Fig. 1.

Translation of Ccnb1 and Ccnb2 mRNAs is differentially regulated during meiotic maturation in mouse oocytes. (A,B) RNA-Seq was performed using mRNA extracts from total cell lysate (total mRNA) or after immunoprecipitation of HA-tagged ribosomes (ribosome-bound mRNA) from oocytes arrested in prophase with cilostamide (time 0) or collected 2, 4, 6 and 8 h after meiotic resumption. Counts per million (CPM) of mapped reads are reported for Ccnb1 (A) and Ccnb2 (B); average CPMs of two independent biological replicates with range are reported. (C) Poly(A) tail lengths of the Ccnb1 and Ccnb2 mRNAs in GV oocytes. The data were mined from PMID: 28792939 and are reported as binned values of up to 80 (A) nucleotides. (D) Rates of translation of Ccnb1 and Ccnb2 mRNA variants in prophase I. Oocytes were injected with a 1:1 mix of oligo-adenylated YFP-(Ccnb2-short, Ccnb2-long, Ccnb1-short or Ccnb1-long) 3′UTR and polyadenylated mCherry. Rate of translation in GV-arrested oocytes were calculated with a 3 h window at a sampling rate of 15 min. Student's t-tests were performed for statistical significance; ***P<0.0001. (E) Translational efficiency of Ccnb1 and Ccnb2 mRNA was calculated by dividing the CPMs of ribosome-bound mRNA by the CPMs of total mRNA. Four biological replicates were used for these calculations. (F) To evaluate the absolute concentration of cyclins in GV-arrested oocytes, western blots were performed using cell lysates and cyclin levels were quantified by interpolating from a standard curve of known concentrations of CCNB1 and CCNB2 recombinant proteins. Calculated concentrations are reported as the mean and s.d. of three independent biological replicates. ns, not significant.