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[
West Coast Worm Meeting,
2000]
One of the goals of our lab is to determine the function of UNC-119, a protein involved in axon guidance and outgrowth. In the course of this study we have chosen to look at C27H5.1, a weak homologue of UNC-119 predicted by the C. elegans Genome Sequencing Project. Like UNC-119, C27H5.1 appears to be expressed early in the developing embryo and pan-neuronally in all subsequent stages of development, suggesting that it too plays a role in neural development. Unlike UNC-119, C27H5.1 shows additional expression in pharyngeal and vulval muscles suggesting other roles for this protein. Unfortunately there are no known mutations in C27H5.1. We are attempting to determine the phenotype of a C27H5.1 knockout through both deletion screening and in vivo RNA interference. While C27H5.1 is weakly similar to UNC-119, it is highly conserved (70% identity) with the delta subunit of mammalian rod phosphodiesterase (PDE6d). This protein was first identified in the retina as a subunit of rod phosphodiesterase (PDE). However, it has since been shown to be ubiquitously expressed and to interact with a number of other proteins including Rab13, RPGR, and Arl2. These interactions have not been fully characterized, but two general observations can be made. PDE6d solubilizes some proteins and affects the cGMP or GTP binding properties of others. These data suggest that PDE6d plays a regulatory role through interactions with other proteins. Interestingly, C27H5.1 interacts with and solubilizes PDE in the same manner as PDE6d suggesting functional conservation between these two proteins. To determine the role of the C27H5.1 protein in C. elegans, we are in the process of performing a yeast 2-hybrid screen of a C. elegans cDNA library using C27H5.1 as bait. We are especially curious to see if C27H5.1 interacts with proteins similar to those which interact with PDEd in mammals. As well, we are producing antibodies against C27H5.1 which will allow us to further characterize the function and expression of this protein.
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[
International C. elegans Meeting,
1999]
C27H5.1 is a weak homologue of UNC-119, a gene involved in axon guidance and outgrowth. We first became interested in C27H5.1 because of its similarity to UNC-119. We have since learned that there is a mammalian homologue of C27H5.1; rod phosphodiesterase delta subunit PDE6D. These two proteins are very highly conserved exhibiting 70% identity and 85% similarity. UNC-119 also has a close mammalian homologue; retinal protein HRG4/RRG4. Both C27H5.1 and UNC-119 are pan-neuronal in C. elegans while HRG4 and PDE6D are expressed in the mammalian retina. The conservation of these genes has led us to speculate that they may constitute a new protein family. While UNC-119 has been fairly well characterized, very little is known about C27H5.1. Based upon its conservation with UNC-119 and PDE6D, we suspect that C27H5.1 is playing an important role, perhaps in nervous system development. However, C27H5.1 may play a more general role. Unlike UNC-119, C27H5.1 expression is not limited to the nervous system. There is also expression in pharyngeal and vulval muscle. To determine this role, we have performed RNA interference against C27H5.1. Unfortunately, no phenotype was observed. Subsequently we performed RNAi against GFP under the control of the C27H5.1 promoter. There was no reduction in fluorescence suggesting that the expression of C27H5.1 is not affected by RNAi. We are currently screening for a deletion in C27H5.1. Although we have not yet been successful in determining the C27H5.1 mutant phenotype, studies with its mammalian homologue, PDE6D, suggest a possible role for this protein. PDE6D has been shown to bind to and solubilize rod phosphodiesterase (PDE). Since PDE6D expression is not limited to the retina, PDE d may regulate the membrane binding of a variety of proteins. Interestingly, C27H5.1 also binds human rod phosphodiesterase, suggesting that there is at least some functional conservation between PDE d and C27H5.1.
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[
West Coast Worm Meeting,
1998]
UNC-119 has a homologue expressed in the mammalian retina (HRG4/RRG4). We have noted a second UNC-119 homologue in C. elegans, C27H5.1, predicted from the Genome Sequencing Project. While only weakly similar to UNC-119, this gene is predicted to encode a 159 amino acid protein which is highly conserved throughout a number of other species. In particular, C27H5.1 is 70% identical and 85% similar to the rod phosphodiesterase delta subunit (PDE-delta) in mammalian retina. In turn, PDE-delta has been shown to be highly conserved throughout a number of vertebrates and invertebrates. Interestingly, two of the genes that share homology with C27H5.1, PDE-delta and HRG4, are retina specific while C. elegans has no demonstrated phototransduction ability. Therefore, it would seem that the role of this protein family (and specifically UNC-119/C27H5.1 in worms) is not specific to vision but is more fundamental in nature. The functions of PDE-delta, UNC-119, and HRG4 are known to varying degrees. PDE-delta has been shown to solubilize the rod phosphodiesterase complex, although the function or effect of this is not yet clear. UNC-119 is believed to play a role in development of the nervous system, particularly in axon guidance and growth cone outgrowth, while the function of its mammalian homologue RG4 is unknown. The function of C27H5.1 is not yet known, but based on its conserved relationship with PDE-delta, it is likely that C27H5.1 plays some role in signal transduction. We have identified, and are currently in the process of cloning out, a mutation in this gene. The in vivo expression of a green fluorescent protein: C27H5.1 promoter construct demonstrates that the gene is expressed throughout the nervous system in the same manner as UNC-119. Therefore, there appears to be a new family of proteins that are pan-neuronal in Caenorhabditis (and perhaps in other invertebrates) but retinal in mammals.