The CPEB RNA-binding domain is required for dimerization. (A) Serial deletions of CPEB used in this study. (B) mRNAs encoding the proteins noted (with HA epitopes at the carboxyl ends) in panel A were injected into oocytes, which was followed by Western blotting analysis. Arrows indicate monomers and asterisks indicate dimers. (C) Quantification of the results from four experiments similar to those presented in panel B. **: P-value < 5 × 10−3; ***: P-value < 5 × 10−4 as determined by paired t-test comparing to the full-length CPEB (FL). CPEB-AA and DD are alanine and phosphomimetic mutations of aurora A phosphorylation sites, respectively. (D) Depiction of dimer and monomer CPEB constructs. The solid line indicates N terminal half of CPEB; the closed circles indicate RRMs and the dashed line indicates ZFs. A curved gray line indicates the linker; a coiled-coil structure indicates the dimerization domain from GCN4, and small A's are alanine mutations. Dimeric CPEBs are (i) CPEB-CPEB: tandem CPEB in single peptide, and (ii) Coil-CPEB: CPEB fused with GCN4 dimerization domain (coiled-coil domain) at the N terminus. Monomeric CPEBs are (i) CPEB-RRM2: CPEB fused with an additional RRM to introduce intramolecular interaction and blocks intermolecular interaction, (ii) ΔRRM: deletion of the dimerization domain characterized in A–C, and (iii) 6A: six-point mutations of cdk1 phosphorylation sites that may change the surface charge and thus the conformation of CPEB to favor monomeric status. (E) Various CPEB proteins expressed in oocytes were assayed by mild denaturing SDS-PAGE (0.5% SDS with no reducing agent in the sample buffer), which shows that the monomeric constructs depicted in D indeed possess lower dimerization potential compared with CPEB WT. In the representative Western blot, arrows indicate monomers and asterisks indicate dimers. The lower panel shows the quantification of three replicates. * indicates statistical significance (**: P < 5 × 10−3; *: P < 0.05, Student's t-test).
