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[
International Worm Meeting,
2017]
The RNA virus Orsay is the only known natural viral pathogen infecting the intestine cells of the nematode C. elegans. The C. elegans intestine cells resemble human intestinal epithelial cells not only in morphological features such as microvilli, but also in molecular mechanisms such as antibacterial innate immune response. As Orsay was discovered only six years ago, the C. elegans antiviral innate immune system remains largely unknown. Only two pathways, RNAi and ubiquitin-mediated protein degradation, have been discovered to be involved in the C. elegans antiviral innate immunity. To explore the genetic landscape of the C. elegans antiviral innate immunity, we conducted a genome-wide RNAi screen for genes whose inactivation sensitizes worms to Orsay infection. 110 genes were identified to be required for C. elegans antiviral innate immunity. 72 of these genes have human orthologs. Tissue-specific RNAi experiments showed that the majority of these genes function in the intestine cells to modulate antiviral innate immunity. In addition to RNAi and ubiquitin-mediated protein degradation, these genes encompass pathways in autophagy, mitochondrial unfolded protein responses, collagens, cytoskeletal organization, RNA processing, and transcription. To identify the gene products that are druggable, we screened 2000 chemicals for drugs that can alleviate Orsay infection symptoms, and discovered four innate immunity enhancing drugs, resorcinol monoacetate (RM), berberine (BBR), bismuth subsalicylate, 3,3'- diindolymethane (DIM). BBR and bismuth subsalicylate are known antidiarrhea drugs, with possible antiviral functions against human gastrointestinal viruses. Chemical-genetic experiments revealed that these drugs have different mechanism of actions: RM strengthens the collagen barrier for viral entry; BBR and DIM enhances the ubiquitin-mediated protein degradation pathway. Together, these data revealed a multifaceted antiviral innate immune system in the C. elegans intestine. Aspects of genetic and chemical modulation of this system may be conserved in human innate immunity against gastrointestinal viruses.
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[
International Worm Meeting,
2003]
Why does C. elegans, unlike other animals, have a substantial fraction of its genes transcribed in multigene clusters? A microarray analysis of the C. elegans genome has identified about 90% of all polycistronic transcription units. (This data is accessible from Wormbase and in Blumenthal et al., 2002, Nature 417: 851) An analysis of this database can provide clues as to what roles the operons play in C. elegans biology (Blumenthal and Gleason, 2003, Nature Rev. Genet. 4: 110). Although only ~15% of all genes are found in operons, some classes of genes are represented far more frequently in operons than are others. The operons preferentially contain genes with two kinds of functions. Almost half of nuclear genes annotated as performing a mitochondrial function are in operons. Similarly, genes that encode the basic machinery of gene expression including transcription, splicing, translation, and especially RNA stability are found preferentially in operons (40-80%). In contrast there are many kinds of genes that are never, or almost never, found in operons. e.g. tissue-specific transcription activators, collagens, major sperm proteins, F-box proteins, cyclins, intermediate filament proteins, peroxisomal proteins. One hypothesis to explain these observations is that the operons are a part of a global regulatory process that allows the worms to respond to changes in environmental conditions. Alternatively, they may contain mostly genes that do not need to be coordinately regulated at the transcriptional level. If so, the operons may have accumulated genes that are transcribed ubiquitously but are regulated at some subsequent stage of gene expression, e.g. mRNA stability or translation. The fact that most operons are expressed in the female germ line (V. Reinke, personal communication), and that so many encode the basic gene expression machinery and mitochondrial proteins required for energy generation, may indicate that they provide a mechanism for the efficient production of the proteins needed to allow the high level of protein synthesis required for development. Many operons co-express genes that are known to function together, presumably resulting in a selective advantage due to co-regulation. However, many operons contain genes of apparently unrelated function, and these gene arrangements may provide only the advantages of a more compact genome and utilization of a single promoter by many genes.
