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[
2006]
RNA interference (RNAi) describes a conserved biological response to double-stranded RNA (dsRNA) resulting in the degradation of homologous messenger RNA. In the last few years, this process of sequence-specific, post-transcriptional gene silencing has become a key technique for rapidly assessing gene function in species ranging from plants to mammals. Fire et al. provided the first insight into the RNAi mechanism by identifying dsRNA as the trigger of RNAi in Caenorhabditis elegans in 1998 [1]. However, a similar gene-silencing phenomenon was reported in earlier studies in both plants and Neurospora [2,3]. The basic RNAi response starts with long dsRNA being processed into small interfering RNAs (siRNAs) by a ribonuclease (RNase) III enzyme, Dicer. Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC). For target RNA recognition to occur, the siRNA duplex must be unwound, allowing binding of one siRNA strand to the target mRNA. This is followed by RISC cleavage of the homologous mRNA. Recent work has shown that the RNAi machinery is also involved in antiviral responses, transposon silencing, development and heterochromatin formation [4].
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[
2005]
RNA interference (RNAi) is a recently discovered phenomenon in which doublestranded RNA (dsRNA) silences endogenous gene expression in a sequencespecific manner (Fire et al., 1998). Since its discovery, the use of RNAi has become widely employed in many organisms to specifically knock down gene function. RNAi shares a remarkable degree of similarity with silencing phenomena in other organisms (Cogoni et al., 1999a; Sharp, 1999). For instance, RNAi, posttranscriptional gene silencing in plants and cosuppression in fungi can all be activated by the presence of aberrant RNAs (Maine, 2000; Tijsterman et al., 2002a). Additionally, plant, worm, and fly cells or extracts undergoing RNA-mediated interference all contain small dsRNAs, around 25 nucleotides in length, identical to the sequences present in the silenced gene (Baulcombe, 1996; Hammond et al., 2000; Zamore et al., 2000; Catalanotto et al., 2000). The high degree of similarity between these RNA-mediated silencing phenomena supports the notion that they were derived from an ancient and conserved pathway used to regulate gene expression, presumably to eliminate defective RNAs and to defend against viral infections and transposons. (Zamore, 2002). Components of RNAi have also been implicated in developmental processes, suggesting that RNAi may play a broader role in regulating gene expression (Smardon et al., 2000; Knight et al., 2001; et al., Ketting et al., 2001). Although we have learned much about the general mechanisms underlying RNAi, a detailed understanding of how RNAi works remains to be elucidated. In this chapter we will discuss first the biology of RNAi, then the genes required for its function, and we will end with a discussion on recent findings that have implicated chromatin silencing in the mechanism of RNAi.
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[
Adv Exp Med Biol,
2010]
Nematode neuropeptide systems comprise an exceptionally complex array of approximately 250 peptidic signaling molecules that operate within a structurally simple nervous system of approximately 300 neurons. A relatively complete picture of the neuropeptide complement is available for Caenorhabditis elegans, with 30 flp, 38 ins and 43 nlp genes having been documented; accumulating evidence indicates similar complexity in parasitic nematodes from clades I, III, IV and V. In contrast, the picture for parasitic platyhelminths is less clear, with the limited peptide sequence data available providing concrete evidence for only FMRFamide-like peptide (FLP) and neuropeptide F (NPF) signaling systems, each of which only comprises one or two peptides. With the completion of the Schmidtea meditteranea and Schistosoma mansoni genome projects and expressed sequence tag datasets for other flatworm parasites becoming available, the time is ripe for a detailed reanalysis ofneuropeptide signalingin flatworms. Although the actual neuropeptides provide limited obvious value as targets for chemotherapeutic-based control strategies, they do highlight the signaling systems present in these helminths and provide tools for the discovery of more amenable targets such as neuropeptide receptors or neuropeptide processing enzymes. Also, they offer opportunities to evaluate the potential of their associated signaling pathways as targets through RNA interference (RNAi)-based, target validation strategies. Currently, within both helminth phyla, theflp signaling systems appear to merit further investigation as they are intrinsically linked with motor function, a proven target for successful anti-parasitics; it is clear that some nematode NLPs also play a role in motor function and could have similar appeal. At this time, it is unclear if flatworm NPF and nematode INS peptides operate in pathways that have utility for parasite control. Clearly, RNAi-based validation could be a starting point for scoring potential target pathways within neuropeptide signaling for parasiticide discovery programs. Also, recent successes in the application of in planta-based RNAi control strategies for plant parasitic nematodes reveal a strategy whereby neuropeptide encoding genes could become targets for parasite control. The possibility of developing these approaches for the control of animal and human parasites is intriguing, but will require significant advances in the delivery of RNAi-triggers.
