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. 2009 Jul;5(7):e1000561.
doi: 10.1371/journal.pgen.1000561. Epub 2009 Jul 10.

Defects in tRNA modification associated with neurological and developmental dysfunctions in Caenorhabditis elegans elongator mutants

Changchun Chen  1 , Simon Tuck, Anders S Byström
Affiliations

Defects in tRNA modification associated with neurological and developmental dysfunctions in Caenorhabditis elegans elongator mutants

Changchun Chen et al. PLoS Genet. 2009 Jul.

Abstract

Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5'methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic drawing of the tRNA secondary structure and modified wobble uridines.
(A) Secondary structure of tRNA with wobble position shown (•). (B) Wobble uridines can be modified to 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyl (mcm5U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U).
Figure 2
Figure 2. C. elegans elpc-1 is required for mcm5 and ncm5 side chain formation at wobble uridines.
(A) Schematic drawing of the distribution of exons and introns of elpc-1. The exons and introns are depicted as boxes and lines, respectively. At top, the line underneath represents the location of the deletion in elpc-1(tm2149). Below, the structure of the elpc-1::gfp transgene. (B–I) Total tRNA isolated from wild-type and elpc-1(tm2149) worms was analyzed by HPLC. Wild-type (N2) profiles are shown in the left panels. elpc-1(tm2149) and elpc-1(tm2149); elpc-1::gfp profiles are in the right panels. Chromatograms were monitored at 254 nm unless otherwise stated. (B,C) The parts of the chromatograms between retention times 46.4 and 51.5 min are displayed. The arrow in C indicates the expected retention time of mcm5s2U. (D,E) The parts of the chromatograms between retention times 10.5 and 18 min are displayed. The arrow in E indicates the expected retention time of ncm5U. (F,G) The parts of the chromatograms between retention times 33 and 39 min are displayed. The arrow in F indicates the expected retention time of s2U. These chromatograms were monitored at 314 nm. (H,I) The parts of the chromatograms between retention times 48 and 54 min are displayed.
Figure 3
Figure 3. ELPC-1 is differentially expressed in C. elegans.
(A–C) Confocal fluorescence micrographs of an hermaphrodite of the genotype elpc-1(tm2149); svEx557[Pelpc-1::elpc-1::gfp]. The large arrow in A denotes the posterior bulb of the pharynx. The smaller arrows denote sensory neurons in the head. The arrowhead indicates a body muscle. In B and C, specific sensory neurons in the head are indicated. (D–O) Micrographs of elpc-1(tm2149); svEx557[Pelpc-1::elpc-1::gfp] worms viewed with either Nomarski differential contrast (DIC) (D,F,H,J,L,N) or fluorescence (E,G,I,K,M,O) optics. The arrows in D and E indicate an ASK sensory neuron; in F and G, an HSN; in H and I, a CAN cell; in J and K, a PLM neuron; in L and M, a cell in the intestine. The green fluorescence in O is from cells in the developing vulva. Scale bars denote 10 microns.
Figure 4
Figure 4. The elp-1(tm2149) and elpc-3(tm3120) mutants are defective in fluorescence recovery after photobleaching.
(A) Images of mec-4::gfp reporter in wild type, elpc-1, elpc-3 and tuc-1 backgrounds before photobleaching (left), after photobleaching (middle) and 5 hours recovery (right). The images at the bottom are of the mec-4::gfp reporter strain treated with the cycloheximide. (B) Quantification of GFP pixel intensities before photobleaching, after photobleaching and 5 hours recovery. The number of worms examined of each strain is given under the graph. Error bars represent standard deviations. Two asterisks indicate a significant fluorescence recovery after 5 hours incubation by student's t test (**p<0.001).
Figure 5
Figure 5. The elp-1(tm2149) and elpc-3(tm3120) mutants show a salt chemotaxis learning defect.
For each strain there are three conditions. ‘NaCl’ indicates worms that were preconditioned on a plate containing 100 mM NaCl for 4 hours prior to the chemotaxis assay. ‘Mock’ indicates that worms were pretreated on NaCl-free plate for 4 hours before assay. ‘Naive’ indicates worms that were assayed without any preconditioning. The chemotaxis index after 30 min of assay is displayed. The assay was repeated four times. Error bars denote standard deviations. Asterisk indicates a significant difference from wild type N2 (*p<0.01 by student's t test). (A) Young adult worms that had been raised at 20°C. (B) Temperature shifted animals. Synchronized eggs were grown at 20°C to the 2nd larval stage (L2). L2 larvae were then shifted to 25°C and cultured until they had become young adults. (C) Young adult worms from a strain that had been maintained at 25°C for several generations.
Figure 6
Figure 6. Neurons in the elpc-1(tm2149) and elpc-3(tm3120) mutants show reduced production of neuropeptide.
(A) Fluorescence micrographs showing the nerve ring in worms harboring a transgene encoding ANF::GFP. (B) At top, western blot of protein extracts from worms of the indicated genotypes that contained the ANF::GFP transgene. The same amount of protein was loaded in each lane. The blot was probed with an antibody against GFP. Below, ANF::GFP transcripts were quantified by Real-time PCR. No significant difference was observed in the levels of ANF::GFP mRNA (Student's t test, p>0.05), which were normalized to tbb-2 mRNA. (C) Micrographs showing coelomocytes in worms carrying the ANF::GFP transgene. Those on the left were viewed with DIC optics. Those on the right are of the same worms viewed with fluorescence optics. Dashed lines indicate the locations of the coelomocytes. Note that the intensity of GFP fluorescence in EG3344 and tuc-1(tm1297) coelomocytes is higher than that in elpc-1(tm2149) and elpc-3(tm3120) mutant worms. (D) Graph showing the normalized pixel intensities of confocal images of coelomocytes (CC). The number of coelomocytes measured for each strain is shown on the bar. The strongest pixel intensity per coelomocyte of ANF::GFP in any worm tested was arbitrarily set to 1. Error bars represent standard deviations. Two asterisks indicate the significant difference from control worms by student's t test (**p<0.001). (E) Aldicarb sensitivity assays. The proportion of worms still able to move is plotted against time for the six different genotypes. N2 is the wild-type control; aex-6(sa24) is a strain previously shown to display increased resistance to aldicarb, and tom-1(ok285) is hypersensitive to aldicarb. (F) Levamisol sensitivity assays were performed in the same way as aldicarb assays. N2 is the wild-type control. lev-11 is a strain previously shown to be resistance to levamisol.
Figure 7
Figure 7. Defects seen in temperature-shifted elpc-1; tuc-1 double mutants.
Micrographs of eggs and larvae viewed with Nomarski DIC optics. (A–D) Embryos arrested prior to (A), during (B,C) or after (D) morphogenesis. (E,F,G,H) Parts of the germline in young adult hermaphrodites. The arrows in E and F indicate oocytes. Note that those in the elpc-1; tuc-1 worm have not matured. The arrows in G and H denote sperm. Those in the elpc-1; tuc-1 worm have grossly abnormal morphology. (I,J) The vulva during the L4 stage. In the elpc-1; tuc-1 double mutant, fewer cells are present and morphogenesis of the vulva to form the tube through which the eggs are laid is abnormal. Scale bars denote 10 microns.
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