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author:("Ashe, allyson")
1.  A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity 
eLife  2013;2:e00994.
RNA interference defends against viral infection in plant and animal cells. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model of host-virus interaction. Using a genome-wide association study in C. elegans wild populations and quantitative trait locus mapping, we identify a 159 base-pair deletion in the conserved drh-1 gene (encoding a RIG-I-like helicase) as a major determinant of viral sensitivity. We show that DRH-1 is required for the initiation of an antiviral RNAi pathway and the generation of virus-derived siRNAs (viRNAs). In mammals, RIG-I-domain containing proteins trigger an interferon-based innate immunity pathway in response to RNA virus infection. Our work in C. elegans demonstrates that the RIG-I domain has an ancient role in viral recognition. We propose that RIG-I acts as modular viral recognition factor that couples viral recognition to different effector pathways including RNAi and interferon responses.
DOI: http://dx.doi.org/10.7554/eLife.00994.001
eLife digest
Most organisms—from bacteria to mammals—have at least a rudimentary immune system that can detect and defend against pathogens, particularly viruses. This defense mechanism, which is known as the innate immune system, uses sensor proteins to recognize viral RNA, and then mobilizes other immune components to attack the invaders.
The specific mechanisms used to destroy viruses differ between species. In mammals, a protein called RIG-1 binds to viral RNA and activates a signaling pathway that leads to the production of interferons: immune proteins named after their ability to ‘interfere’ with viral replication. Plants and insects do not use interferons, but instead use a mechanism called RNA interference, in which long double-stranded RNAs are cleaved into shorter fragments.
The nematode worm C. elegans also deploys RNA interference against viruses but, in contrast to insects and plants, worms do not possess a specific set of RNA interference enzymes that participate solely in the antiviral response. They do, however, express a protein called DRH-1 that is related to the RIG-I protein found in mammals.
To investigate whether DRH-1 contributes to innate immunity in C. elegans, Ashe et al. infected 97 strains of C. elegans from around the world with a virus, and showed that some strains were more sensitive to the virus than others, with certain strains showing complete resistance. By comparing a sensitive strain with a resistant one, Ashe et al. revealed that viral sensitivity was caused by a mutation in the gene encoding DRH-1.
Further experiments showed that DRH-1 is required for the first step in RNA interference. Ashe et al. have thus identified a conserved role for RIG-1 in initiating antiviral responses, and propose that the protein couples virus recognition to distinct defense mechanisms in different evolutionary groups.
DOI: http://dx.doi.org/10.7554/eLife.00994.002
doi:10.7554/eLife.00994
PMCID: PMC3793227  PMID: 24137537
RNA interference; immunity; virus infection; C. elegans
2.  An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse 
Genome Biology  2013;14(9):R96.
Background
We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes).
Results
We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly, abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance.
Conclusions
Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.
doi:10.1186/gb-2013-14-9-r96
PMCID: PMC4053835  PMID: 24025402
3.  Mutations in mouse Ift144 model the craniofacial, limb and rib defects in skeletal ciliopathies 
Human Molecular Genetics  2012;21(8):1808-1823.
Mutations in components of the intraflagellar transport (IFT) machinery required for assembly and function of the primary cilium cause a subset of human ciliopathies characterized primarily by skeletal dysplasia. Recently, mutations in the IFT-A gene IFT144 have been described in patients with Sensenbrenner and Jeune syndromes, which are associated with short ribs and limbs, polydactyly and craniofacial defects. Here, we describe an N-ethyl-N-nitrosourea-derived mouse mutant with a hypomorphic missense mutation in the Ift144 gene. The mutant twinkle-toes (Ift144twt) phenocopies a number of the skeletal and craniofacial anomalies seen in patients with human skeletal ciliopathies. Like other IFT-A mouse mutants, Ift144 mutant embryos display a generalized ligand-independent expansion of hedgehog (Hh) signalling, in spite of defective ciliogenesis and an attenuation of the ability of mutant cells to respond to upstream stimulation of the pathway. This enhanced Hh signalling is consistent with cleft palate and polydactyly phenotypes in the Ift144twt mutant, although extensive rib branching, fusion and truncation phenotypes correlate with defects in early somite patterning and may reflect contributions from multiple signalling pathways. Analysis of embryos harbouring a second allele of Ift144 which represents a functional null, revealed a dose-dependent effect on limb outgrowth consistent with the short-limb phenotypes characteristic of these ciliopathies. This allelic series of mouse mutants provides a unique opportunity to uncover the underlying mechanistic basis of this intriguing subset of ciliopathies.
doi:10.1093/hmg/ddr613
PMCID: PMC3313797  PMID: 22228095
4.  The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking 
The Journal of Cell Biology  2012;197(6):789-800.