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[
WormBook,
2006]
Through genetic analyses, the function of genes is investigated by studying organisms where gene function is altered. In classical forward genetic screening, individuals are treated with mutagens to induce DNA lesions and mutants with a phenotype of interest are sought. After a mutant is found, the gene mutated is identified through standard molecular techniques. Detailed studies of the mutant phenotype coupled with molecular analyses of the gene allows elucidation of the gene's function. Forward genetics has been responsible for our understanding of many biological processes and is an excellent method for identifying genes that function in a particular process.In reverse genetics, the functional study of a gene starts with the gene sequence rather than a mutant phenotype. Using various techniques, a gene's function is altered and the effect on the development or behaviour of the organism is analysed. Reverse genetics is an important complement to forward genetics. For example, using reverse genetics, one can investigate the function of all genes in a gene family, something not easily done with forward genetics. Further, one can study the function of a gene found to be involved in a process of interest in another organism, but for which no forward genetic mutants have yet been identified. Finally, the vast majority of genes have not yet been mutated in most organisms and reverse genetics allows their study. The availability of complete genome sequences combined with reverse genetics can allow every gene to be studied.This chapter gives detailed protocols for the two main methods of perturbing gene function in C. elegans: RNA interference and the creation of deletion mutants. Either technique can be applied to the study of individual genes. With less than a day of actual work, RNAi creates a knockdown of gene function without altering the organism's DNA (see below). In contrast, with about a month of work, a deletion mutation permanently removes all gene function. Deciding which technique to use will depend on the nature of the experiment. The techniques can also be combined, where RNAi is used for rapid screening of loss of function phenotypes and then deletion mutants are made to study genes of particular interest. RNAi can also be carried out on a global scale, where knockdown of (nearly) every gene is tested for inducing a phenotype of interest. In this case, the reverse genetics technique of RNAi can be thought of as a forward genetic screening tool.
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[
Methods Mol Biol,
2014]
Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment.
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[
WormBook,
2006]
The completion of the C. elegans genome sequence permits the comprehensive examination of the expression and function of genes. Annotation of virtually every encoded gene in the genome allows systematic analysis of those genes using high-throughput assays, such as microarrays and RNAi. This chapter will center on the use of microarrays to comprehensively identify genes with enriched expression in the germ line during development. This knowledge provides a database for further studies that focus on gene function during germline development or early embryogenesis. Additionally, a comprehensive overview of germline gene expression can uncover striking biases in how genes expressed in the germ line are distributed in the genome, leading to new discoveries of global regulatory mechanisms in the germ line.
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[
Methods Cell Biol,
2008]
The Caenorhabditis elegans gonad and early embryo have recently emerged as an attractive metazoan model system for studying cell and developmental biology. The success of this system is attributable to the stereotypical architecture and reproducible cell divisions of the gonad/early embryo, coupled with penetrant RNAi-mediated protein depletion. These features have facilitated the development of visual assays with high spatiotemporal resolution to monitor specific subcellular processes. Assay development has relied heavily on the emergence of methods to circumvent germline silencing to allow the expression of transgenes encoding fluorescent fusion proteins. In this chapter, we discuss methods for the expression and imaging of fluorescent proteins in the C. elegans germline, including the design of transgenes for optimal expression, the generation of transgenic worm lines by ballistic bombardment, the construction of multimarker lines by mating, and methods for live imaging of the gonad and early embryo.
