KHDC1B Interacts with CPEB Figure 7. KHDC1 proteins colocalize with mCPEB1 in Xenopus embryos, mammalian cells, and mouse oocytes. 1 ng of HA-mCpeb1 RNA was injected together with (A) 1 ng of Flag-Khdc1a or (B) 1 ng of Flag-Khdc1b mRNA into one cell of two-cell stage Xenopus em- bryos. Embryos were allowed to develop to stage 6, and protein localization was exam- ined by immunostaining for the Flag (FITC, green) and HA (Cy5, magenta) tags. Images are single optical sections taken via confocal and are representative of the expression pat- tern seen in the majority of cells. A single blastomere from an injected region is shown. mCPEB1 and KHDC1 proteins accumulated around the spindle in dividing cells. Note the tripolar spindle structure resulting from KHDC1B expression. Scale bar in A 10 m. Scale bar in B 14 m. (C) Flag-KHDC1A and HA-mCPEB1 were coexpressed in HeLa cells and protein localization was detected as above. Scale bar 6 m. (D) Flag-KHDC1B and HA-mCPEB1 were coexpressed in HeLa cells, and their distribution was detected by immunostaining. Scale bar 10 m. (E) Im- munostaining using the pan-KHDC1 anti- body (Cy5, magenta) and a mouse mAb against mCPEB1 (FITC, green) reveal that the endogenous proteins colocalize in GV oo- cytes. Scale bar 40 m. (F) KHDC1 and mCPEB1 also partially colocalize in MI stage oocytes. Note that mCPEB1 is enriched on the spindle, but endogenous KHDC1 proteins are not. Scale bar 40 m. expressed in HeLa cells (Figure 7). It appears that the C-termi- nal extension of KHDC1A blocks nuclear localization (Supple- mental Figure 2) either through tethering the protein in the cytoplasm or by facilitating nuclear export. We favor a tether- ing mechanism as the C-terminal extension does not contain a known export sequence. Instead, there is a potential transmem- brane motif that may anchor the protein to intracellular mem- brane compartments such as the ER or golgi. In the cytoplasm of oocytes, KHDC1 proteins were enriched in cytoplasmic granules, in the cortex of oocytes, and within the perinuclear region. The pattern of KHDC1 protein localization underwent subtle shifts during oocyte Vol. 21, September 15, 2010 maturation with more KHDC1 localized to the perinuclear region during GVBD, MII stages, and the ﬁrst embryonic cell division. This may hint at a role of KHDC1 proteins in regulating progression through the meiotic cell cycle. KH domain proteins can exhibit both RNA and ssDNA binding and participate in many aspects of nucleic acid biology including transcription, DNA repair, splicing, translation, and RNA stability (Valverde et al., 2008). We have demonstrated that both KHDC1A and KHDC1B have nucleic acid binding activity despite having a variant KH domain lacking the GXXG motif. This is consistent with previous studies on the nucleic acid binding activity 3145