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[
International Worm Meeting,
2019]
Progress in ascribing phenotypic characteristics to genetic identity has moved at a fast pace since the annotation of genomes. However, a comprehensive annotation of metabolomes has moved at a woefully slow pace. Recent advances made in high resolution accurate mass spectrometry (HRAM) now provide the opportunity for small molecule research to adopt that same discovery rate that has been seen for genomes. While traditional metabolomic analysis measures dozens or, at best, hundreds of metabolites, common HRAM analysis detects tens of thousands of features with largely unknown identities. In this work, we are pursuing a comprehensive annotation of the entire C. elegans metabolome. By using Furrier Transform Mass Spectrometry (FTMS), we have developed the methods to systematically annotate molecular formulae and structural characteristics of the C. elegans metabolome across all developmental stages. We have evidence for the presence of more than 10,000 different compounds, and thus genome and metabolome of C. elegans may be of similar numerical sizes. Among several thousand novel molecules we have identified or partially characterized, some represent compound classes that have not previously been described in C. elegans but are known from other organisms, and others are entirely novel. Many of the detected compounds suggest the presence of yet unexplored biosynthetic networks. For example, we identified over 80 N-acyl amino acids, a class of endogenous molecules not previously described in worms, whereas in mammals these compounds are well known for their anti-inflammatory and analgesic functions. Biosynthesis and function of these small molecules and many other new compound families in C. elegans we will present on is currently unknown and remains a focus of this work. In parallel, we are developing a timeline for the production of all metabolites throughout larval stages and adulthood. We believe that our comprehensive structural and functional annotation of the C. elegans metabolome will become an invaluable resource to the worm community and beyond, allowing researchers to connect metabolomic information with genetic tractability. Lastly, the first draft annotation of the C. elegans metabolome may become an example for other metazoan model systems, akin to the sequencing of the C. elegans genome over 20 years ago.
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[
International Worm Meeting,
2013]
In mammals, endocannabinoids (EC) and synthetic cannabinoids (CB) mediate their effects through CB receptors. We have previously demonstrated that eicosapentaenoyl ethanolamine (EPEA), an N-acylethanolamine (NAE) which is structurally most similar to the mammalian EC anandamide, prevents dauer formation in dauer constitutive mutants. Despite the biological activity of endocannabinoids in worms, there is abundant evidence that they do not possess orthologs of the canonical CB receptors. We have therefore performed a forward genetic screen to identify components of NAE / cannabinoid signaling pathways in the worm. EPEA prevents dauer formation in daf-c mutants at semi-permissive temperatures, but it is not fully penetrant, thus limiting its utility in a forward genetic screen. However, we have found that the CB receptor inverse agonist AM251 rescues dauer formation in
daf-2(
e1368) mutants at the fully restrictive temperature (25 deg C) and acts synergistically with EPEA. Conversely, the CB1 receptor agonist O-2545 induces dauer formation in
daf-2(
e1368) at semi-permissive temperatures and competes with AM251 and EPEA at the restrictive temperature, suggesting shared molecular targets. While performing structure activity studies, we identified an AM251 derivative, SR10589, which not only prevents dauer entry at lower doses but also causes a fully penetrant L1 arrest phenotype at higher concentrations. Importantly, the L1 arrest is competed by O-2545, and SR10589 at low doses acts synergistically with AM251 and EPEA to prevent dauer formation. We have conducted an F1 forward mutagenesis screen and isolated mutations that suppress the L1 arrest of SR10589 treated animals. The mutants have different degrees of resistance to SR10589, and, interestingly, all the mutants characterized so far also display resistance to AM251 and EPEA, indicating that these molecules act through common factors. We are currently cloning the mutations and believe that their identification will help understand the non-canonical actions of N-acylethanolamines and cannabinoids in worms and higher organisms.
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Czerwieniec, Gregg, Gaman, Emily A., Lithgow, Gordon J., Hughes, Robert E., Rodrigues, Pedro R., Evani, Uday S., Peters, Theodore, Lucanic, Mark, Alavez, Silvestre, Mooney, Sean D., Gibson, Bradford W.
