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.

unc-10 encodes the C. elegans homolog of the rab3 effector Rim

Liping Wei1, Gayla Hadwiger1, James B. Rand2, Michael L. Nonet1

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.