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[
East Coast Worm Meeting,
2004]
We are interested in studying organ development and function in order to understand both the normal processes and how improper organ development and function can lead to birth defects or cancer. The research in this project focuses on how an organ's structure, function, and response can be modified by external stimuli, such as environmental conditions. The C. elegans excretory system is a good model for organ development and function because of its simplicity. The excretory system is composed of only four cell types, yet it performs all of the necessary functions of osmotic regulation and waste removal, similar to mammalian kidneys. The simple structure of the excretory system makes it possible to study development and function at the cellular level. Dauer formation is an example of a change induced by environmental conditions in C. elegans . During dauer formation, the morphology of numerous cells and organs is altered, including the pharynx, hypodermis, and excretory system (Riddle, Blumenthal, Meyer, and Priess, 1997). It was previously shown that the excretory system might play a role in the C. elegans ' morphological transition into dauer state (Nelson and Riddle, 1984). We are following up on this research using GFP markers to help visualize each component of the excretory system. We currently have markers for the excretory cell and excretory duct and are working on markers for the excretory pore and gland cells. Using the markers, we are able to observe the morphology of the excretory system during its development. As an initial experiment, we compared the morphology of the excretory duct in normal L3 larvae and L3 larvae that had entered dauer. Our studies showed there is a difference in the excretory duct morphology between dauer and non-dauer larvae. In dauer larvae, the duct's position had shifted, the duct cell body was elongated, and the appendages were hyper-extended as compared to normal L3 larvae. We plan to expand these studies to other excretory cell types, and to investigate how these changes alter the excretory system function. Riddle, D.L., Blumenthal, T., Meyer, B.J., and Priess, J.R., C. elegans II (1997): 739-768 Nelson, F.K and Riddle, D.L., J. Exp. Zool. (1984) Jul; 231 (1): 45-56
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[
Japanese Worm Meeting,
2000]
abf-1 and
abf-2 encode ASABF-type antimicrobial peptides. In this study, we explored the transcriptional properties of these genes.
abf-1 and
abf-2 are adjacently located in tandem and in the same direction. Because the distance between the poly(A) additional site of
abf-1 and the SL1 acceptor site of
abf-2 was only 78 bp,
abf-1 and
abf-2 were predicted to form an operon. A polycistronic precursor RNA encoding both ORFs of
abf-1 and
abf-2 despite loss of the
abf-1 intron was detected by RT-PCR, suggesting that
abf-1 and
abf-2 actually form an operon. In contrast, the 5' ends of the
abf-1 and
abf-2 transcripts, determined by 5' RACE, were detected at a more proximal position than the SL1-acceptor site. The TATA-box was found at the appropriate position in each gene, suggesting that these could be produced as the transcripts of single genes. As a conclusion,
abf-1 and
abf-2 could be expressed in two separated way, i.e., (1) synchronous expression as an operon, and (2) independent expression as multiple single genes. We thank Prof. Thomas Blumenthal (University of Colorado Health Science Center) for his technical suggestions and helpful discussion.
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[
West Coast Worm Meeting,
2004]
We are studying the role of an alpha-tubulin gene,
tba-1 , in motor neuron development.
tba-1 , is one of nine C. elegans alpha-tubulins, and is widely expressed in the nervous system (1).
tba-1 is also the first gene of a C. elegans operon (2). The second gene of this operon, F26E4.10, is predicted to encode a protein containing double stranded RNA binding motifs and several RNAse III domains, consistent with a role in RNA processing. Although genes within C. elegans operons may not always be related by function, the presence of an RNA regulatory gene in the same operon with
tba-1 is intriguing. Evidence from both vertebrate and invertebrate systems suggests that synaptic plasticity, axon outgrowth and axon regeneration all require local protein synthesis. A recent study demonstrated that alpha-tubulin mRNA is present in the synaptic regions of Aplysia neurons and is translated locally in response to serotonin signaling (3). Thus,it is possible that selective activation or suppression of specific alpha-tubulin mRNAs is a mechanism for modulating axon or synapse growth. We are interested in determining the function and targets of the F26E4.10 protein in the C. elegans nervous system. As a first step, we have constructed GFP and dsRed reporter genes to examine the subcellular localization and developmental expression of this protein in C. elegans motor neurons. (1) Fukushige et al., J. Mol. Biol.1993; Biochim.Biophys.Acta 1995. (2) Blumenthal et al., Nature 2002. (3) Moccia et al. J. Neurosci, 2003.
