Miska E A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Novel Viruses Infecting Caenorhabditis Nematodes Trigger a Small RNA Response"

Goldstein L D, Jiang Y, Wang D, Felix M, Sanroman M, Zhao G, Piffaretti J, Wu G, Ashe A, Miska E A, Nuez I, Belicard T

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

C. elegans has served as a laboratory model organism due to its ease of manipulation and the availability of both forward and reverse genetics. In recent years, C. elegans has been used to study host-pathogen interactions. For example, analysis of infections by bacteria such as Pseudomonas, Salmonella or Serratia has revealed the existence of innate immune pathways in C. elegans that are conserved in vertebrates. To date, no virus that naturally infects either C. elegans or C. briggsae has been described. Here we report the isolation and partial sequencing of two viruses that were detected in wild isolates of C. elegans and C. briggsae. Phylogenetic analysis demonstrated that the two novel RNA viruses - tentatively named Orsay nodavirus and Santeuil nodavirus - are most closely, but distantly, related to viruses in the family Nodaviridae. Filtrates of Orsay and Santeuil nodaviruses prepared by passing lysates from infected nematodes through 0.2 microm filters specifically infected strains of C. elegans and C. briggsae, respectively, and each further showed intraspecific specificity. The viruses triggered a specific small RNA response by the RNAi machinery. Infection by Orsay nodavirus of mutants defective in small RNA pathways yielded higher levels of viral RNA and infection symptoms as compared to infection of isogenic N2 reference animals. These data demonstrate that nodaviruses are natural parasites of nematodes in the wild and that the RNAi response is a physiological antiviral defense mechanism. Further study of the interactions between these viruses and nematodes is likely to provide insight into the natural ecology of nematodes and the evolution of the small RNA response and may reveal novel innate immune mechanisms that respond to viral infection.

Alejandra Clark et al. (2006) European Worm Meeting "Elucidating the Role of MicroRNAs in C. elegans Development"

Eric Miska, Alejandra Clark

[European Worm Meeting, 2006]

MicroRNAs (miRNAs) are small non-coding RNAs found in all metazoa studied so far. In animals miRNAs regulate gene expression by binding with incomplete complementarity to the 3UTR of target mRNAs inhibiting their translation. Although a number of miRNAs have now been cloned and identified, in most cases their functions remain elusive. This is partly due to the difficulty in identifying target genes based on partial complementarity between miRNAs and target mRNAs. To investigate the biological roles of miRNAs in C. elegans development we are using an algorithm that predicts miRNA targets based on miRNA seed complementarity and conservation of the mRNAs 3UTR between C. elegans and C. briggsae (Stark A. et al., 2005, Brennecke J. et al., 2003). To validate the targets proposed by this method and gain new insight into the biological roles of specific miRNAs, C. elegans miRNA deletion strains will examined for the expected up-regulation of a number of predicted targets (Miska E. et al., unpublished). Antibody stainings and GFP::Target 3UTR reporter constructs in wild-type and miRNA deletion strains will be investigated. In addition, predicted target genes will be tested by RNAi knockdown screening for suppression of abnormal phenotypes of miRNA deletion strains.

W. Robert Shaw et al. (2007) International Worm Meeting "Role of the mir-51 Family of microRNAs in Caenorhabditis elegans Development."

W. Robert Shaw, Eric A. Miska

[International Worm Meeting, 2007]

MicroRNAs (miRNAs) are a class of small regulatory RNA that inhibit target gene expression post-transcriptionally. Several hundreds of miRNAs have been discovered in diverse organisms but their biological roles are largely unknown. Caenorhabditis elegans </I> mutants with deletions in most miRNA genes have been generated by a deletion screen (1) as a resource for the study of miRNA function. The mir-51 family of miRNAs is a highly conserved family of six miRNAs that share a ''seed'' region (seven continuous base pairs at their 5'' end at bases 2 to 8). Animals lacking individual members of the mir-51 family of miRNAs show no obvious abnormal phenotypes, likely because these six miRNAs act redundantly. Mutant strains in which multiple mir-51 family members have been deleted show gradually more severe defects in growth rate and development as further family members are deleted, culminating in embryonic or early larval lethality. This series correlates with the relative expression levels of the miRNAs (1). Mutant phenotypes can be rescued by short genomic regions encoding mir-51 family miRNAs. Bioinformatic miRNA target prediction has provided lists of candidate miRNA target mRNAs. These targets are being tested using a combination of RNAi, fluorescent protein reporter constructs and antibodies. In addition, we are carrying out enhancer and suppressor screens to identify direct targets of the mir-51 miRNA family. (1) Miska, E. A., Alvarez-Saavedra, E. A., Abbott, A. L., Lau, N. P., Hellman, A. B., McGonagle, S., Bartel, D. P., Ambros, V. R., Horvitz, H. R. - unpublished results.

