Huang X-Y et al. (1991) International C. elegans Meeting "BIOCHEMICAL CHARACTERIZATION OF TWO PROTEINS SPECIFICALLY ASSOCIATED WITH THE TRANS-SPLICED LEADER"

Hirsh DI, Huang X-Y

[International C. elegans Meeting, 1991]

RNA trans-splicing in nematodes involves the transfer of an evolutionarily conserved 22-nucleotide spliced-leader (SL) and a trimethylguanosine (TMG) cap to the 5'-end of the recipient mRNA. The physiological role of trans-splicing, of the SL and the biological effect of TMG cap at the 5' ends of mRNAs are not yet understood. The possibility has been raised that SL itself has a catalytic role in splicing. The TMG cap has recently been shown to be an essential nuclear targeting signal. We hope to learn the functions of SL or transsplicing by identifying proteins specifically bound to SL and elucidating their functions. Electrophoretic mobility shift assays combined with competition analysis showed two proteins SLBP1 and SLPB2, bind specifically to SL1. The binding can be stimulated by a cap structure at the 5' end of SL. Although SLBP2 can bind SL1 RNA, SLBP1 seems unable to bind SL1 RNA. This may be due to the highly structured SL1 RNA interfering SLBP1 binding. We are presently testing the binding of SLBP1 and SLBP2 to SL2. UV cross-linking and SDS-PAGE revealed that SLBP1 has a molecular mass of 57kD and SLBP2 of 30kD. Nitrocellulose filter retention assay showed that the binding between SLBP1 and SL1, SLBP2 and SL1 conforms to a simple biomolecular reaction. SLBP1 has been purified to homogeneity as judged by silver staining on SDS-PAGE. Gel filtration analysis indicates that SLBP1 exists as a monomer and a dimer in solution. However we do not know whether dimerization is required for RNA binding. We intend to isolate their genes and generate antibodies against SLBP1 and SLBP2 to further characterize their biochemical properties and biological functions.

D Combes et al. (1999) International C. elegans Meeting "Acetylcholinesterase genes in C. elegans.II. Tandem organization of the third and fourth genes."

Y Fedon, M Arpagaus, M Grauso, E Culetto, D Combes, J -P Toutant

[International C. elegans Meeting, 1999]

In an attempt to clone ace-3, a sequence (clone 48) was obtained by RT-PCR on total RNAs from C. elegans ace-1 null mutants. This clone hybridized to YACs Y48B6 and Y59C8 indicating a site on chromosome II near unc-52, the expected location of ace-3. Blast examination of genomic sequences covering this region in C. briggsae showed that two sequences on chromosome II in this species were related to clone 48. One was the homologous of clone 48 and the other had also the characteristics of an ace gene. Those two sequences were located in very close proximity on chromosome II with only 372 bp between the stop of the upstream ORF and the ATG of the downstream ORF. A similar tandem organization of these two ace genes was also found in C. elegans by PCR (with 356 bp between the two ORFs and 200 bp separating the polyadenylation site of the 5' gene and the trans-splicing site of the 3' gene). Due to the close proximity of the two genes on chromosome II it was not possible to decide which sequence corresponded to ace-3 (defined as encoding AChE of class C) and those genes were provisionally named ace-x (upstream ORF) and ace-y (downstream ORF). Using RT-PCR, both genes were shown to be transcribed in both species. In both C.elegans and C. briggsae, ace-x contains 16 introns and ace-y 7 introns. We found FGQSAG (ace-x) and VGESAG (ace-y) for sequences around the active serine: both sequences suggest a low catalytic activity. The C-terminal sequences are of the H type suggesting that ACE-X and ACE-Y are glycolipid-anchored membrane proteins. We then sequenced ace-x and ace-y in the mutant ace-3 (strain dc2) in order to identify ace-3. We found that the only change was a deletion (580 nt-long) covering the non coding region between ace-x and ace-y as well as two small portions of coding sequence including the stop codon of ace-x and the initiator ATG of ace-y. The deletion did not shift the reading frame, so that a single long ORF was present in the mutant dc2. A single large mRNA was found by RT-PCR in dc2 instead of two distinct mRNAs in N2 strain. It is likely that the folding of the large encoded protein prevents catalytic activity of ace-x and ace-y. Thus the dc2 mutant did not allow the identification of ace-x or ace-y to ace-3.

Hirsh DI et al. (1989) International C. elegans Meeting "characterization of a new trans-spliced leader in C elegans."

Hirsh DI, Huang X-Y

[International C. elegans Meeting, 1989]

Albritton, Sarah et al. (2011) International Worm Meeting "Regulation of X chromosome transcription in Caenorhabditis species."

