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Darzynkiewicz E, Jones DN, Stepinski J, Jankowska-Anyszka M, Dickson L, Davis RE, Liu W, Piecyk K, Niedzwiecka A, Wallace A, Kieft J, Zhao R, Stolarski R
[
Nucleic Acids Res,
2011]
Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an mG-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate mG-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the mG-cap. Nematode eIF4E binding to the mG-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.
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[
Acta Biochim Pol,
2002]
Translation initiation factor eIF4E binds the m(7)G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m(3)(2'2'7)G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m(7) GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m(7)G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m(7)G and m(3)(2,2,7)G-containing caps (K(as)2X10(6) M-1 to 7X10(6) M-1) wheras IFE-3 and IFE-4 discriminated strongly in favor of m(7)G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of K-as on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous
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[
FASEB J,
2012]
Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase C. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.
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[
J Cell Biol,
2000]
The Caenorhabditis elegans genome encodes one alpha spectrin subunit, a beta spectrin subunit (beta-G), and a beta-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans alpha spectrin is reproduced by tandem depletion of both beta-G and beta-H spectrins. We propose that alpha spectrin combines with the beta-G and beta-H subunits to form alpha/beta-G and alpha/beta-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode beta-G spectrin and vertebrate beta spectrins exhibit three striking parallels including: (1) beta spectrins are associated with the sites of cell-cell contact in epithelial tissues; (2) the highest levels of beta-G spectrin occur in the nervous system; and (3) beta spectrin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode beta-G spectrin associates with plasma membranes at sites of cell-cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode beta-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, beta-G spectrin is not associated with internal membranes and depletion of beta-G spectrin was not associated with any detectable defects in secretion. Instead beta-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans beta-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.
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[
Nematology,
1999]
The secondary metabolites, 3,5-dihydroxy-4-isopropylstilbene (ST) and indole, from the culture filtrate of Photorhabdus luminescens MD, were shown to have nematicidal properties. ST caused nearly 100% mortality of 54 and adults of Aphelenchoides rhytium, Bursaphelenchus spp. and Caenorhabditis elegans at 100 mu g/ml, but had no effect on J2 of Meloidogyne incognita or infective juveniles (IJ) of Heterorhabditis megidis at 200 mu g/ml. Indole was lethal to several nematode species at 300 mu g/ml, and caused a high percentage of Bursaphelenchus spp. (54 and adults), M, incognita (J2) and Heterorhabditis spp. (IJ) to be paralysed at 300, 100 and 400 mu g/ml, respectively. Both ST and indole inhibited egg hatch of M, incognita. ST repelled IJ of some Steinernema spp. but not IJ of Heterorhabditis spp., and indole repelled IJ of some species of both Steinernema and Heterorhabditis. ST, but not indole, was produced in nematode-infected larval Galleria mellonella. after 24 h infection.
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[
Chem Sci,
2018]
Reductive cleavage of alkenes is rarely reported in synthetic chemistry. Here we report a unique H<sub>2</sub>S-mediated reductive cleavage of C[double bond, length as m-dash]C bonds under mild conditions, which is a successful new strategy for the design of probes for effective sensing of H<sub>2</sub>S with turn-on dual-color fluorescence. A short series of phenothiazine ethylidene malononitrile derivatives were shown to react with H<sub>2</sub>S, <i>via</i> reductive cleavage of C[double bond, length as m-dash]C bonds with intramolecular cyclization reactions to form thiophene rings. Enlightened by this new reaction mechanism, four effective probes with turn-off to turn-on fluorescence switches were successfully applied for sensing H<sub>2</sub>S, an important gaseous signalling molecule in living systems, among which PTZ-P4 exhibited two fluorescent colors after reductive cleavage. The dual-color probe was applied for imaging endogenous H<sub>2</sub>S and showed distinct differences in brightness in living <i>C. elegans</i> for wild type N2, <i>
glp-1</i> (<i>
e2144</i>) mutants (higher levels of endogenous H<sub>2</sub>S), and <i>
cth-1</i> (<i>
ok3319</i>) mutants (lower levels of endogenous H<sub>2</sub>S). The discovery of H<sub>2</sub>S-mediated reductive cleavage of C[double bond, length as m-dash]C bonds is expected to be valuable for chemical synthesis, theoretical studies, and the design of new fluorescent H<sub>2</sub>S probes.
