Identification of the C. elegansanaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast
© Dong et al; licensee BioMed Central Ltd. 2007
Received: 26 September 2006
Accepted: 20 March 2007
Published: 20 March 2007
RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes.
Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Δ mutant.
These data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.
A major goal in biology is to understand the function of each gene. For many organisms, complete genome sequences are now available. Combined with knockdown techniques such as RNAi or morpholino oligos, genes can be quickly assayed for in vivo function [1, 2]. In C. elegans, genome-wide knockdown of gene activity through RNAi has provided important phenotypic information for thousands of genes [3–8]. Although a useful starting point, much of the phenotypic information lacks detail; for example, many genes are only annotated as essential for viability.
Several studies have carried out additional analyses to identify more precise gene functions. In particular, RNAi videorecording screens have uncovered very detailed defects allowing genes to be grouped into more specific phenotypic classes [4, 7–9]. However, within each class there still exist groups of genes with different functions. Analysing the phenotypes of individual genes in more depth is important for assigning genes to specific functions. Through an interest in embryonic polarity, we investigated the function of B0511.9, previously identified as having embryonic polarity defects in a large-scale RNAi videorecording screen . Through phenotypic analyses, we show that B0511.9 shares functions with components of the cell cycle regulator, the anaphase promoting complex/cyclosome (APC/C).
The APC/C is a complex of 12 subunits in animal cells (13 in yeast) that regulates destruction of key cell cycle regulators at the appropriate times by targeting them for degradation by the 26 S proteasome through its E3 ubiquitin ligase activity (reviewed in [10–12]). In C. elegans, nine of the 12 APC/C subunits have been identified based on sequence analysis [13–17]; Cdc26, Apc7, and Apc13 were not identified. For seven subunits, loss of function using mutants or RNAi causes an arrest at metaphase of meiosis I; for two (apc-5 and apc-10), weak embryonic lethality was seen along with germline maintenance problems consistent with mitotic defects (reviewed in ).
Results and Discussion
B0511.9 is required for the metaphase to anaphase transition of meiosis I
In large scale RNAi videorecording screens, RNAi of B0511.9 was shown to cause different phenotypes: loss of asymmetry in the first cell division or one cell arrest due to failure to pass through meiosis [8, 9]; To understand the role of B0511.9 in cell division, we examined the RNAi phenotype in detail.
Time course of RNAi of B0511.9
Time post RNAi
Dead embryos (n)
Hatched embryos (n)
Phenotype of dead embryos
One cell arrest or multicellular
Predominantly one cell arrest
> 56 hours
One cell arrest
Weak depletion of B0511.9 causes embryonic polarity defects
We next examined the phenotype of embryos laid after shorter maternal RNAi of B0511.9, which should cause a weaker depletion. In wild-type embryos, the first cell division is asymmetric, occurring at 56% embryo length (range of 54–57%, n = 20). In contrast, we found that first division in B0511.9(RNAi) embryos is much more symmetric, occurring on average at 52% embryo length (range of 51–54%, n = 10). This suggests that B0511.9(RNAi) embryos have a defect in embryonic polarity.
In wild-type embryos, polarity is initiated during the first cell cycle, leading to the asymmetric localisation of PAR polarity proteins, with a complex of PAR-3/PAR-6/PKC-3 at the anterior cortex and PAR-1 and PAR-2 at the posterior cortex (reviewed in ). These proteins are required for the posterior displacement of the first mitotic spindle, which leads to an asymmetric cell division. In par mutant embryos, the first division is symmetric instead of asymmetric and the remaining PAR proteins are often mislocalized [19–26].
B0511.9 shows homology to the APC/C subunit Cdc26
There are two alternatively spliced isoforms of B0511.9 inferred from the sequence of ESTs, called B0511.9a and B0511.9b, which are predicted to encode proteins of 175aa and 187aa, respectively . The final intron predicted in Wormbase  is not removed in the ESTs, making the proteins shorter than originally proposed due to an earlier stop codon. Homology searches using Blastp  with standard settings against the non-redundant protein database (NCBI) did not uncover any similarity of B0511.9 to any known protein. However, Blastp searches against the S. cerevisiae protein database revealed homology of the N-terminus of B0511.9 to the N-terminus of Cdc26.