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[
International Worm Meeting,
2005]
UNC-18/nSec1 appears to have many roles in regulating synaptic vesicle release. Studies in Drosophila and cell culture have shown that UNC-18 may act as an inhibitor of synaptic transmission 2. In C. elegans,
unc-18 mutant animals show a decrease in neurotransmitter secretion at the neuromuscular junction and in both worms and mice there is a decrease in the number of docked vesicles 1,4,5. One contributing factor in how UNC-18 positively regulates synaptic release may be the availability of UNC-64/Syntaxin-1 for forming SNARE complexes. If UNC-18/nSec1 is functioning to maintain levels of UNC-64/Syntaxin at the plasma membrane, this may explain some of the synaptic transmission defects seen in
unc-18 mutant worms. Studies using non-neuronal vertebrate cell types have shown that UNC-18/nSec1 is required for UNC-64/Syntaxin-1 to be properly trafficked to the plasma membrane 3.To address the role of UNC-18 in regulating anterograde transport in neurons, we looked to see if UNC-64 is properly localized in
unc-18 mutant worms. Using both antibodies against UNC-64 and an UNC-64a::GFP fusion protein we showed that a larger fraction of UNC-64 is retained in the cell bodies of neurons in
unc-18 animals when compared to wild type. Trafficking of other synaptic proteins such as SNB-1/Synaptobrevin, RIC-4/SNAP-25, UNC-10/RIM-1 and SYD-2 is unaffected in
unc-18 mutants, suggesting that this is not a general defect in intracellular transport. Current experiments are testing whether UNC-18 regulates ER or Golgi exit of UNC-64.1. Gengyo-Ando K et al. Neuron 19932. Schulze KL et al. Neuron 19943. Rowe J, et al. J Cell Sci 20014. Voets T, et al. Neuron 20015. Weimer RM, et al. Nature Neuroscience 2003
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[
International C. elegans Meeting,
1991]
Microtubules are cylindrical filamentous structures composed mainly of alpha and beta tubulin proteins, and serve a variety of functions in multicellular organisms. In C. elegans, like other higher organisms, alpha and beta tubulins are coded by a family of genes, and individual isotypic variants of tubulins can be separated on isoelectric focusing gels (Siddiqui et al. 1989). Here we report characterization of two members of the alpha tubulin gene family in C. elegans. Tubulin alpha -1 was cloned by direct screening of a C. eleFans cDNA expression library with a anti-alpha tubulin McAb 3A5 (M. Fuller), and Tubulin alpha -2 was originally identified by L. Gremke and J. Culotti, by screening C. eleFans genomic library with chicken alpha - tubulin as a probe. We have determined the physical map and the nucleotide sequence of the two alpha tubulin genes. The amino acid sequence deduced from the nucleotide sequence data has been compared with tubulin sequences from other organisms, and shows strong similarity with mammalian tubulins. A novel feature (pointed out to us by Dr. T. Blumenthal) in the 5' flanking sequence of both tubulin genes was the presence of typical eight nucleotide C. elegans acceptor sequence in the trans-splicing reaction of mRNA. We have now found that indeed a 22 nucleotide leader sequence is trans-spliced to 5'-flanking region of both alpha tubulin mRNA in C. elegans. Finally, Northern hybridization using alpha tubulin probes, has shown that these genes are expressed increasingly from embryonic to Ll-L3 stages. Experiments to determine the in situ expression of these genes are in progress. We thank the support to our work provided by the laboratories of Drs. J. Miwa and G. Eguchi.
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[
Midwest Worm Meeting,
2000]
How does the nucleus retain pre-mRNAs until the last intron has been removed? We believe we may have found an answer to that question by studying an alternatively spliced exon of the U2AF65 gene,
uaf-1, that contains an array of 10 3' splice sites, U4CAG/R. When we placed this exon in the 3' UTR of a gfp reporter gene, it prevented expression of that gene by preventing release of the RNA from the nucleus (MacMorris et al., 1999, PNAS 96:3813). Since both U2AF subunits have been shown to bind to U4CAG/R (Zorio and Blumenthal, 1999, Nature 402, 835), we used RNAi depletion of each U2AF subunit to determine that both are required to prevent expression of the gfp reporter gene. We inferred that by eliminating binding of U2AF, we allowed transport out of the nucleus. Now we have confirmed by RNAi of C. elegans TAP, a mammalian gene required for mRNA transport, that the increase in GFP expression is due to mRNA transport out of the nucleus. RNAi of TAP eliminates the GFP seen with
uaf-1 RNAi. We have also found that U2AF alone is not sufficient for nuclear retention: both PUF60 and
p54, which in vertebrates form a tight complex with each other and also bind to U2AF, are also required. When PUF60 or
p54 is depleted by RNAi, the reporter gene is released from the nucleus and expressed, even though U2AF levels presumably remain high. Control RNAi experiments included other vital RNA processing proteins: U1 70K, SRp20, SAP49, PRP8, CstF64, U1A/U2B'' and SF1, none of which resulted in RNA release. We suggest that a complex of U2AF65/35 with PUF60/p54 binds to the array of 3' splice sites in the reporter (without splicing) to prevent transport, and we propose that all four of these polypeptides are required components of a 3' splice site recognition complex. It is possible that only when this complex is released from a transcript by splicing is the resulting mature mRNA licensed for export.