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[
FEBS J,
2006]
Signal transduction mediated by heterotrimeric G proteins regulates a wide variety of physiological functions. We are interested in the manipulation of G-protein-mediating signal transduction using G-protein-coupled receptors, which are derived from evolutionarily distant organisms and recognize unique ligands. As a model, we tested the functionally coupling GOA-1, Galpha(i/o) ortholog in the nematode Caenorhabditis elegans, with the human muscarinic acetylcholine receptor M(2) subtype (M(2)), which is one of the mammalian Galpha(i/o)-coupled receptors. GOA-1 and M(2) were prepared as a fusion protein using a baculovirus expression system. The affinity of the fusion protein for GDP was decreased by addition of a muscarinic agonist, carbamylcholine and the guanosine 5''-[3-O-thio]triphosphate ([(35)S]GTPgammaS) binding was increased with an increase in the carbamylcholine concentrations in a dose-dependent manner. These effects evoked by carbamylcholine were completely abolished by a full antagonist, atropine. In addition, the affinity for carbamylcholine decreased under the presence of GTP as reported for M(2)-Galpha(i/o) coupling. These results indicate that the M(2) activates GOA-1 as well as Galpha(i/o).
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[
Arch Environ Contam Toxicol,
1997]
The toxicity of many chemicals depends on the physical conditions of the test environment, and any change or adjustment made to the tests can alter the results. Therefore it is important to establish the sensitivity of the test organism over a range of test conditions to determine when it is necessary to make adjustment and to what extent. In this study, we established the tolerance range of the nematode Caenorhabditis elegans for pH, salinity and hardness using 24- (without food source) and 96-h (with food source) aquatic toxicity tests. The tests were performed in two media: K-medium and moderately hard reconstituted water (MHRW). C.elegans has high tolerance under these test conditions. In K-medium worms survived a pH range of 3.1 to 11.9 for 24 h and 3.2 to 11.8 for 96 h without significant (p > 0.05) lethality. In MHRW the pH range was 3. 4 to 11.9 for 24 h and 3.4 to 11.7 for 96 h. Salinity tolerance tests were approximated with NaCl and KCl individually. Up to 15.46 g/L NaCl and 11.51 g/L KCl were tolerated by C. elegans in K-medium without significant lethality (p> 0.05). In MHRW higher salt concentrations were tolerated; about 20.5 g/L NaCl and 18.85 g/L KCl did not show any adverse effect compared to control. Hardness tolerance was tested by adding NaHCO3. The nematode could tolerate 0. 236 to 0.246 g/L of NaHCO3. The high tolerance of C. elegans to these test conditions (pH, salinity, and hardness) allows more versatility than other organisms commonly used in aquatic toxicity tests. It also allows the monitoring of effluents and receiving waters from freshwater or estuarine sources without dilution or adjustment.
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[
Genes Cells,
2001]
BACKGROUND: ERA (Escherichia coli Ras-like protein) is an E. coli GTP binding protein that is essential for proliferation. A DNA database search suggests that homologous sequences with ERA exist in various organisms including human, mouse, Drosophila, Caenorhabditis elegans and Antirrhinum majus. However, the physiological function of eukaryotic ERA-like proteins is not known. RESULTS: We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. CONCLUSION: Our data suggest that H-ERA plays an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain.
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[
J Biol Chem,
2000]
RX, a homeodomain-containing protein essential for proper eye development (Mathers, P. H. Grinberg, A., Mahon, K. A., and Jamrich, M. (1997) Nature 387, 603-607), binds to the photoreceptor conserved element-1 (PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter and stimulates gene expression. RX is found in many retinal cell types including photoreceptor cells. Another homeodomain-containing protein, CRX, which binds to the OTX element to stimulate promoter activity, is found exclusively in photoreceptor cells (Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gillbert, D. J. Jenkins, N. A., and Zack, D. J. (1997) Neuron 19, 1017-1030; Furukawa, T., Morrow, E. M., and Cepko, C. L. (1997) Cell 91, 531-541). Binding assay and cell culture studies indicate that both PCE-1 and OTX elements and at least two different regulatory factors RX and CRX are necessary for high level, photoreceptor cell-restricted gene expression. Thus, photoreceptor specificity can be achieved by multiple promoter elements interacting with a combination of both photoreceptor-specific regulatory factors and factors present in closely related cell lineages.