Mutations in mouse intraflagellar transport–A complex genes alter Sonic hedgehog signaling because of their effects on cilia structure and on trafficking of membrane proteins into cilia.
Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking.
doi:10.1083/jcb.201110049
PMCID: PMC3373400  PMID: 22689656
5.  A Forward Genetic Screen Identifies Eukaryotic Translation Initiation Factor 3, Subunit H (eIF3h), as an Enhancer of Variegation in the Mouse 
G3: Genes|Genomes|Genetics  2012;2(11):1393-1396.
We have used a forward genetic screen to identify genes required for transgene silencing in the mouse. Previously these genes were found using candidate-based sequencing, a slow and labor-intensive process. Recently, whole-exome deep sequencing has accelerated our ability to find the causative point mutations, resulting in the discovery of novel and sometimes unexpected genes. Here we report the identification of translation initiation factor 3, subunit H (eIF3h) in two modifier of murine metastable epialleles (Mommes) lines. Mice carrying mutations in this gene have not been reported previously, and a possible involvement of eIF3h in transcription or epigenetic regulation has not been considered.
doi:10.1534/g3.112.004036
PMCID: PMC3484669  PMID: 23173090
mouse; epigenetics; forward genetic screen; eIF3h
6.  piRNAs Can Trigger a Multigenerational Epigenetic Memory in the Germline of C. elegans 
Cell  2012;150(1):88-99.
Summary
Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.
Graphical Abstract
Highlights
► Multigenerational inheritance and piRNAs converge on same nuclear silencing pathway ► HRDE1/WAGO-9 and chromatin factors required for inheritance of piRNA silencing ► piRNAs can induce multigenerational silencing for more than 20 generations. ► Long-term memory independent of piRNA triggers but remains dependent on nuclear pathway
Multigenerational inheritance and piRNAs converge on same silencing pathway, in which both nuclear WAGOs and chromatin factors are required. The piRNA trigger can be lost, but the nuclear silencing pathway maintains the silencing for more than 20 generations.
doi:10.1016/j.cell.2012.06.018
PMCID: PMC3464430  PMID: 22738725
7.  Modifiers of epigenetic reprogramming show paternal effects in the mouse 
Nature genetics  2007;39(5):614-622.
There is increasing evidence that epigenetic information can be inherited across generations in mammals, despite extensive reprogramming both in the gametes and in the early developing embryo. One corollary to this is that disrupting the establishment of epigenetic state in the gametes of a parent, as a result of heterozygosity for mutations in genes involved in reprogramming, could affect the phenotype of offspring that do not inherit the mutant allele. Here we show that such effects do occur following paternal inheritance in the mouse. We detected changes to transcription and chromosome ploidy in adult animals. Paternal effects of this type have not been reported previously in mammals and suggest that the untransmitted genotype of male parents can influence the phenotype of their offspring.
doi:10.1038/ng2031
PMCID: PMC3199608  PMID: 17450140
8.  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
9.  Reduced levels of two modifiers of epigenetic gene silencing, Dnmt3a and Trim28, cause increased phenotypic noise 
Genome Biology  2010;11(11):R111.
Background
Inbred individuals reared in controlled environments display considerable variance in many complex traits but the underlying cause of this intangible variation has been an enigma. Here we show that two modifiers of epigenetic gene silencing play a critical role in the process.
Results
Inbred mice heterozygous for a null mutation in DNA methyltransferase 3a (Dnmt3a) or tripartite motif protein 28 (Trim28) show greater coefficients of variance in body weight than their wild-type littermates. Trim28 mutants additionally develop metabolic syndrome and abnormal behavior with incomplete penetrance. Genome-wide gene expression analyses identified 284 significantly dysregulated genes in Trim28 heterozygote mutants compared to wild-type mice, with Mas1, which encodes a G-protein coupled receptor implicated in lipid metabolism, showing the greatest average change in expression (7.8-fold higher in mutants). This gene also showed highly variable expression between mutant individuals.
Conclusions
These studies provide a molecular explanation of developmental noise in whole organisms and suggest that faithful epigenetic control of transcription is central to suppressing deleterious levels of phenotypic variation. These findings have broad implications for understanding the mechanisms underlying sporadic and complex disease in humans.
doi:10.1186/gb-2010-11-11-r111
PMCID: PMC3156950  PMID: 21092094
10.  A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development 
Genome Biology  2008;9(12):R182.
An extended ENU screen for modifiers of transgene variegation identified four new modifiers, MommeD7-D10.
Background
Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.
Results
We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.
Conclusions
These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
doi:10.1186/gb-2008-9-12-r182
PMCID: PMC2646286  PMID: 19099580

Results 1-10 (10)