Albritton, Sarah, Ercan, Sevinc

[International Worm Meeting, 2011]

Animals with different numbers of X chromosomes in males and females possess mechanisms to compensate for the difference in the X-linked gene dose between the two sexes. In addition to the X chromosome dose difference between the sexes, the presence of a single-copy X chromosome per two-copy diploid autosomes creates an important problem for males, because all X-linked genes are haploinsufficient compared to the autosomal genes. In C. elegans, hermaphrodites (XX) contain two Xes, whereas males (XO) contain a single X, therefore facing X haploinsufficiency. By performing microarray analysis of RNA abundance in XX and XO worms, we observed that the overall transcript levels from the X chromosome in both XX and XO animals is similar to that of overall expression from autosomes. This suggests that transcription from the single X in XO L3 hermaphrodites (TY2205, her-1(e1520) sdc-3(y126) V; xol-1(y9) X) is increased approximately two-fold. The mechanism of this upregulation is unclear. We had shown that the X chromosome promoters have higher GC content compared to the autosomes (Ercan et al 2010), suggesting a DNA-encoded mechanism of transcriptional regulation. We will study X upregulation by comparing transcription of orthologus genes that are on the X versus autosomes in four Caenorhabditis species. Ercan S, Lubling Y, Segal E, Lieb JD. High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans. Genome Res. 2011 Feb;21(2):237-44. PMID:21177966.

Hecht RM et al. (1989) International C. elegans Meeting "colocalization of nematode myofilaments and mRNAs for GAPDH, actin, and myosin."

Huang X-Y, Hecht RM

[International C. elegans Meeting, 1989]

Zhen M et al. (2000) West Coast Worm Meeting "syd-8, a new player in axon guidance."

Huang X-Y, Zhen M, Jin YS

[West Coast Worm Meeting, 2000]

Growth cones sense and respond to various extracellular cues to be guided to their targets. Many such cues and their receptors have been identified in recent years, including netrins, slits, and ephrins. However, the signal transduction pathways of these cues are not well understood. We describe here a gene that may function in the downstream signaling of axon guidance. syd-8 (ju39) was isolated in a genetic screen for abnormal synapse mutants 1. syd-8 (ju39) animals have wild-type mating, moving and egg-laying behaviors. In syd-8 (ju39) animals, the synaptic GFP puncta are discontinuous in the dorsal cord. Analysis of axonal morphology in syd-8 mutant revealed several axonal defects in DD and VD neurons: 1) commissures terminate prematurely before reaching the dorsal cord, 2) commissures branch or wander to the lateral region, 3) axons defasciculate in both dorsal and ventral cords. Similar defects are also observed in the DA and DB axons, but to lesser extents. We also observed defects in pathfinding by the HSNs. In wild type animals, HSNs send out processes along the ventral nerve cord to the nerve ring . Normally, HSNL extends along the left small bundle of ventral cord. HSNR extends along the large right bundle of ventral cord. In syd-8(ju39) animals, HSNs have two types of defects: 1) HSN, mostly the HSNL, wander in the lateral region, and 2) HSNs fasciculate on the ventral cord. In 20% of syd-8 (ju39) animals, the HSNs are in the large right bundle of ventral cord. The touch neurons appear to be normal as judged by the Pmec-7-GFP marker. ju39 is a recessive, partial loss-of-function mutation. We have mapped syd-8 on Chromosome V between lin-25 and him-5. arDf1, itDf1 and yDf8 complement syd-8; yDf12 and nDf42 fail to complement it. Cloning of syd-8 is underway and we will report further molecular analysis of syd-8 at the meeting. 1. Zhen, M and Jin, Y. Nature 1999

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.

Hodgkin, Jonathan et al. (2009) International Worm Meeting "Analysis of breakage and meiotic instability in attached-X-chromosome strains."

Hodgkin, Jonathan, O'Rourke, Delia, Jethwa, Nirmal

[International Worm Meeting, 2009]

The isolation of an attached X chromosome (X^X), consisting of two apparently intact X chromosomes joined at their left telomeres, was previously reported (Hodgkin and Albertson 1995, Genetics 141: 527). In contrast to attached X chromosome constructs in other organisms, and also to X-autosome fusions in C. elegans, this X^X configuration is unstable, frequently breaking down to give an apparently normal X chromosome, or else an X chromosome with a deletion of the left end, or an X chromosome carrying a duplication of the left end. The extent of these deletions and duplications is variable, and they are generated at high frequency (in at least 5% of all X^X oogenic meioses). X^X strains therefore provide a useful source of aberrations in this region of the genome, which contains both a meiotic pairing site and numerator sites for sex determination. It seemed likely that X^X breakage was occurring at meiosis, and might be dependent on the meiotic recombination machinery. To test this hypothesis, X^X was crossed onto a series of meiosis-defective backgrounds (him-1, him-3, him-5, him-6, him-8, him-14, xnd-1, etc.). X^X breakage, as measured by the production of self-progeny males, was significantly or greatly reduced in most of these backgrounds, indicating that the breakage events are partly or wholly dependent on meiotic recombination. Previous FISH studies using YAC probes indicated that the end junction between the two X chromosomes was symmetrical and involved no deletion. We attempted to clone the junction fragment by single primer PCR, in order to determine its exact structure. However, PCR amplification was not successful, perhaps because the junction may include a substantial stretch of telomere repeats or local rearrangements. Further investigation of the structure and properties of the end junction is in progress.

