Amy Tong et al. (2005) International Worm Meeting "The role of C. elegans DAP kinase MOR-3 in post-embryonic epidermal development"

Andrew CHISHOLM, Amy Tong

[International Worm Meeting, 2005]

Mutations in mor-3 cause an unusual late-onset epidermal morphogenesis defect (Tong et al, 2004 WCWM abstract 193). mor-3 mutants display vacuolation, thickening, and encrustation of specific regions of the epidermis (notably the nose, dorsal midline, vulva, and tail) that become apparent from later larval stages onwards. Using EM we have found that the cuticle in such regions is up to 4 times thicker than normal and has an aberrant ultrastructure. Markers for the cuticle cortex (COL-19::GFP) and struts (BLI-1::GFP) show mislocalization and accumulation in mor-3 mutants. Speculatively, MOR-3 might act in negative feedback control of cuticle secretion in the epidermis. mor-3 encodes the C. elegansortholog of death-associated protein kinase (DAP kinase, DAPK), a protein previously implicated in apoptotic and autophagic cell death. Preliminary analysis indicates that the epidermal defects in mor-3 mutants do not reflect aberrant activation of known cell death pathways. However mor-3 mutants do have subtle defects in apoptosis (Yi-Chun Wu, personal communication) suggesting that CeDAPK/MOR-3 may have separable roles in apoptosis and epidermal development. A functional MOR-3::GFP is first detectable in the epidermis in late embryonic stages. In larvae and adults MOR-3::GFP is punctate within epidermal cells. We are trying to determine the identity of these puncta. MOR-3 reporter genes are also highly expressed in body wall and vulval muscles and in some neurons; however, genetic mosaic analysis suggests mor-3 function in muscles is not required for epidermal morphogenesis. We are using tissue specific promoters to define when and where MOR-3 function is required in epidermal development. We are also trying to determine which domains of MOR-3 are important in its epidermal function, and to identify interacting partners of MOR-3 using genetic modifier screens.

Amy Tong et al. (2004) West Coast Worm Meeting "mor-3 encodes a C. elegans death-associated protein kinase (DAP kinase) that functions in post-embryonic epidermal morphogenesis"

Amy Tong, Sarah Moseley, Yishi Jin, Andrew CHISHOLM, Alexandr Goncharov

[West Coast Worm Meeting, 2004]

From forward genetic screens for epidermal morphogenesis mutants we identified two mutations defining a new locus, mor-3 . Mutations in mor-3 cause a unique epidermal morphology defect. mor-3 mutant animals are indistinguishable from wild type until L3 larval stage when vacuoles appear in the anterior lateral epidermis. L4 larvae and adult animals display a blunt nose morphology defect. This phenotype is fully penetrant at 23-25 C. All alleles of mor-3 are temperature-sensitive (ts) for this phenotype, suggesting that the process becomes ts in the absence of mor-3 . Between 30 to 50% of mutants develop refractile encrustations of the cuticle that become progressively more severe with age. Underneath these encrustations the epidermis appears highly vacuolated. Strikingly, these encrustations are most prominent at the dorsal midline of the epidermis between the pharynx and the mid-body. Electron microscopy of mor-3 mutants revealed massive thickening of the cuticle in affected regions. By genetic mapping and allele sequencing we found that mor-3 encodes the C. elegans death-associated protein kinase (DAP kinase) ortholog, K12C11.4. The worm DAP kinase is a serine/threonine kinase with a kinase domain, a set of 7 ankyrin repeats, and a death domain. In our forward screens we found two alleles of mor-3 : ju4 and ju469 . ju4 causes a missense change in a non-conserved amino acid in the kinase domain and ju469 is a small deletion. A third allele, gk219 , isolated by the knockout consortium, results in an intermediate Mor phenotype. In summary, existing alleles of mor-3 likely reduce but may not eliminate gene function. We are currently screening for additional alleles by non-complementation. DAP kinase has been implicated in several cell death pathways, including apoptosis and autophagy. Downregulation of human DAP kinase has been found in various tumor cell lines, indicating that DAP kinase acts as a tumor suppressor gene. We have begun to test whether mor-3 functions in apoptosis or autophagy. mor-3 mutants do not display obvious apoptosis defects and so far have not shown genetic interactions with apoptosis mutants. Conversely, known apoptosis and autophagy mutants do not display epidermal defects resembling those of mor-3 . Thus, our preliminary studies suggest that DAP kinase may not regulate cell death in C. elegans . Our additional genetic and molecular characterization of mor-3 will be presented.

