Worm Breeder's Gazette 15(2): 22 (February 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Washington University School of Medicine, St. Louis, MO 63110|
|2||Oklahoma Medical Research Foundation, Oklahoma City OK 73104|
unc-10 mutants exhibit a variety of behavioral defects including locomotory, pharyngeal pumping and defecation abnormalities. Futhermore, these mutants are resistant to the acetylcholinesterase inhibitor aldicarb. In concert, these phenotypes suggested that unc-10 could encode a synaptic component. Previous genetic mapping of unc-10 positioned it left of center on chromosome X. However, the gene was only roughly mapped relative to cloned genes. Our interest in a gene encoding a rab3 GTP-binding protein effector which physically mapped in the unc-6 unc-18 interval led us to test if the gene might be mutated in unc-10 animals. pRIM4, a 17.5 kb genomic subclone of C34H12 containing the C. elegans homolog of Rab3 interacting molecule (Rim) rescued the behavioral phenotypes of both unc-10(e102) and unc-10(md330) . unc-10 + pRIM4 trangenic animals also exhibited aldicarb sensitivity comparable to wild type animals. Additionally, we examined ten unc-10 alleles (e102, js244, md293, md330, md1117, md1972, md1995, md2122, ox64 and ox67) for polymorphic lesions using PCR. md330 and md1117 both delete large portions of the coding region of the Rim gene. Specifically, md1117 consists of an 8.6 kb deletion that removes the entire coding region of the gene (500 bp upstream of the ATG to 100 bp downstream of the termination codon). Finally, we refined the map position of unc-10, positioning it between unc-6 and dpy-7; a position that corresponds to the physical location of the Rim gene. Specifically, from unc-10(e102)/unc-6(n102) dpy-7(e88) 6 of 16 Dpy non Unc recombinants carried unc-10 and 6 of 10 Unc non Dpy recombinants carried unc-10. Thus, we conclude that unc-10 encodes the C. elegans homolog of rab3 effector Rim. Rim was isolated by Thomas Sudhof's group in a two-hybrid screen using an activated form of rab3 as bait (Wang et al. 1997. Nature 388:593). Vertebrate Rim binds to activated (GTP-bound forms of Rab3), but not to GDP-Rab3 or other Rab proteins. Most interestingly, Rim is localized to the presynaptic nerve terminal. Structurally, Rim is a very large protein containing a zinc finger domain, a proline rich domain, a PDZ domain, and two C2 domains. The highest conservation between the C. elegans gene and vertebrate Rim occurs in the C2 domains. Similar C2 domains are also found in synaptotagmin (snt-1), unc-13, and rabphilin-3A. Our great interest in the gene stems for its potential use as a novel synaptic marker. Since PDZ domains have been demonstrated to localize components synaptically, we engineered GFP::Rim-PDZ domain constructs. Unfortunately, these fusion proteins containing only the Rim-PDZ domain fail to localize to synaptic regions. Production of antisera and other GFP constructs are in progress. Interestingly, the phenotype of unc-10 Rim mutants are much more severe than those of rab-3 (Nonet et al. 1997. J. Neurosci 17:8061) or aex-3 mutants (a nucleotide exchange factor for rab-3; Iwasaki et al. 1997. Neuron 18:613). This suggests that some Rim functions are probably independent of rab-3 activity. Through deletion of various domains of Rim, we intend to define the functional regions of this protein. For example, is the rab-3 binding domain (zinc finger) required for Rim function? We thank Terese Rakow and Bill Schafer for sharing unc-10 mapping data and Erik Jorgensen for unc-10 alleles.