BC Prasad et al. (1999) International C. elegans Meeting "The unc-3 locus encodes an O/E transcription factor, CeO/E, which is required for axonal guidance and chemosensory function."

BC Prasad, RR Reed

[International C. elegans Meeting, 1999]

The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI, unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several unc-3 target genes ( daf-7, unc-17/cha-1, unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.

Yuen, Grace J. et al. (2013) International Worm Meeting "Enterococcus infection of C. elegans as a model of innate immunity."

Yuen, Grace J., Ausubel, Frederick M.

[International Worm Meeting, 2013]

Enterococcus is a Gram-positive commensal that is also an important opportunistic pathogen. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both species. We have previously shown that infection with either species leads to gut distention, but only E. faecalis is able to establish a persistent and lethal infection in the nematode. We now provide evidence that at least three canonical C. elegans immune signaling pathways are important for survival during infection with E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by E. faecium infection, mutations in the PMK-1, FSHR-1, and BAR-1 immune signaling pathways lead to an immunocompromised phenotype. This new finding suggests that an active host response is required to keep E. faecium infection "in check" in the worm intestine. To further characterize the C. elegans host response to Enterococcus infections, we used genome-wide transcriptional profiling of nematodes feeding on E. faecalis and E. faecium, as well as two controls, heat-killed E. coli and live Bacillus subtilis, a non-pathogenic Gram-positive. We found that relative to B. subtilis, E. faecalis and E. faecium caused the upregulation of 249 and 166 genes, respectively, of which 105 genes were common to both, comprising the Enterococcus gene signature. Shared by both Enterococcus infection signatures were genes relating to oxidation/reduction, acyl-CoA dehydrogenase/oxidase activity, fatty acid metabolism, and C-type lectins. Additionally, the Enterococcus infection gene signature is fairly distinct from the P. aeruginosa, S. aureus, and C. albicans infection signatures. Furthermore, of the 91 genes that are upregulated in E. faecalis (more virulent) relative to E. faecium (less virulent), 22 are shared with the 77 genes upregulated in worms infected with virulent Microbacterium nematophilum relative to avirulent M. nematophilum (O'Rourke et al., 2006), suggesting that these genes may comprise a "virulence response signature." Studies are underway in understanding the biology of Enterococcus infection in C. elegans and identifying novel Enterococcus-activated pathways.

Kaufman E J et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "MicroRNA Control of Excretory Cell Morphogenesis in C. elegans"

Kaufman E J, Miska E A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

MicroRNAs (miRNAs) are short (18-26 nt), non-coding RNAs that function as post-transcriptional silencers of gene expression by binding to complementary sequences in 3'UTRs of target mRNAs. This sequence complementarity suggests that it should be possible to predict which genes might be targeted by a given miRNA and thus deduce its biological function. While it is true that predicted targets, in particular those whose target sites are conserved in closely related species, are typically enriched in the class of genes upregulated following disruption of the cognate miRNA, the biological significance of these minor changes in target expression (generally &lt;2-fold) remains an open question for nearly all miRNAs. Very few studies approach the question of target recognition genetically, identifying targets by their suppression or phenocopy of the miRNA mutant phenotype. This approach would seem better suited to identifying biologically and evolutionarily relevant targets. >In the C. elegans community, we are fortunate to have access to a comprehensive library of miRNA knockout strains. Here, we use one of these mutant strains to study the function of mir-232, a previously uncharacterized miRNA gene conserved within ecdysozoa and lophotrochozoa. From observations of the mir-232 knockout strain, we have found that mir-232 is cell-autonomously required for the normal morphogenesis of the excretory canal cell, one quarter of the four-cell osmoregulatory apparatus of the animal and, as we have found, the primary site of miR-232 expression. Using a candidate approach, we have identified the glucuronyltransferase SQV-8, part of the glycosaminoglycan biosynthetic pathway, as a direct target of miR-232 in the excretory cell, whose ectopic expression at least partially accounts for abnormal canal extension in mir-232 mutant animals. Consistent with the targeting of sqv-8, we reproducibly detect changes to the profile of chondroitin-modified proteins in mir-232 mutant animals. Ongoing work seeks to address additional aspects of this biology, including the temporal requirements of mir-232 during excretory cell morphogenesis, the functional consequences of this abnormal morphogenesis with respect to osmoregulation, and the physiological consequences of hyperactive glycosaminoglycan biosynthesis in the excretory cell, in an attempt to integrate the phenotypic and targeting data into a coherent model of miR-232 function. Progress on each of these fronts will be presented.