Wang D et al. (2019) Science "Genetic behavioral screen identifies an orphan anti-opioid system."

Johnston C, Zucca S, Grill B, Gill MS, Dao M, Wang D, Lundquist EA, Song C, Giles AC, Martemyanov KA, Orlandi C, Stoveken HM, Opperman KJ, Masuho I

[Science, 2019]

Opioids target the -opioid receptor (MOR) to produce unrivaled pain management but their addictive properties can lead to severe abuse. We developed a whole animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in <i>C. elegans,</i> we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR and inhibits signaling to G proteins. Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of <i>C. elegans</i> as a scalable platform for genetic discovery of GPCR signaling principles.

Perreault J et al. (2007) Mol Biol Evol "Ro-associated Y RNAs in metazoans: evolution and diversification."

Perreault J, Perreault JP, Boire G

[Mol Biol Evol, 2007]

The Y genes encode small non-coding RNAs whose functions remain elusive, whose numbers vary between species, and whose major property is to be bound by the Ro60 protein (or its ortholog in other species). To better understand the evolution of the Y gene family, we performed a homology search in 27 different genomes along with a structural search using Y RNA specific motifs. These searches confirmed that Y RNAs are well conserved in the animal kingdom and resulted in the detection of several new Y RNA genes, including the first Y RNAs in insects and a second Y RNA detected in Caenorhabditis elegans. Unexpectedly, Y5 genes were retrieved almost as frequently as Y1 and Y3 genes, and, consequently are not the result of a relatively recent apparition as is generally believed. Investigation of the organization of the Y genes demonstrated that the synteny was conserved among species. Interestingly, it revealed the presence of six putative "fossil" Y genes, all of which were Y4 and Y5 related. Sequence analysis led to inference of the ancestral sequences for all Y RNAs. In addition, the evolution of existing Y RNAs was deduced for many families, orders and classes. Moreover, a consensus sequence and secondary structure for each Y species was determined. Further evolutionary insight was obtained from the analysis of several thousand Y retropseudogenes among various species. Taken together, these results confirm the rich and diversified evolution history of Y RNAs.

Yamaguchi M et al. (2016) Biochim Biophys Acta "NF-Y in invertebrates."

Yoshida H, Ly LL, Yamaguchi M, Ali MS, Yoshioka Y

[Biochim Biophys Acta, 2016]

BothDrosophila melanogaster and Caenorhabditis elegans (C. elegans) are useful model organisms to study in vivo roles of NF-Y during development. Drosophila NF-Y (dNF-Y) consists of three subunits dNF-YA, dNF-YB and dNF-YC. In some tissues, dNF-YC-related protein Mes4 may replace dNF-YC in dNF-Y complex. Studies with eye imaginal disc-specific dNF-Y-knockdown flies revealed that dNF-Y positively regulates the sevenless gene encoding a receptor tyrosine kinase, a component of the ERK pathway and negatively regulates the Sensless gene encoding a transcription factor to ensure proper development of R7 photoreceptor cells together with proper R7 axon targeting. dNF-Y also controls the Drosophila Bcl-2 (debcl) to regulate apoptosis. In thorax development, dNF-Y is necessary for both proper Drosophila JNK (basket) expression and JNK signaling activity that is responsible for thorax development. Drosophila p53 gene was also identified as one of the dNF-Y target genes in this system. C. elegans contains two forms of NF-YA subunit, CeNF-YA1 and CeNF-YA2. C. elegans NF-Y (CeNF-Y) therefore consists of CeNF-YB, CeNF-YC and either CeNF-YA1 or CeNF-YA2. CeNF-Y negatively regulates expression of the Hox gene egl-5 (ortholog of Drosophila Abdominal-B) that is involved in tail patterning. CeNF-Y also negatively regulates expression of the tbx-2 gene that is essential for development of the pharyngeal muscles, specification of neural cell fate and adaptation in olfactory neurons. Negative regulation of the expression of egl-5 and tbx-2 by CeNF-Y provides new insight into the physiological meaning of negative regulation of gene expression by NF-Y during development. In addition, studies on NF-Y in platyhelminths are also summarized.

