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[
Chromosome Res,
2013]
While initial studies of small RNA-mediated gene regulatory pathways focused on the cytoplasmic functions of such pathways, identifying roles for Argonaute/small RNA pathways in modulating chromatin and organizing the genome has become a topic of intense research in recent years. Nuclear regulatory mechanisms for Argonaute/small RNA pathways appear to be widespread, in organisms ranging from plants to fission yeast, Caenorhabditis elegans to humans. As the effectors of small RNA-mediated gene regulatory pathways, Argonaute proteins guide the chromatin-directed activities of these pathways. Of particular interest is the C. elegans Argonaute, chromosome segregation and RNAi deficient (CSR-1), which has been implicated in such diverse functions as organizing the holocentromeres of worm chromosomes, modulating germline chromatin, protecting the genome from foreign nucleic acid, regulating histone levels, executing RNAi, and inhibiting translation in conjunction with Pumilio proteins. CSR-1 interacts with small RNAs known as 22G-RNAs, which have complementarity to 25% of the protein coding genes. This peculiar Argonaute is the only essential C. elegans Argonaute out of 24 family members in total. Here, we summarize the current understanding of CSR-1 functions in the worm, with emphasis on the chromatin-directed activities of this ever-intriguing Argonaute.
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[
Worm,
2014]
Endogenous small RNA pathways related to RNA interference (RNAi) play a well-documented role in protecting host genomes from the invasion of foreign nucleic acids. In C. elegans, the PIWI type Argonaute, PRG-1, through an association with 21U-RNAs, mediates a genome surveillance process by constantly scanning the genome for potentially deleterious invading elements. Upon recognition of foreign nucleic acids, PRG-1 initiates a cascade of cytoplasmic and nuclear events that results in heritable epigenetic silencing of these transcripts and their coding genomic loci. If the PRG-1/21U-RNA genome surveillance pathway has the capacity to target most of the C. elegans transcriptome, what mechanisms exist to protect endogenous transcripts from being silenced by this pathway? In this commentary, we discuss three recent publications that implicate the CSR-1 small RNA pathway in the heritable activation of germline transcripts, propose a model as to why not all epialleles behave similarly, and touch on the practical implications of these findings.
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[
Dev Cell,
2013]
In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.
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[
Dev Cell,
2013]
Genomes are constantly challenged by invaders, so determining what belongs is crucial. Small RNAs silence alien DNA, but Conine etal. (2013), Seth etal. (2013), and Wedeles etal. (2013) now report in Cell and Developmental Cell that these tiny transcripts can also license trusted DNA for expression.
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[
J Helminthol,
1989]
The callitrichid primates, Callithrix jacchus jacchus (the marmoset) and Saguinus labiatus (the tamarin) were inoculated with infective larvae of Brugia malayi and B. pahangi. Microfilaraemia at low levels developed in 3 out of 4 C.j. jacchus infected with B. malayi and living or dead adult worms found in all 4. Only one of 4 C.j. jacchus became microfilaraemic (mf + ve) when given B. pahangi and adults were found in two. Of 4 S. labiatus given B. pahangi one became very lightly mf + ve and adults were found in 3. It is concluded that these animals are not suitable hosts for chemotherapeutic experiments.
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Tyc, K.M., Wedeles, C.J., Claycomb, J.M., Wu, M.Z., Nabih, A., Sobotka, J.A.
[
International Worm Meeting,
2017]
Proper regulation of the germline transcriptome is essential for the maintenance of fertility and survival of a species. In C. elegans, germline transcriptome homeostasis hinges on a complex repertoire of small RNA pathways that act in both activating and silencing capacities. Our understanding of how fundamental RNA processing steps, such as splicing, capping, nuclear export, and poly-adenylation intersect with these small RNA machineries in the germline remains relatively limited. Here, we link the conserved intron binding protein and splicing factor, EMB-4/AQR/IBP160 to two key 22G-RNA pathways in the C. elegans germline. EMB-4 associates with the Argonautes CSR-1 and HRDE-1, and is enriched at the genomic loci of CSR-1 and HRDE-1 target genes. Loss of
emb-4 leads to distinct alterations in CSR-1 vs. HRDE-1 small RNA and mRNA transcriptomes. Our transcriptome-wide analysis shows that EMB-4 is enriched along pre-mRNAs of nearly 10,000 transcripts. For a subset of these genes, including mostly CSR-1 pathway targets, EMB-4 enriches for intronic, but not exonic, sequences. Notably, loss of
emb-4 or the Argonautes with which it associates leads to similar defects in germline chromatin resetting in the primordial germ cells of embryos. Together these data point to EMB-4 as a factor that may help to enable the biogenesis of small RNAs and distinguish the targets of these two germline nuclear small RNA pathways in the worm. In turn, these activities are important for proper germline development and contribute to fertility over multiple generations.