Kosalka, J. et al. (2015) International Worm Meeting "Mechanism and regulation of piRNA biogenesis in the C. elegans germline."

Weick, EM., Miska, E., Kosalka, J.

[International Worm Meeting, 2015]

Mechanism and regulation of piRNA biogenesis in the C. elegans germlineJoanna Kosalka, Eva-Maria Weick, Eric A MiskaWellcome Trust/CRUK Gurdon Institute/Department of Genetics, University of Cambridge, Cambridge, United KingdomPiwi-interacting RNAs (piRNAs) are the most recently identified and by far the largest class of endogenous small regulatory RNAs (sRNAs). In C. elegans, piRNAs are synthesised as single strand precursors from specific genomic loci known as piRNA clusters, processed into mature 21 nucleotides long, 5'U sRNAs that are recognised by the PIWI clade of Argonaute proteins. piRNAs are germline-expressed and are essential for fertility and genome stability. piRNAs can align to transposons and repetitive sequences in an antisense orientation, targeting them for silencing and the loss of the piRNA pathway results in reactivation of transposons. In addition, some endogenous protein-coding genes are under control of the piRNA pathway.Many aspects of the C. elegans piRNA pathway remain poorly understood and elucidating the biogenesis of 21U RNAs is of particular interest. Importantly, the fundamental questions of how the synthesis of piRNA precursors is initiated and directed to specific loci, and how the maturation of the precursor molecules is executed, remain unanswered. Furthermore, the developmental regulation of piRNA expression is unknown.In a forward genetic screen our lab has identified prde-1 (piRNA defective) as an essential germline-expressed factor for piRNA biogenesis (Weick et al. 2014). prde-1 is involved in the initial steps of piRNA biogenesis and is indispensable for piRNA precursor synthesis and/or stability. Interestingly, PRDE-1 protein localises specifically to parts of chromosome IV coinciding with the large piRNA cluster. Although the molecular mechanisms of prde-1 function are currently unknown, these findings provide an exciting starting point to elucidate the mechanisms of piRNA cluster transcription and regulation.Here we will present our most recent progress on understanding the molecular machinery required for the biogenesis of piRNAs. We used a range of biochemical approaches to identify and characterise PRDE-1-interacting proteins and their role in piRNA biogenesis.

Yuen, Grace J. et al. (2011) International Worm Meeting "Enterococcus infection of Caenorhabditis elegans as a model of innate immunity."

Ausubel, Frederick M., Pukkila-Worley, Read, Yuen, Grace J.

[International Worm Meeting, 2011]

Enterococcus is a Gram-positive commensal that is found in the gastrointestinal and biliary tracts of all healthy humans. It is also an important opportunistic pathogen, causing nosocomial urinary tract and wound infections that are often complicated by antibiotic drug resistance. Most human enterococcal infections are caused by either E. faecalis or E. faecium. Our laboratory has modeled Enterococcus infection in C. elegans using both strains. We have previously shown that infection with either strain leads to gut distention, but only E. faecalis is able to establish a persistent infection and kill the nematode. We now provide evidence that at least two canonical C. elegans immune signaling pathways are important for survival during infection with both E. faecalis and E. faecium. While the lifespan of wild-type worms is unaffected by an E. faecium infection, mutations in the PMK-1 and FSHR-1 immune signaling pathways lead to an immunocompromised phenotype, where pmk-1 and fshr-1 mutants die rapidly upon E. faecium feeding. This new finding suggests that an active immune response is required to keep E. faecium infection "in check" in the worm intestine, and that E. faecium is indeed pathogenic to the nematode. We are now using microscopy and genetic studies to understand the biology of the Enterococcus infection in C. elegans and to identify novel Enterococcus-activated immune signaling pathways.

Fuentes, A. et al. (2015) International Worm Meeting "Diet affects neurodegenerative processes in C. elegans."

Fuentes, A., Calixto, A., Garcia, V., Palominos, MF.

