C. Cai et al. Figure 4. KHDC1A and 1B bind polyU and can multimerize like typical KH domain containing proteins. (A) GST-KHDC1A and GST-KHDC1B bind PolyU agarose. GST alone is shown as a control. (B) HA-KHDC1A coimmunoprecipitates with Flag-KHDC1A (lane 1) or Flag-KHDC1B (lane 3) expressed in 293T cells. HA-KHDC1B also coimmunoprecipitates with Flag-KHDC1B (lane 5). This dem- onstrates that KHDC1 proteins can form homo or hetero multimers. arrest phenotype, and only 5.4% of these embryos survive past MBT. However, coinjection of mCPEB1 RNA reversed this phenotype resulting in a nearly complete rescue of survival (91.6%) (Figure 5, D and E). Curiously, coexpression of mCPEB1 was also able to partially rescue the cell death phenotype caused by KHDC1A expression. Only 1.3% of embryos expressing KHDC1A survived to MBT. However, 55.3% of embryos coinjected with mCPEB1 survived. Note that this rescue was not as efﬁcient as that achieved for KHDC1B. Also, note that expression of the weakly related KH-domain protein ESG1/DPPA5 (Tanaka et al., 2006; Pierre et al., 2007) did not perturb Xenopus development indicating that the KHDC1 phenotypes are speciﬁc and not just the result of expressing high levels of an RNA-binding protein. KHDC1 Proteins Are Part of the CPEB Protein Complex Analysis of the KHDC1 proteins in Xenopus indicate that these proteins may regulate CPEB activity. To further test this hypothesis, we examined whether mCPEB1 and KHDC1 proteins can physically interact. Both Flag- KHDC1A and Flag-KHDC1B were able to coimmunopre- cipitate with HA-mCPEB1 when expressed in Xenopus em- bryos or in 293T cells (Figure 6A). The interaction between Flag-KHDC1A and mCPEB1 in Xenopus appeared to be slightly weaker than the interaction between KHDC1B and mCPEB1. This is consistent with the fact that the KHDC1A phenotype was only partially rescued by mCPEB1 expres- sion (Figure 5E). The interaction between KHDC1B and mCPEB1 was not dependent on RNA (Figure 6B). However, it is the C-terminal RNA-binding domain of mCPEB1 that is required for association (Figure 6, C and D). 3142 KHDC1 Proteins Colocalize with mCPEB1 in Xenopus Embryos, Mammalian Cell Lines, and Mouse Oocytes The interaction between KHDC1 and CPEB was further analyzed by coimmunostaining. Flag-tagged KHDC1 pro- teins and HA-tagged-mCPEB1 were expressed in Xenopus embryos (Figure 7, A and B) or HeLa cells (Figure 7, C or D), and the subcellular distribution of the proteins were examined by indirect immunoﬂuorescence using anti Flag antibodies (FITC, green) and anti HA antibodies (Cy5, magenta). The distribution of endogenous KHDC1 proteins and mCPEB1 were also examined in mouse oocytes (Figure 7, E and F) by indirect immunoﬂuorescence using the pan-KHDC1 antibody (Cy5, magenta) and a mouse mAb against mCPEB1 (FITC, green). In Xenopus, CPEB was reported to be a microtubule binding protein and localizes to the mitotic spindle (Groisman et al., 2000). Coimmunostaining for KHDC1 proteins and mCPEB1 revealed colocalization of these proteins in structures resembling the spindle in Xenopus blastomeres (Figure 7, A and B). KHDC1B expression disrupts cell division, and the spindle shown in Figure 7B is tripolar. In HeLa cells, overexpressed KHDC1A formed large aggregates in the cytoplasm that only partially overlapped with mCPEB1 expression. KHDC1B colo- calized with mCPEB1 in small cytoplasmic granules. However, a portion of KHDC1B was found to accumulate in the nucleus where it did not colocalize with mCPEB1. In germinal vesicle stage oocytes, KHDC1 proteins were found in cytoplasmic granules as previously shown (Figure 3E), and these structures overlapped with the distribution of mCPEB1. The staining intensities for cytoplasmic KHDC1 proteins and mCPEB1 both drop in MI stage oocytes, and the proteins do not colocalize as well as in GV oocytes (Figure 7F). Also, in MI stage oocytes, mCPEB1 staining was enriched on the meiotic spindle similar to its distribution in Xenopus (Figure 7A), but endogenous KHDC1 staining was absent from the spindle. KHDC1A and 1B, despite sharing 65% identity (Figure 1), have distinct subcellular localizations (Figure 7, C and D) and activities (Figure 5B) when ectopically expressed. To test the structural basis for this, we performed domain swap experiments (Supplemental Figure 2). We tested the activity and subcellular localization of a truncated form of KHDC1A lacking the C-terminal extension. Additionally, we fused the KHDC1A extension to the C terminus of KHDC1B. The truncated form of 1A behaved like 1B (Supplemental Figure 2). It caused cell cycle arrest in Xenopus and was able to accumulate in the nucleus when expressed in HeLa cells. Likewise the KHDC1B-fu protein behaved like 1A. This demonstrates that the unique C-terminal extension of 1A represents an important structural motif. KHDC1B Levels and Binding Partners Fluctuate with the Cell Cycle The exact protein components of the cytoplamic polyade- nylation complex depend on the stage of the cell-cycle. For instance, the overall protein levels of the CPEB-inhibitor Maskin were shown to oscillate during the cell-cycle (Gro- isman et al., 2000). Additionally, phosphorylation of CPEB by the M-phase kinase aurora A is proposed to regulate its ability to bind to CPSF and mediate polyadenylation (Gro- isman et al., 2000). To test whether the KHDC1 proteins are cell cycle-regulated, we generated a KHDC1 stable cell lines and examined KHDC1 protein levels at different cell cycle stages. The cells were synchronized with double thymidine at G1/S stage and were released by addition of fresh media. KHDC1A protein levels remained steady throughout the cell cycle (Figure 8A) whereas KHDC1B levels oscillated (Figure 8B) with low levels in S phase and peak levels at the M/G1 transition. Molecular Biology of the Cell