E Jan et al. (1999) International C. elegans Meeting "GLD-1 represses tra-2 translation through a poly (A) tail-dependent mechanism"

EB Goodwin, E Jan

[International C. elegans Meeting, 1999]

The C. elegans sex determination gene, tra-2 , is translationally regulated by elements in the 3'untranslated region (3'UTR) called TGEs. tra-2 activity is required for female development. Male development in both somatic and germline tissues requires that tra-2 translation is repressed via the TGEs. The TGE control is a conserved mechanism present in invertebrates and vertebrates that controls the translation of other mRNAs. Recently, we have demonstrated that the C. elegans GLD-1 protein binds specifically to the TGEs and represses translation in a TGE-dependent manner in vitro and in vivo . GLD-1 is a cytoplasmic germline specific protein and is a member of the STAR family of RNA binding proteins. GLD-1 is essential for oogenesis and is also necessary for inhibiting premeiotic proliferation and promoting hermaphrodite spermatogenesis. In general, the mechanism of 3'UTR regulation and the function of STAR proteins are not well understood. Polysome analysis suggest the TGEs repress translation by inhibiting ribosome initiation in C. elegans . To better understand the mechanism of GLD-1 repression, we developed a yeast in vitro translation assay. We find that addition of GLD-1 protein to yeast extracts represses translation of reporter RNAs in a TGE-dependent manner. We also find that this repression occurs only when reporter RNAs carry a poly (A) tail, suggesting that GLD-1 represses translation through a poly (A) tail-dependent mechanism. Furthermore, deadenylation is not necesssary for GLD-1 repression, since GLD-1 can inhibit translation of RNAs carrying a stretch of 30 nucleotides 3' to the poly (A) tail which blocks deadenylation. Lastly, the 5'cap of an mRNA is not required for GLD-1 repression suggesting that the mechanism of TGE control is not acting through the cap-binding protein, eIF4E. Our results suggest a model by which binding of GLD-1 to the TGEs represses translation by influencing the effect of the poly (A) tail on ribosome initiation.

Jan E et al. (1998) Midwest Worm Meeting "Searching for tra-2 3'UTR TGE regulators: We've struck GLD-1!"

Graves LE, Goodwin EB, Motzny CK, Jan E

[Midwest Worm Meeting, 1998]

The two first authors contributed equally to this study. The C. elegans sex determination gene, tra-2, is translationally regulated by elements in the 3' untranslated region (3'UTR) called TGEs. tra-2 activity is required for female development. Male development in both somatic and germ line tissues requires that tra-2 translation is repressed via the TGEs. The TGE control is a conserved mechanism present in invertebrates and vertebrates that controls the translation of other mRNAs. A factor, called DRF, in crude worm extract binds the TGEs and may be a repressor of translation. Our working model is that binding of DRF to the TGEs represses tra-2 translation. Using the yeast three hybrid screen, we have identified a protein, GLD-1, that binds specifically to the TGEs and which may be a repressor of tra-2 translation. GLD-1 is a cytoplasmic germ line specific protein and is a member of the STAR family of RNA-binding proteins. The RNA targets of the STAR proteins have not been previously identified. GLD-1 is essential for oogenesis, and is also necessary for spermatogenesis and inhibition of germ cell proliferation. Our results suggest that GLD-1 promotes spermatogenesis by repressing the translation of tra-2 through the TGEs. Six lines of evidence support this conclusion. First, GLD-1 represses the translation of reporter RNAs via the TGEs both in vitro and in vivo. Second, GLD-1 is required to maintain low TRA-2 protein levels in the adult hermaphrodite gonad. Third, ectopic expression of GLD-1 in the soma can repress the development of female cell fates, causing masculinization of somatic structures in hermaphrodite animals. Fourth, genetic analysis indicates that GLD-1 acts upstream of the TGE control. Fifth, antibody experiments show that endogenous GLD-1 is a component of DRF. Sixth, purified bacterially expressed GLD-1 fusion protein binds specifically to the TGEs. These results are consistent with the idea that GLD-1 represses tra-2 translation by acting through the TGEs and that it may be a component of DRF. The conservation of both the TGE control and the STAR family raises the possibility that a subset of STAR proteins may control RNA activity by repressing translation via TGEs.

Jan E et al. (1996) Midwest Worm Meeting "THE TRANSLATIONAL REGULATION VIA THE tra-2 3'UTR MAY BE CONSERVED ACROSS SPECIES"