Burns RG (1996) Trends in Cell Biology "Reply: Defining the gamma-, delta- and epsilon-tubulins."

Burns RG

[Trends in Cell Biology, 1996]

Keeling and Logsdon propose that the y-like sequences from Caenorhabditis elegans and Saccharomyces cerevisiae are bona fide y-tubulins that have undergone rapid evolutionary divergence. Indeed, genetic and localization studies with the yeast epsilon-tubulin (encoded by the TUB4 gene) reveal striking similarities to the bona fide y-tubulins, whereas there is no apparent human analogue to the C. elegans delta-tubulin among the 60 available human y-tubulin expressed-sequence tags. (ESTs).

Gardiner TJ et al. (2009) RNA "A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication."

Langley AR, Gardiner TJ, Krude T, Christov CP

[RNA, 2009]

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.

Erickson DL et al. (2006) J Bacteriol "Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis."

Hinnebusch BJ, Erickson DL, Wren BW, Jarrett CO

[J Bacteriol, 2006]

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.

Sato, Manami et al. (2015) International Worm Meeting "Expression pattern of C. elegans Y RNA homologs."

Ushida, Chisato, Kihara, Shinya, Sato, Manami

[International Worm Meeting, 2015]

Y RNA is a small structured ncRNA of about 100 nt in length. This RNA binds to Ro60 protein, which is a target of autoimmune disease antibody in patients with systemic lupus erythematosus and Sjogren's syndrome. Several lines of evidence suggest that the role of Y RNA and Ro60 function in the quality control of structured ncRNAs in cells under stress conditions. It is also indicated that vertebrate Y RNAs function in the initiation of DNA replication without Ro60. However, the molecular mechanisms of these functions and the contribution of Ro60/Y RNP to the autoimmune disease are still unclear. C. elegans genome encodes one Ro60 homolog (ROP-1) and 19 Y RNA homologs (1 CeY RNA and 18 sbRNAs). Other animals also have several Y RNA homologs, but C. elegans is the first example which has more than 5 Y RNA homologs encoded in the genome. Here we show the expression pattern and the cellular localization of these Y RNA homologs in C. elegans examined by the RNA fluorescent in situ hybridization (RNA-FISH). The signals of 14 homologs were detected in the intestinal cytoplasm. The signals of two other homologs were detected in the germ cytoplasm. The remaining three could not be detected, probably because they present in too low abundance to be detected by RNA-FISH. All 19 Y RNA homologs have the structural elements required for the binding of ROP-1. In other organisms, Ro60 binding stabilizes Y RNAs in cells. To know whether C. elegans Y RNA homologs also stabilized by the presence of ROP-1, we examined RNA-FISH of the Y RNA homologs against a mutant strain MQ470, which has a transposon insertion in the middle of the ROP-1 gene and lacks ROP-1 proteins in the cell. As expected, all Y RNAs examined so far decreased extensively. These were confirmed by northern hybridization. The results suggest that several C. elegans Y RNA homologs are expressed in a tissue-specific manner and most Y RNA homologs are stabilized by ROP-1 binding as well as those in other organisms.

Zhou D et al. (2011) Protein Cell "Formation and regulation of Yersinia biofilms."

Yang R, Zhou D

[Protein Cell, 2011]

Flea-borne transmission is a recent evolutionary adaptation that distinguishes the deadly Yersinia pestis from its progenitor Y. Pseudotuberculosis, a mild pathogen transmitted via the food-borne route. Y. Pestis synthesizes biofilms in the flea gut, which is important for fleaborne transmission. Yersinia biofilms are bacterial colonies surrounded by extracellular matrix primarily containing a homopolymer of N-acetyl-D-glucosamine that are synthesized by a set of specific enzymes. Yersinia biofilm production is tightly regulated at both transcriptional and post-transcriptional levels. All the known structural genes responsible for biofilm production are harbored in both Y. Pseudotuberculosis and Y. Pestis, but Y. Pestis has evolved changes in the regulation of biofilm development, thereby acquiring efficient arthropod-borne transmission.