We have previously described a role for
lin-35/Rb,
ubc-18 (a ubiquitin conjugating enzyme), and
pha-1 (a novel protein) in the regulation of pharyngeal morphogenesis (Fay et al., 2003, 2004). To discover the ubiquitin-pathway genes acting downstream of
ubc-18 we have carried out a yeast 2-hybrid screening using the UBC-18 as the bait molecule. Among three putative ubiquitin ligases isolated, one (
ari-1/C27A12.8) enhanced pharyngeal defects in a manner identical to that of
ubc-18 when inactivated by RNAi in various genetic backgrounds. Based on its homology with the fly ariadne protein, we term this gene
ari-1.
ari-1 encodes a protein with an RBR signature motif (two RING finger domains separated by an IBR/DRIL domain), a motif shared by the E3 ligase Parkin. Given the fact that UBC-18 physically interacts with ARI-1 (in both bait and prey configurations) and that RNAi feeding generates identical synthetic phenotypes, we believe that ARI-1 functions downstream UBC-18 to coordinately regulate an early step of pharyngeal morphogenesis with LIN-35 and PHA-1. Current efforts are directed towards studying the expression pattern of several ARI-1::GFP fusion constructs, characterizing a potential
ari-1 deletion mutant, and studying other members of the
ari-1 family in C. elegans. To identify additional ubiquitin pathway components (as well as potential target substrates), a second 2-hybrid screen was carried out using ARI-1 as bait. Along with UBC-18, the ARI-1 bait identified CSN-5.
csn-5 codes a subunit of the COP9 /signalosome complex, which was previously shown to positively regulate E3 ubiquitin ligases, physically interact with SCF-type E3 ubiquitin ligases, and affect protein degradation (Pintard etal., 2003). Inactivation of
csn-5 (and other signalsome components) also enhances the phenotypes of certain mutant combinations, indicating a role for this complex controlling pharyngeal development with ARI-1 and UBC-18. 1. Lu, X., and H.R. Horvitz. Cell 95: 981-91, 1998 2. Fay et al. Genes & Dev. 16: 503-17, 2002 3. Fay et al. Development 130: 3319-3330, 2003 4. Fay et al. Dev. Biol. 271:11-25, 2004 5. Aguilera et al. Genetics 155: 1231-44, 2000 6. Pintard et al. Current Biology, 13: 911-921, 2003