Raymond Y L Yu et al. (2001) International C. elegans Meeting "Cloning of a collagen which functions specifically during sensory organ morphogenesis in C. elegans."

Raymond Y L Yu, King L Chow

[International C. elegans Meeting, 2001]

Extracellular matrix assembly is an important component for cell signaling and morphogenesis during animal development. Mutations in genes encoding the extracellular matrix components could result in failure of cellular communication, differentiation and thus mutant phenotypes. Collagen is one of the most abundant ECM proteins. It is an important structural component of the extracellular matrix in the animals that it has been shown to play significant role in body morphogenesis. Cloning of ram-4 gene reveals that it encodes a cuticular collagen. ram-4 mutants show a male specific phenotype: abnormal morphology of the 9 pairs of male sensory rays in the tail. Each ray shows tapered shape in wild-type male but has amorphous shape in ram-4 males (lumpy ray). A ray is composed of two neuronal cells, a hypodermis and a structural cell. All these ray cell components gave abnormal morphology in a lumpy ray, which suggest that the expression of this molecule has major impact on all ray cells during the retraction stage in late L4 larvae. Expression analysis revealed that RAM-4 was highly expressed specifically in the male hypodermis at the onset of ray retraction. The molecular features of this cuticular collagen is similar to other collagen protein with Gly-X-Y tripeptide repeat. It consists of three domains: amino and carboxyl non-Gly-X-Y domains flanking the central Gly-X-Y domain. Domain-deletion analysis has been performed to address the function of these domains in terms of rescue activity. Preliminary results suggesting that replacement of amino non-Gly-X-Y domain with synthetic signal sequence, which was used to ensure proper secretion of modified RAM-4 to the extracellular matrix, resulted in dominant negative effect. Interestingly, expression of amino non-Gly-X-Y domain alone also produced lumpy ray in WT background. This observation suggests a novel function of this domain for molecular interaction. The detail of this analysis will be discussed in the poster.

Zhen M et al. (2000) West Coast Worm Meeting "RPM-1, a conserved novel protein, regulates presynaptic terminal formation"

Jin YS, Huang X-Y, Bamber BA, Zhen M

[West Coast Worm Meeting, 2000]

Presynaptic terminals contain highly specialized subcellular structures to facilitate neurotransmitter release. We used the synaptic vesicle tagged GFP marker under the unc-25 promoter to visualize the presynaptic varicosities of the GABAergic DD and VD motor neurons 1. In wild-type animals GFP expression is observed as distinct fluorescent puncta, corresponding to individual neuromuscular junctions along the dorsal and ventral nerve cords. rpm-1 mutations were isolated in a screen for abnormal morphology of the GFP puncta. In rpm-1 mutants the total number of these GFP puncta is reduced to various extents depending on the strength of the mutation, and regions along the dorsal and ventral nerve cords often contained no GFP puncta. Moreover, the remaining GFP puncta were variable in size and shape. The overall axonal morphology of the DD and VD neurons appeared normal. At the ultrastructural level, we observed two defects at GABAergic NMJs in the rpm-1 mutants: 1) "over-developed" synapses that contained more than two individual electron-dense presynaptic active zones within the same varicosity, 2) "under-developed" synapses that had few synaptic vesicles and instead were filled with electron-dense debris-like material. In cholinergic NMJs, we observed the presynaptic terminals with longer active zones. rpm-1 encodes a large protein similar to Drosophila Highwire (Hiw) and mammalian Pam. All three proteins have a putative GEF domain and a highly conserved C-terminal with several zinc finger domains. RPM-1 is localized to the presynaptic region independently of synaptic vesicles. Mosaic analysis indicated that RPM-1 functions cell-autonomously. Using a temperature sensitive allele of rpm-1, we found that RPM-1 is required around the time of the formation of presynaptic terminals. Our results suggest that RPM-1 may regulate the distribution of presynaptic terminals, or function to prevent the formation of excessive presynaptic structures. 1. Zhen, M and Jin, Y. Nature 1999