Goodwin EB, Jan E

[Midwest Worm Meeting, 1996]

tra-2 promotes female cell fates in C. elegans. tra-2 loss of function mutants transform XX animals into males whereas tra-2 gain of function (gf) mutants cause XX animals to develop as females (they do not make sperm). tra-2(gf) mutants map to two 28 nt direct repeat elements (DRE) separated by a 4 nt spacer located in the 3' untranslated region (3'UTR). Previous evidence suggests that the DREs regulate tra-2 activity by inhibiting the translation of tra-2. The DREs are a binding site for a factor (DRF) that is present in crude worm extracts, and which we hypothesize is a repressor of translation. Our working model is that the binding of DRF to the DREs represses translation of tra-2. To investigate the biological importance of the translational regulation of tra-2, we asked whether this 3'UTR control is evolutionarily conserved by assaying for the presence of the regulation in the nematode C. briggsae and mammalian tissue culture cells. Previously, we developed an assay in which the translation of a b-gal reporter transgene is controlled by the presence of the wild-type tra-2 3'UTR (both DREs are present) but not by mutant 3'UTRs (where one or both of the DREs have been deleted) (ref 1). In this assay, little b-gal activity is detected in the soma of animals carrying a transgene with the wild-type 3'UTR, but strong b-gal activity is detected in animals that carry a transgene with a mutant 3'UTR. RNAse protection analysis demonstrated that the different transgenes produce similar amounts of RNA, indicating that the differences in b-gal activity is likely due to translational control. We used the identical transgenes to address whether the 3'UTR control mechanism is present in C. briggsae. C. briggsae is estimated to have diverged from C. elegans approximately 50 million years ago. Similar to C. elegans, the wild-type tra-2 3'UTR was able to repress the translation of b-gal but mutant 3'UTRs could not, suggesting that the molecular mechanism that mediates the translation of tra-2 is present in C. briggsae. To ask whether the 3'UTR regulation is conserved in mammals, we tested whether the wild-type C. elegans 3'UTR can repress translation of a reporter transgene in Hela cells. In these experiments a reporter construct that contained the SV40 promoter and the luciferase gene was used. Preliminary experiments show that the presence of the wild-type 3'UTR can repress luciferase expression, but a mutant 3'UTR can not. Therefore, the fact that the C. elegans 3'UTR can control translation of reporter transgenes both in C. briggsae and Hela cells suggests that the molecular mechanism that governs the 3'UTR regulation is a fundamental process that is conserved across species. 1. Goodwin E.B., Okkema P.G., Evans T.C., and Kimble J. (1993) Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C. elegans. Cell 75: 329-339

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.

Y Yoo et al. (1999) International C. elegans Meeting "The C. elegans sex-determining gene tra-1 may be translationally regulated by its 3'UTR"

Y Yoo, EB Goodwin, E Jan

[International C. elegans Meeting, 1999]

tra-2 , which is necessary for determining female cell fate in C. elegans , has been shown to be translationally regulated by two elements in its 3'UTR. These elements are also found in C. briggsae tra-2 and the human oncogene GLI. This suggests that these elements, termed TGEs for tra-2 and GLI elements, are involved in a more broadly conserved mechanism of translational regulation. We are currently investigating whether C. elegans tra-1 , which is homologous to the human oncogene GLI and is the terminal regulator in the sex determination pathway, may also be regulated by TGEs. Translational regulation of tra-1 would suggest an additional form of regulation for determining sexual cell fate. Moreover, regulation of tra-1 at the translational level may also provide insight into the control of the human oncogene GLI in tumor growth. To determine whether tra-1 expression is controlled on a translational level through TGEs, we initially identified potential TGEs through sequence analysis. This search revealed one potential TGE in the 3'UTR of tra-1 which later was shown to bind GLD-1 in RNA gel shift analyses. It has been previously established that GLD-1 recognizes and binds tra-2 TGEs in a specific manner. Presently, we are using reporter transgenes to functionally test this putative TGE and examine whether there are other TGEs in the 3'UTR. We are planning to use immunofluorescence and RNA in situ experiments to reveal whether indeed tra-1 is regulated in this manner.

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.

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.

Huayue Ye et al. (2008) "Molecular control of the retrieval and prevention functions in stress-induced learning defects from vitamin E administration in Caenorhabditis elegans"

Dayong Wang, Huayue Ye

[2008]

"Vitamin E (α-tocopherol), a potent radical chain breaking antioxidant, is popularly used as ancillary drug for neurodegeneration disease, oxidative stress-related impairment and cognitive dysfunction. In the current work, we explored the thermosensation model to investigate the effects of vitamin E administration on learning behaviors and the related molecular mechanism in Caenorhabditis elegans. In the assay model, administration of 100 μg/mL and 200 μg/mL vitamin E had no significant effects on learning behaviors, whereas relatively high concentration (400 μg/mL) of vitamin E exposure shortened the acquisition period of the association paradigm. Pre-treatment or post-treatment with 200 μg/mL vitamin E could prevent or retrieve and even enhance the UV irradiation- and heavy metal (Al and Pb) exposure- induced learning defects. Moreover, treatment with 200 μg/mL vitamin E could restore the learning formed in the ncs-1 and hen-1 mutant worms, suggesting that vitamin E can largely mimic the function of NCS-1 and HEN-1 in regulating the learning behaviors. In addition, vitamin E could also mimic the function of DEG-1, which plays a key role in the regulation of neurodegeneration, and MEV-1, whose mutation will result an altered sensitivity to oxidative stress. Therefore, our data suggest that the direct association may exist between trace dietary vitamin E intake and learning impairment induced by oxidative stress, and a specific response mechanism may be activated or even be amplified after the administration of vitamin E. Moreover, trace vitamin E administration may ameliorate the metal exposure- and/or the UV irradiation-induced learning impairment at least partially by regulating the NCS-1 functions in learning control in C. elegans."