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[
International Worm Meeting,
2007]
Synapses are asymmetric structures that facilitate the transmission of signals between neurons and their targets. The mature synapse consists of a presynaptic terminal in direct apposition to a postsynaptic terminal. Neurotransmission occurs when neurotransmitter-filled presynaptic vesicles dock and fuse with the plasma membrane. The neurotransmitter contents are released into the synaptic cleft and subsequently bind to receptors at the postsynapse. The active zone is an electron-dense region of the synapse that is required for the docking and fusion events of synaptic transmission. Our lab is interested in identifying genes that regulate the formation of active zones. In our study we are using the GABAergic nervous system of C. elegans as our model system. The active zones in this nervous system are visualized using a unique active zone specific marker SYD-2::GFP that was developed in our laboratory (Yeh et al., 2006). A forward genetic screen isolated an
unc-7 loss-of-function mutant that shows a significant decrease in number of SYD-2::GFP puncta. We have determined that the innexin UNC-7 localizes to perisynaptic regions in addition to gap junctions. Subsequently, we showed that loss-of-function
unc-9 mutants, which encodes an innexin protein that is 56% identical to UNC-7, shows the same synaptic defects as
unc-7 mutants (Yeh and Ng, unpublished), suggesting a novel regulatory role for innexins at chemical synapses. We further investigated the role of innexins at the synapse by performing an
unc-7 suppressor screen. We identified 10 dominant alleles of
unc-1, a gene that encodes a stomatin homologue, that suppress the kinker behavior as well as the synaptic phenotype of
unc-7 loss-of-function mutants. We found that UNC-7 and UNC-1 co-localize at peri-synaptic regions, indicating that the two proteins may functionally interact. We are investigating the physical properties of the potential UNC-7, UNC-9, and UNC-1 channel complexes. Current progress will be presented at the meeting. Yeh E, Kawano T, Weimer RM, Bessereau JL, Zhen M. 2005. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J Neurosci. 25(15):3833-41.
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[
International C. elegans Meeting,
1999]
Understanding how the aging process can be regulated is a fascinating and fundamental problem in biology. We have found that signals from the reproductive system influence the lifespan of the nematode C. elegans . If the cells that give rise to the germline are killed with a laser microbeam, the lifespan of the animal is extended (Hsin and Kenyon, Nature , in press). Our findings suggest that germline signals act by modulating the activity of an insulin/IGF-1-like pathway known to regulate the aging of this organism. Mutants with reduced activity of the insulin/IGF-1 receptor homolog DAF-2 have been shown to live twice as long as normal (1,2,3), and their longevity requires the activity of DAF-16, a member of the forkhead/winged-helix family of transcriptional regulators (1,2,4,5). We find that in order for germline ablation to extend lifespan, DAF-16 is required, as well as a putative nuclear hormone receptor, DAF-12 (6,7). In addition, our findings suggest that signals from the somatic gonad have an opposite effect on lifespan, and that this effect appears to require DAF-2 activity. Together our findings imply that the C. elegans insulin/IGF-1 system integrates multiple signals to define the rate of aging of the animal. This study demonstrates an inherent relationship between the reproductive state of this animal and its lifespan, and may have implications for the co-evolution of reproductive capability and longevity. 1. C. Kenyon, et al., Nature 366 , 461-4 (1993). 2. P. Larsen, P. Albert, D. Riddle, Genetics 139 , 1567-83 (1995). 3. K. Kimura, H. Tissenbaum, Y. Liu, G. Ruvkun, Science 277 , 942-6 (1997). 4. K. Lin, J. Dorman, A. Rodan, C. Kenyon, Science 278 , 1319-22 (1997). 5. S. Ogg, et al., Nature 389 , 994-9 (1997). 6. W-H. Yeh. Ph.D. Thesis, University of Missouri, Columbia (1991). 7. D. Riddle, P. Albert, in C. elegans II D. Riddle, T. Blumenthal, B. Meyer, J. Priess, Eds. (Cold Spring Harbor Laboratory Press, 1997) pp. 739-68.