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[
WormBook,
2006]
The C. elegans embryo is a powerful model system for studying the mechanics of metazoan cell division. Its primary advantage is that the architecture of the syncytial gonad makes it possible to use RNAi to generate oocytes whose cytoplasm is reproducibly (typically > 95%) depleted of targeted essential gene products via a process that does not depend exclusively on intrinsic protein turnover. The depleted oocytes can then be analyzed as they attempt their first mitotic division following fertilization. Here we outline the characteristics that contribute to the usefulness of the C. elegans embryo for cell division studies. We provide a timeline for the first embryonic mitosis and highlight some of its key features. We also summarize some of the recent discoveries made using this system, particularly in the areas of nuclear envelope assembly/ dissassembly, centrosome dynamics, formation of the mitotic spindle, kinetochore assembly, chromosome segregation, and cytokinesis.
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[
WormBook,
2005]
Ubiquitin is a highly conserved 76 amino acid polypeptide, which is covalently attached to target proteins to signal their degradation by the 26S proteasome or to modify their function or localization. Regulated protein degradation, which is associated with many dynamic cellular processes, occurs predominantly via the ubiquitin-proteasome system. Ubiquitin is conjugated to target proteins through the sequential actions of a ubiquitin-activating enzyme, ubiquitin-conjugating enzymes, and ubiquitin-protein ligases. The nematode Caenorhabditis elegans has one ubiquitin-activating enzyme, twenty putative ubiquitin-conjugating enzymes, and potentially hundreds of ubiquitin-protein ligases. Research in C. elegans has focused on the cellular functions of ubiquitin pathway components in the context of organismal development. A combination of forward genetics, reverse genetics, and genome-wide RNAi screens has provided information on the loss-of-function phenotypes for the majority of C. elegans ubiquitin pathway components. Additionally, detailed analysis of several classes of ubiquitin-protein ligases has led to the identification of their substrates and the molecular pathways that they regulate. This review presents a comprehensive overview of ubiquitin-mediated pathways in C. elegans with a description of the known components and their identified molecular, cellular, and developmental functions.
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[
2009]
Although the worms may not be universally accepted as having a direct relevance for AD pathology, they are well suited for validation of target A toxicity in vivo [12,21]. The absence of endogenous A production in the worms offers an opportunity to find a direct role of the A involvement in pathological behaviors [22]. In addition, predominantly intracellular expression of A provides another tool to address specific roles of intracellular A in relation to its toxicity. Substantial evidence implicates intracellular A oligomers in early events related to AD [16]. Intracellular A has also been observed in human brain neurons [23] and in triple transgenic AD mouse models, where its accumulation preceded neurofibrillary tangle formation [24]. This evidence supports the notion that A toxicity assayed in the worm model reflects A toxicity in mammalian neurons. A recent study indicates that the transgenic C. elegans model may be generally relevant to the proteotoxicity underlying neurodegenerative diseases [25]. Additionally, the strain has been used to investigate the role of insulin-like signaling and heat-shock factor in A proteotoxicity [26,27], providing excellent examples for the relevance of the C. elegans model to AD. There are several advantages of C. elegans over the mouse model for initial drug screening and target characterization. First, there are highly conserved biochemical pathways between worms and humans. Second, established transgenic mutant linking of human A expression with pathological behavioral phenotypes are easy to score. The worms have a relatively low cost of cultivation because of their small size, rapid life cycle, and short life span [28], which allow screening of thousands of animals over multiple generations on microtiter plates. The simple structure of its nervous system, consisting of only 302 neurons in an adult nematode, makes it valuable for screening drugs against age-associated neurodegeneration and the ease of genetic manipulations, which is evident in the availability of mutants and application of RNA interference (RNAi) knockdown. Several examples illustrate the power of C. elegans in screening for new drugs [29], including many known human drugs [30,31]. Some lead molecules originating from worm-based screening assays are in advanced stages of drug discovery [11]. Using the C. elegans model in the past years, we have uncovered effects of natural compounds on extension of the worms' life span; [32] on a stress response protein, the small heat-shock protein
hsp-16.2; [33] on age-related behavioral declines; [34] on muscle degeneration; [35] and on A-expression-induced pathological behaviors [22]. Most of those experiments would be difficult and might be impossible to perform in mice. In this chapter, we describe methods we have employed for compound screening and pharmacological evaluations of potential AD drugs using the C. elegans model.