B0511.9 complements a budding yeast cdc26mutant
Our functional and phenotypic assays indicate that B0511.9 encodes the C. elegans APC/C subunit Cdc26 and accordingly we have named it cdc-26. Inhibition of cdc-26 activity leads to the same range of defects as seen after inhibition of other APC/C subunits, namely embryonic polarity defects after weak knockdown, and meiotic metaphase I arrest following strong knockdown. Consistent with the role of yeast Cdc26 in stabilizing the association of Cdc16 with other subunits, a large-scale two-hybrid study in C. elegans showed that CDC-26 binds to APC/C component EMB-27/Cdc16 .
Previous studies had identified C. elegans homologs of 9 of the 12 known human APC/C subunits (reviewed in ). No sequences with significant matches to Cdc26, Apc7, or Apc13 had been found. This study illustrates the power of RNA interference screens coupled with detailed phenotypic analyses in assigning gene function. RNAi embryo videorecording data for hundreds of genes are available [4, 7–9]. In many cases, the defects observed in these movies give insight into the biological process affected. Further study of genes in different phenotypic classes will provide a deeper understanding into the mechanisms of shared cell division processes. For example, careful analyses of these data may lead to the identification of C. elegans Apc7 and Apc13.
RNAi was carried out by feeding  as in  using RNAi feeding clones from [3, 5]. Sequences of the clones are available in Wormbase  as sjj_B0511.9 for B0511.9 and as sjj_F10B5.6 for emb-27. For the time course in Table 1, wild-type N2 L4 hermaphrodites were placed on RNAi feeding plates containing the same bacterial strain at 20°C for 24 hours, then moved to new RNAi feeding plates for the indicated collection times. Embryos laid on each plate were scored the next day for embryo hatching and terminal phenotype. Fertilized embryos were easily distinguished from unfertilized oocytes by their oblong rather than rounded shape and presence of an eggshell, visible in the dissecting microscope. For strong RNAi of B0511.9 in Figure 1, GFP::H2B L4 hermaphrodites were placed on feeding plates for 30 hours at 25°C and then scored by DIC and GFP microscopy; for RNAi of emb-27/Cdc16, RNAi feeding was for 20 hours. For weak RNAi of B0511.9 in Figure 2 and for videorecording analyses, wild-type N2 L4s were placed on feeding plates for 40 hours at 20°C, then embryos dissected and processed for antibody staining or videorecording.
Videorecordings were done as in . Antibody staining was done as in . The PAR-3 antibody was raised in rat using GST-PAR-3 described in  and then affinity purified against the same protein after preclearing the serum of anti-GST antibodies. The PAR-2 antibody was raised in rabbit against N-terminally His-tagged full length PAR-2 and then affinity purified against the His-tagged N-terminus of PAR-2 (amino acids 1–100). Secondary antibodies were from Jackson Immunoresearch.
The cdc26Δ::KanMX4 strain and the corresponding wild-type are in the BY4741 genetic background (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and were obtained from the European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF). The doc1Δ::KanMX4 deletion allele was introduced into the W303 background (MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3-1).
To express C. elegans B0511.9a in yeast, a cDNA in the donor vector pDONR201 was obtained from  and then transferred to the expression destination vector pVV214 using GATEWAY recombinational cloning . The resulting plasmid pJA189 contains an URA3 marker, the yeast 2-micron origin, and expresses B0511.9a from the PGK promoter. Translation starts at the second methionine of the original B0511.9a sequence where the homology with other Cdc26 orthologues begins (see Figure 3). To construct positive controls for the complementation of yeast mutants, yeast CDC26 and DOC1 were cloned into the vectors YCplac33  and pRS426 , respectively. Standard protocols were used to transform yeast and to prepare growth media . To determine the budding index > 200 cells were counted after brief sonication. To measure cellular DNA content cells were stained with propidium iodide and analyzed on a Becton Dickinson FACScan flow cytometer.
Thanks to Richard Durbin, Bruno Fievet, Gino Poulin, and Josana Rodriguez and anonymous reviewers for helpful comments on the manuscript and Ken Kemphues for reagents. Some strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). YD and JA are supported by the Wellcome Trust (054523). Work in the WZ lab is supported by the Max Planck Society.
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