Hawkinson D et al. (1999) Worm Breeder's Gazette "exc-7 Encodes an RNA-Binding and -Processing Protein"

Hawkinson D, Buechner M

[Worm Breeder's Gazette, 1999]

Our lab studies genes that affect the morphology of the excretory canals. Mutations in nine exc genes cause the canals to swell into fluid-filled cysts of varying size and placement. Mutations in let-4, let-653, and sma-1 cause similar phenotypic effects. The allele rh252 of exc-7 characteristically exhibits smaller septate cysts along the length of the canal, and terminates in a bolus of cysts. The length of these mutant canals varies from about 1/3 to 1/2 the length of wild-type canals. In addition, mutant worms often exhibit a slightly deformed tail whip. We previously mapped the exc-7 gene between the left ends of mnDf58 and mnDf59 on LGII. This area is spanned by approximately 13 cosmids. Microinjection of single cosmids along with the rol-6 marker showed rescue by cosmid E02H1. The genome project predicts 10 genes on this cosmid. We restriction-digested E02H1 into fragments; two fragments did not rescue, while a 3rd fragment, containing three predicted genes, rescued both the canal and tail whip phenotypes. We microinjected PCR-amplified fragments of each of these predicted genes. Only one fragment rescued; the predicted gene F35H8.5 encodes a homologue of the paraneoplastic encephalomyelitis HuD antigen. We are currently injecting RNAi to this gene to phenocopy the canal cysts. The HuD antigen and homologues have been characterized in Drosophila, Xenopus, rat, mouse, and human as members of a family of RNA-binding proteins that contain multiple 80-amino-acid RNA recognition motifs. The EXC-7 protein is most closely homologous to the Drosophila Elav14 protein, which is expressed early in development in neuronal cells. The excretory cell is an ectodermally-derived epithelial cell whose sister is a neuron; in addition, the excretory canals respond to neural outgrowth and guidance cues such as UNC-6, UNC-53, and UNC-73 to extend their tubular processes full-length. Our previous electron micrographs (with Ed Hedgecock and Dave Hall) of canals in exc-7 mutants show abnormal concentrations of endosomes at the tips of the cells, which could reflect abnormal accumulations of incorrectly processed RNA. We believe that the exc-7 gene could therefore be involved in RNA processing in many neurons during development, although the EXC-7 protein is most clearly needed in the excretory cell. We are currently making GFP constructs to see the location of its expression, as well as sequencing the rh252 mutation and screening for more exc-7 mutants.

D Hawkinson et al. (1999) International C. elegans Meeting "exc-7 Encodes a Homologue to the RNA-Processing Protein ELAV"

KV King, D Hawkinson, M Buechner

[International C. elegans Meeting, 1999]

Mutations in nine exc genes, and in let-4 , let-653 , and sma-1 , cause the excretory canals to swell into fluid-filled cysts of varying size and placement. The exc-7 allele rh252 characteristically exhibits smaller septate cysts along the canal terminating in a bolus of cysts. The length of these mutant canals varies from about 1/3 to 1/2 the length of wild-type canals. Mutant worms also often exhibit a slightly deformed tail whip. We previously mapped the exc-7 gene between the left ends of mnDf58 and mnDf59 on LGII, an area spanned by 13 cosmids. Both canal and tail whip phenotypes were rescued by microinjection of cosmid E02H1, in which GeneFinder predicts 10 genes. The position was narrowed by microinjection of cosmid restriction fragments, and located by microinjection of a PCR product containing the promoter and coding regions of the predicted gene F35H8.5. F35H8.5 encodes a homologue of the paraneoplastic encephalomyelitis HuD antigen. This protein and its homologues have been characterized in Drosophila, Xenopus, and mammals as RNA-binding proteins containing multiple 80-amino-acid RNA recognition motifs. The predicted EXC-7 protein is most closely homologous to Drosophila Elav14, which is expressed early in neuronal cell development. Although the excretory cell acts as the C. elegans osmoregulatory organ, it has many neuronal characteristics: It is an ectodermally-derived cell whose sister is a neuron; in addition, extension of canal processes requires neural guidance cues such as UNC-6, UNC-53, and UNC-73. Our previous electron micrographs (with Ed Hedgecock and Dave Hall) of canals in exc-7 mutants show abnormal concentrations of endosomes at the tips of the cells, which could reflect abnormal accumulations of incorrectly processed RNA. Surprisingly, although the phenotype of ELAV mutants in other creatures shows strong neural defects, our RNAi results, as well as the phenotype of the rh252 allele placed over a deficiency, suggest that the null phenotype shows strong effects in the excretory canal and the tail whip, with negligible effects on neuronal processes. We are currently making GFP constructs to identify the location of EXC-7 expression, and are sequencing the rh252 mutation in order to understand the molecular basis of the Exc-7 phenotype.

aldose beta-D-fructosyltransferase activity

Catalysis of the reaction: alpha-D-aldosyl1 beta-D-fructoside + D-aldose2 = D-aldose1 + alpha-D-aldosyl2 beta-D-fructoside.

lacto-N-biosidase activity

Catalysis of the reaction: H2O + beta-D-Gal-(1,3)-beta-D-GlcNAc-(1,3)-beta-D-Gal-(1,4)-D-Glc = beta-D-Gal-(1,4)-D-Glc + beta-D-Gal-(1,3)-D-GlcNAc.

mannotetraose 2-alpha-N-acetylglucosaminyltransferase activity

Catalysis of the reaction: 1,3-alpha-D-mannosyl-1,2-alpha-D-mannosyl-1,2-alpha-D-mannosyl-D-mannose + UDP-N-acetyl-D-glucosamine = 1,3-alpha-D-mannosyl-1,2-(N-acetyl-alpha-D-glucosaminyl-alpha-D-mannosyl)-1,2-alpha-D-mannosyl-D-mannose + UDP.

globoside alpha-N-acetylgalactosaminyltransferase activity

Catalysis of the reaction: N-acetyl-D-galactosaminyl-(1,3)-D-galactosyl-(1,4)-D-galactosyl-(1,4)-D-glucosylceramide + UDP-N-acetylgalactosamine = N-acetyl-D-galactosaminyl-N-acetyl-D-galactosaminyl-(1,3)-D-galactosyl-(1,4)-D-galactosyl-(1,4)-D-glucosylceramide + UDP.

dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase activity

Catalysis of the reaction: an alpha-D-man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl D-mannosyl phosphate = H+ + alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->3)-alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl phosphate.

glucosaminylgalactosylglucosylceramide beta-galactosyltransferase activity

Catalysis of the reaction: N-acetyl-D-glucosaminyl-(1,3)-D-galactosyl-(1,4)-D-glucosylceramide + UDP-galactose = D-galactosyl-N-acetyl-D-glucosaminyl-(1,3)-D-galactosyl-(1,4)-D-glucosylceramide + UDP.

glucose-fructose oxidoreductase activity

Catalysis of the reaction: D-fructose + D-glucose = D-glucitol + D-glucono-1,5-lactone.

dol-P-Man:Man(6)GlcNAc(2)-PP-Dol alpha-1,2-mannosyltransferase activity

Catalysis of the reaction: alpha-D-man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->3)-alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl D-mannosyl phosphate = H+ + alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-(alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-alpha-D-Man-(1->6))-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-D-GlcNAc-diphosphodolichol + dolichyl phosphate.