[
International Worm Meeting,
2011]
While it is generally recognized that misfolding of specific proteins can cause late-onset disease states, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging. SDS Insoluble proteins purified from young and aged C. elegans were chemically labelled by isobaric tagging for relative and absolute quantitation (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Transgenic nematodes expressing three proteins identified in the insoluble fraction, DAF-21, RPS-0 and EFT-3 fused to Green Fluorescent Protein (GFP) showed the formation of visible aggregates by fluorescence microscopy. In the case of RPS-0 and EFT-3, these aggregates appeared immobile as measured by Fluorescence Recovery after Photobleaching (FRAP). Expression of genes encoding insoluble proteins observed in aged nematodes was knocked-down in using RNAi and effects on lifespan were measured. Forty of 100 genes tested were shown to extend lifespan after RNAi. These data indicate that genes encoding proteins that become insoluble with age are modifiers of lifespan. These data also demonstrate that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that aggregation of a diverse range of proteins may be a general feature of aging.
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[
Zootaxa,
2022]
Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.
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[
International Worm Meeting,
2021]
The PIWI/piRNA pathway is a highly conserved small RNA pathway. Central to the pathway is an RNA silencing complex made of an Argonaute protein of the PIWI clade and PIWI-interacting RNAs (piRNAs), which target transposons in animal germlines. In C. elegans, piRNAs also target endogenous protein-coding genes, making the PIWI/piRNA pathway an important regulator of gene expression. C. elegans piRNAs are referred to as 21U RNAs since they are 21 nucleotides long and have a 5' bias for uridine monophosphate. We recently identified a protein complex driving 21U RNA biogenesis, which we named PETISCO (Cordeiro Rodrigues et al., 2019). However, we found that PETISCO can have different functions depending on whether it interacts with the proteins PID-1 (PiRNA Induced silencing Defective) or TOST-1 (Twenty-One U antagoniST). PETISCO:PID-1 complex participates in the 21U RNA biogenesis by interaction and stabilization of the 21U RNA precursors. While being important for gene regulation, the loss of PID-1 is not embryonic lethal. In contrast, PETISCO:TOST-1 is not involved in 21U RNA biogenesis but is nevertheless essential for embryonic development of the subsequent generation (Cordeiro Rodrigues et al., 2019; Zeng et al., 2019). The molecular function of this complex as well as the observed lethality effect is not yet understood. Based on preliminary data, we hypothesize that the PETISCO:TOST-1 complex regulates replication-dependent histones mRNA expression. In this study we want to characterize the PETISCO::TOST-1 complex and dissect its role in embryogenesis. This study would highlight the role of 21U RNA pathway factors in gene expression and C. elegans embryonic development.
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[
J Biol Chem,
1990]
The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.
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[
Worm Breeder's Gazette,
1994]
R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with
bc1-2, the human homolog to
ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and
bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar
lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with
ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.
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[
Nat Commun,
2021]
R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.
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[
Dev Biol,
2024]
While the nervous system of bilaterian animals is mainly left-right (L-R) symmetric at the anatomical level, some molecular and functional L-R asymmetries exist. However, the extent of these molecular asymmetries and their functional consequences remain poorly characterized. C. elegans allows to study L-R asymmetries in the nervous system with single-neuron resolution. We have previously shown that a neural bHLH transcription factor, HLH-16/Olig, is L-R asymmetrically expressed in the AIY neuron lineage and regulates AIY axon projections in a L-R asymmetric manner. Here, by combining a candidate approach and single-cell RNA sequencing data analysis, we identify the ephrin protein EFN-2 and the Flamingo protein FMI-1 as downstream targets of HLH-16 that are L-R asymmetrically expressed in the AIY lineage. We show that EFN-2 and FMI-1 collaborate in the L-R asymmetric regulation of axonal growth. EFN-2 may act via a non-canonical receptor of the L1CAM family, SAX-7. Our study reveals novel molecular L-R asymmetries in the C. elegans nervous system and their functional consequences.
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[
Commun Integr Biol,
2011]
The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.