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[
International Worm Meeting,
2013]
Self-avoidance, tiling and coexistence are the main mechanisms that enable the best dendritic coverage. C. elegans undergoes aging-associated changes that ultimately lead to decreased functionality of the organism, including its neurological functions. Recent research has shown that the nervous system of C. elegans undergoes changes and alterations during aging including dendritic morphology (Tank et al., 2011). We study dendritic plasticity, aging and spatial dendritic organization of two highly arborized mechanoreceptors in C. elegans, PVD and FLP (Oren-Suissa et al., 2010). PVD dendrites of L4s and young adults show regenerative ability following dendrotomy (laser induced severing of dendrites). Our working hypothesis is that in older ages this ability to regenerate is compromised. Previous studies and our preliminary results indicate that PVD and FLP do not overlap in larval stages (Smith et al., 2010). In addition, dendrites within each bilateral PVD do not overlap through a self-avoidance mechanism (Smith et al., 2012). We found that (1) the coverage fields of the PVD and FLP overlap in adult worms, which indicates coexistence and not tiling. This overlap increases as the worm ages. (2) PVDs show aberrant arborization at the age of 9 days of adulthood. (3) Dramatic increase in self-avoidance defects as animals age. In humans many neurodegenerative diseases as well as generalized cognitive decline are associated with age, aberrant arborization or both (e.g. autism and Alzheimer's disease). However our understanding of how these disorders are triggered and aggravated is scarce. Our research provides an insight into the aging and regeneration process of individual neurons. Oren-Suissa, M., et al. (2010). Science 328, 1285-1288. Smith, C.J. et al. (2010). Developmental Biology 345, 18-33. Smith, C.J., et al. (2012). Nature Neuroscience 15, 731-737. Tank, E.M.H. et al. (2011). Journal of Neuroscience 31, 9279-9288.
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[
International Worm Meeting,
2009]
Several C. elegans mutants have seizures with acute exposure to neurostimulants1,2 (e.g. pentylenetetrazole). Loss-of-function alleles of
unc-43, the worm homologue of CaMKII, have robust full-body convulsions easily induced by elevated temperature and drug exposure. To further describe worm seizures genetically, we have conducted pilot EMS screens for suppressors and enhancers of
unc-43 seizures. Thus far, we have isolated fourteen suppressors and enhancers. Since
unc-43(lf) animals have a range of phenotypes caused by a lack of function in specific tissues, modifiers have been classified by changes in seizures and a variety of other behaviors (e.g. spontaneous reversal rate). Characterization of genetic lesions is currently underway. Though identification can be time consuming, a key advantage of forward genetic screens is to isolate single point mutations that create more subtle variations in gene function. This is exciting since so little is known about modifiers in human epilepsies. Such modifiers may contribute to drug resistance, seizure frequency and developmental timing. 1Williams, S.N. et al. (2004). Hum Mol Genet (13) 2043-59. 2Locke, C.J. et al. (2006). Brains Res (1120) 23-34. .
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[
J Cell Biol,
2002]
gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.
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[
International Worm Meeting,
2003]
Dopamine, a major neurotransmitter in the mammalian central nervous system, acts through two classes of G protein-coupled receptors known as D1-like and D2-like receptors. These two classes of receptor signal to antagonistically regulate neural activity. Despite intensive research, the signaling mechanisms responsible for the antagonistic effects of dopamine in the brain remain debated. We describe the first genetic screen designed to identify proteins required for dopamine signaling. C. elegans uses endogenous dopamine to inhibit locomotion behavior1, and treatment with excess exogenous dopamine paralyzes worms2. We isolated mutants unresponsive to exogenous dopamine and found that they are also defective for endogenous dopamine signaling. Eleven mutations identified five different genes. Four of these genes encode components of the Go and Gq signaling pathways, including the G protein Go itself and subunits of the RGS complex that inhibits Gq signaling. The Go and Gq signaling pathways act antagonistically in C. elegans to control behavior and their components are conserved in humans. Mutations in these signaling components that decrease Go signaling or increase Gq signaling cause dopamine resistance, while mutations that have opposite effects on these two signaling pathways cause dopamine hypersensitivity. Thus the physiological effects of dopamine are mediated by Go in C. elegans and are antagonized by Gq signaling. Such a dopamine signaling mechanism in mammals could explain many of the opposing effects of D1-like and D2-like receptor activation. Our screen also identified one gene that appears to encode a new Go/Gq signaling component. 1. Sawin, E.R., Ranganathan, R., and Horvitz, H.R. (2000). Neuron 26, 619-631; 2. Schafer, W.R., and Kenyon, C.J. (1995). Nature 375, 73-78.