[International Worm Meeting, 2015]

C. elegans has been used to understand the dietary impact on development and behavior (Macneil & Walhout., 2013), but very little is known about the role of diet on neurodegenerative processes. To study the effect of diet on neuronal degeneration, we use animals expressing the hyperactivated degenerins mec-4d expressed in the touch receptor neurons (TRNs) and deg-1 expressed in the PVC neurons. We measured the impact of different bacterial diets on neuronal integrity in the respective degeneration models. mec-4d animals with GFP marked TRNs were fed with E. coli OP50, B, HT115 and K12; Commamonas aquatica, Commamonas testosteronii and Bacillus megaterium and their TRN integrity assessed both morphologically (by visual inspection) and functionally (by the touch response) at 72 hours where most axons are degenerated (Calixto et al., 2012). The maximum protection was elicited by E. coli HT115 diet, with up to 50% wild type axons while the control strain E. coli OP50 and its precursor B only protects 3% at 72 hours post hatching. Like in the mec-4d animals, deg-1 animals PVC show a higher percent of functional response when animals are fed E. coli HT115. E. coli HT115 is capable of protecting neurons during large periods of time after adulthood since animals at 168 hours post hatching still showed 16.5 % of wild type axons. E. coli K12, Comammonas aquatica, testosteronii and Bacillus megaterium protected less (between 10 and 17% of wild type axons) than E. coli HT115 and more than E. coli OP50. E. coli HT115 is still capable of generating neuroprotection when diluted 1:100 in E. coli OP50, showing that E. coli OP50 does not produce neurodegeneration. Additionally, this shows that a very small amount of the protective food is necessary to elicit neuronal protection. E. coli HT115 does not produce dietary restriction and supports well animal growth, with 100% animals reaching adulthood at 72 hours vs 100% in E. coli OP50. Dead bacteria produce the same levels of neuronal protection than live bacteria, indicating that the interaction between bacteria and intestine is not required. Our data suggest that E. coli HT115 is capable of triggering pathways involved in neuroprotective processes early in animal development (first 12 hrs after hatching), highlighting the relevance of developmental time when the protective diet is ingested. We are currently performing transcriptomics analysis to identify the bacterial components from E. coli HT115 that mediate neuroprotection.

Prasad BC et al. (1998) East Coast Worm Meeting "The O/E transcription factor, UNC-3, is required for axonal guidance and ASI function"

Reed RR, Seydoux GC, Prasad BC

[East Coast Worm Meeting, 1998]

The O/E family of vertebrate rHLH transcription factors has been implicated in neurogenesis and axonal pathfinding. The mammalian O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis. A Xenopus O/E homologue, Xcoe2, functions downstream of neurogenin and upstream of neuroD in neurogenesis. The murine O/E members are also expressed in olfactory neurons throughout development and may regulate components of the odorant signal transduction cascade. A C. elegans O/E (CeO/E) homologue has been identified in our laboratory and shown to be encoded by the unc-3 locus, mutations which result in an uncoordinated phenotype. Previous electron microscopic reconstruction of unc-3 mutants revealed that the axons of the ventral cord motor neurons are severely defasiculated, the neuromuscular junctions occur at ectopic sites and the motor neurons receive input from inappropriate interneurons as a result of the defasiculation. Using a GFP fusion to the unc-3 promoter and specific antibodies, we observed that CeO/E is expressed in a large subset of the ventral cord motor neurons and in a pair of chemosensory neurons (ASI amphid neurons). The CeO/E protein is expressed transiently, during times of axonogenesis in the ventral nerve cord and throughout development in the ASI neurons. Several observations suggest that the protein may have distinct roles in the two cell types. Specifically, the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI and the unc-3 mutants enter the dauer pathway under inappropriate conditions. There is also evidence for autoregulation of the gene in ASI neurons - the expression of CeO/E is greatly reduced in unc-3 mutants. CeO/E lacks a conserved second repeat helix found that is highly conserved in the mammalian protein. Experiments suggest that CeO/E protein is able to homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. We are currently identifying the binding site for CeO/E by analysis of sequences essential for regulation of expression in the ASI neurons as well as by specific binding site selection methods (Selex). We are using PCR and differential expression methods to isolate downstream targets of CeO/E that might participate in signaling in the ASI neurons and mediate the axonal pathfinding activities in the ventral nerve cord. The isolation of putative cofactors and downstream targets of CeO/E should provide insight into the function of this transcription factor in neuronal differentiation in C. elegans and vertebrates.