Fig 2: Ribosome loading onto Mos, Ccnb1, and Ccnb2 mRNA and translation of reporters. Oocytes from RiboTag transgenic mice were isolated at the GV stage and matured in vitro for the indicated times. Extracts were then subjected to immunoprecipitation with HA antibody or IgG (RiboTag IP) and the recovery of transcripts in the IP pellet monitored by qPCR. The Mos (A), Ccnb1 (B) and Ccnb2 (C) mRNAs recovery in the IP pellet are reported as solid symbols. Each point is the mean ± SEM, N = 3. In parallel, oocytes from wild type mice were collected and injected with cRNAs coding for the Firefly luciferase and each of the reporters containing Renilla luciferase fused to the Mos, Ccnb1 or Ccnb2 3'UTR. After recovery in media with milrinone, oocytes were released from cell cycle arrest by washing and transferring to media without milrinone. Oocytes were collected at the indicated time points and the accumulation of the luciferase reporter was measured (dashed lines). Each point is the mean ± SEM of at least 5 biological replicates. (D) Accumulation of the endogenous Mos, Cyclin B1 and Cyclin B2 proteins during oocyte maturation was monitored using western blot analysis. Extracts from 100 oocytes per lane were resolved on the SDS PAGE and proteins were detected with specific antibodies, respectively. Tubulin levels were monitored as the loading control. The experiment was repeated twice with identical results.

Fig 3: Proposed model for the regulation of translation of Cyclins during mouse oocyte maturation. Translation of endogenous Cyclin mRNAs was monitored by measuring ribosome loading onto the mRNAs or exogenously injected reporter accumulation. CPEB1 phosphorylation and reporter translation is independent of Aurora A kinase. It is instead directly or indirectly dependent on Cdk1 activation. Phosphorylated CPEB1 modulates the translation of Ccnb1 whereas Ccnb2 translation is largely independent of CPEB1. The rate of synthesis, rather than that of degradation, of the two proteins is a major determinant of Cyclin levels and of MPF levels in mouse oocytes.

Fig 4: FOXM1 regulates KIF4A expression. a, b HEK293T cells were transfected with overexpression plasmids for specific isoforms of FOXM1 for 48 h and then harvested for western blotting with indicated antibodies (a) and quantitative real-time PCR (b). c, d HepG2, Huh7, and Sk-Hep1 cells were transfected with pcDNA 3.1-Myc/HisA-vector or pcDNA 3.1-Myc/HisA-FOXM1c plasmids for 48 h and then harvested for western blotting with indicated antibodies (c) and quantitative real-time PCR (d). e HEK293T cells transfected with shRNA-Control or shRNA-FOXM1 constructs for 48 h were harvested for western blotting with indicated antibodies. f, g Huh7, and Hep3B cells were transfected with sh-Control or sh-FOXM1 for 48 h and then harvested for western blotting (f) with indicated antibodies and quantitative real-time PCR (g). CENPF and CCNB1 were used as a positive control. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01

Fig 5: Cdk1 activity is necessary for translational activation after GVBD. (A) Effect of protein kinase inhibitors on the mobility shift of CPEB1. Loading was monitored by measuring the amount of tubulin (lower panel). The density of fast and slow migrating bands of CPEB1 from three independent blots were quantified and reported in the bar graph. N = 3. (B,C,D) Oocytes were injected with a Mos (B) Ccnb1 (C) or Ccnb2 (D) luciferase reporter and exposed to the Cdk inhibitor roscovitine (100 µM) for the entire 8 h incubation or for shorter time intervals reported in the ordinate. All oocytes were harvested at 8 h and the luciferase activity determined. Data are reported as the ratio of Mos, Ccnb1 or Ccnb2 3'UTR Renilla luciferase activity over Firefly luciferase activity used as a control for the injection. Mos: Bars are the mean ± SEM of 4 biological replicates. CcnB1: Bars are the mean ± SEM of 7 biological replicates. CcnB2: Bars are the mean ± range of 2 biological replicates *P < 0.05; **P < 0.01.