Cullin/Ring finger complexes are a major class of ubiquitin ligase that facilitate the ubiquitin-mediated proteolysis of substrates. The human Cul2/VCB complex was the first CUL-2/RING finger complex identified. It contains Cul2, Elongin C, Elongin B, Rbx1, and the substrate recognition component (SRC) VHL. VHL has a BC-box motif that is required to interact with Elongin C and B. The CUL-2/VCB complex is conserved in C. elegans. However, the inactivation of VHL-1 does not produce any obvious phenotypes. In contrast, loss of CUL-2 produces a host of phenotypes, including: defects in chromatin condensation that leads to the formation of multiple nuclei, unequal DNA distributions, and DNA bridges in
cul-2 embryos; a delay mitotic timing; defective meiotic progression; a G1 phase arrest, and cytoskeletal extensions/blebbing in the embryo. The discordance between the
cul-2 and
vhl-1 phenotypes suggests that C. elegans CUL-2/RING finger complexes contain other SRCs in addition to VHL-1. In humans, three SRCs have been identified in CUL-2/RING finger complexes and all of these contain BC-box motifs to allow interaction with Elongin B and elongin C. C. elegans CUL-2/RING finger SRCs are therefore also likely to contain BC-box motifs. To identify potential CUL-2/RING finger SRCs, we screened the C. elegans genome for genes that encode BC-box motif proteins and identified 1058. We believe that the degenerate BC-box motif would produce a large number of false positives. To narrow the list of BC-box candidates to test, we employed two criteria: first, genes that produced embryonic or larval arrest phenotypes in large-scale RNAi screens that did not have known biochemical functions; and second, BC-box proteins with a second protein binding domain (to allow the binding of substrates). We identified 13 genes in the first category and 12 genes in the second category. We tested whether the inactivation of these genes by RNAi produced aspects of the
cul-2 mutant phenotype, as this would suggest the possibility that they functioned in particular CUL-2-dependent processes. We observed the following RNAi phenotypes: RNAi of six of the BC-box genes produced embryonic arrest; three produced an L1 larval arrest, and the other 16 genes had no visible RNAi phenotypes. Two of the embryonic arrests produced multi-nuclei and, of these, one appeared to have a failure of chromosome condensation, similar to that observed in
cul-2 mutants. Two of the L1 stage arrest RNAi phenotypes had fewer, larger hypodermal cells with 2n DNA content suggesting a G1 phase arrest, similar to
cul-2 mutants. We are currently testing these three genes for physical interaction with Elongin B and Elongin C, and CUL-2.