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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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[
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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[
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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[
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]
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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[
2007]
In the pair of the nematode Caenorhabiditis elegans serotonergic chemosensory neurons ADF, the TRPV channel protein OCR-2 interacts with another TRPV protein, OSM-9, to control the production of the neurotransmitter serotonin. The activity and specificity of OCR-2 in the serotonergic neurons is governed by structural determinants within the channel protein in concert with defined cellular components. The dynamic gating mechanisms, multiple sensory modalities, and functional conservation in diverse organisms make TRPV channels ideal candidates for the long-awaited molecular sensors that underscore the ancient role of the serotonergic system in coupling sensory cues and internal milieu to behavior and physiology.
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[
1992]
Mutants in the
age-1 gene have life spans that are 70% longer than wild type under various environmental conditions. Here we show that the
age-1 gene localizes to chromosome 2 near
unc-4 and that the increase in life span is separable from a previously associated decrease in fertility.
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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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[
WormBook,
2005]
The C. elegans germ line proliferates from one primordial germ cell (PGC) set aside in the early embryo to over a thousand cells in the adult. Most germline proliferation is controlled by the somatic distal tip cell, which provides a stem cell niche at the distal end of the adult gonad. The distal tip cell signals to the germ line via the Notch signaling pathway, which in turn controls a network of RNA regulators. The FBF-1 and FBF-2 RNA-binding proteins promote continued mitoses in germ cells located close to the distal tip cell, while the GLD-1 , GLD-2 , GLD-3 , and NOS-3 RNA regulators promote entry into meiosis as germ cells leave the stem cell niche. In addition to these key regulators, many other genes affect germline proliferation.
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[
WormBook,
2005]
Alternative splicing is a common mechanism for the generation of multiple isoforms of proteins. It can function to expand the proteome of an organism and can serve as a way to turn off gene expression post-transcriptionally. This review focuses on splicing and its regulation in C. elegans. The fully-sequenced C. elegans genome combined with its elegant genetics offers unique advantages for exploring alternative splicing regulation in metazoans. The topics covered in this review include constitutive splicing factors, identification of alternatively spliced genes, examples of alternative splicing in C. elegans, and alternative splicing regulation. Key genes whose regulated alternative splicing are reviewed include
let-2 ,
unc-32 ,
unc-52 ,
egl-15 and
xol-1 . Factors involved in alternative splicing that are discussed include
mec-8 ,
smu-1 ,
smu-2 ,
fox-1 ,
exc-7 and
unc-75 .