Implicating SCF Complexes in Organogenesis in Caenorhabditis elegans

Abstract

Development of the Caenorhabditis elegans foregut (pharynx) is regulated by a network of proteins that includes the Retinoblastoma protein (pRb) ortholog LIN-35; the ubiquitin pathway components UBC-18 and ARI-1; and PHA-1, a cytoplasmic protein. Loss of pha-1 activity impairs pharyngeal development and body morphogenesis, leading to embryonic arrest. We have used a genetic suppressor approach to dissect this complex pathway. The lethality of pha-1 mutants is suppressed by loss-of-function mutations in sup-35/ztf-21 and sup-37/ztf-12, which encode Zn-finger proteins, and by mutations in sup-36. Here we show that sup-36 encodes a divergent Skp1 family member that binds to several F-box proteins and the microtubule-associated protein PLT-1/t. Like SUP-35, SUP-36 levels were negatively regulated by UBC-18-ARI-1. We also found that SUP-35 and SUP-37 physically associated and that SUP-35 could bind microtubules. Thus, SUP-35, SUP-36, and SUP-37 may function within a pathway or complex that includes cytoskeletal components. Additionally, SUP-36 may regulate the subcellular localization of SUP-35 during embryogenesis. We carried out a genome-wide RNAi screen to identify additional regulators of this network and identified 39 genes, most of which are associated with transcriptional regulation. Twenty-three of these genes acted via the LIN-35 pathway. In addition, several S-phase kinaseassociated protein (Skp)1-Cullin-F-Box (SCF) components were identified, further implicating SCF complexes as part of the greater network controlling pharyngeal development.

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development

“…Depletion of skr-10 or skr-17 did not lead to lethality in ubc-18 mutant animals, although these genes have been reported to have synthetic genetic interactions with lin-35 that result in larval lethality (21). We also did not observe genetic interactions with skr-8 or skr-9, which suppress the synthetic lethality of ubc-18; lin-35 animals (19). Based on these findings, we conclude that ubc-18 works with ubc-3, cul-1, and skr-1/2 in pharyngeal development.…”

“…The single SKP1 gene product in humans binds simultaneously to SCF subunits CUL-1 and F-box proteins. SUP-36 was previously found to bind to several F-box proteins (FBXC-20, FBXC-32, and FBXC-53) in yeast two-hybrid (Y2H) experiments (18)(19)(20), supporting the notion that it has SKP1-like properties. To further test the assignment of SUP-36 as a SKP1-like protein, we tested for its ability to interact with Cullins in a Y2H assay, and observed a physical interaction between SUP-36 and CUL-1 (Fig.…”

“…Data are aggregated from three independent experiments. Similar to genetic interactions between ubc-18 and other components of pharynx development, the synthetic interactions between ubc-18 and SCF components ubc-3, skr-1/2, and cand-1 are each suppressed by a sup-36 null allele (19) (Fig. 3A, Table 2, and Fig.…”

“…The candidate genes that we tested included five Cullins (cul-1, -2, -4, -5, -6) and SKP1-related (skr) and F-box genes chosen based on published genetic and physical interactions with members of the pharyngeal attachment pathway (18)(19)(20)(21). Consistent with previous reports (22), we found that only 25% of WT animals grown on cul-1(RNAi) survived to adulthood, which excluded cul-1(RNAi) from our primary screen.…”

“…S3). Based on sequence similarity, sup-36 is predicted to encode a SKP1-like protein (19). The single SKP1 gene product in humans binds simultaneously to SCF subunits CUL-1 and F-box proteins.…”