit flies. Here, the identification of Caenorhabditis elegans W09H1.5/MECR-1 as a 2-trans-enoyl-thioester reductase of mitochondrial FASII is reported. To identify MECR-1, Saccharomyces cerevisiae etr1Δ mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p. These yeast mutants fail to synthesize sufficient levels of lipoic acid or form cytochrome complexes, and cannot respire or grow on non-fermentable carbon sources. A mutant yeast strain ectopically expressing nematode mecr-1 was shown to contain reductase activity and resemble the self-complemented mutant strain for these phenotype characteristics. Since MECR-1 was not intentionally targeted for compartmentalization using a yeast mitochondrial leader sequence, this inferred that the protein represented a physiologically functional mitochondrial 2-trans-enoyl-thioester reductase. In accordance with published findings, RNAi-mediated knockdown of mecr-1 in C. elegans resulted in life span extension, presumably due to mitochondrial dysfunction. Moreover, old mecr-1(RNAi) worms had better internal organ appearance and were more mobile than control worms, indicating a reduced physiological age. This is the first report on RNAi work dedicated specifically to curtailing mitochondrial FASII in metazoans. The availability of affected survivors will help to position C. elegans as an excellent model for future pursuits in the emerging field of mitochondrial FASII research.
doi:10.1371/journal.pone.0007791
PMCID: PMC2774161  PMID: 19924289

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