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Fig 1. Translation of cyclin E1 during oocyte maturation is CPE-dependent.A. Cyclin E1 mRNA is present throughout oogenesis. RT-qPCR was carried out on total RNA extracted from staged oocytes and eggs. The graph represents the relative amount of cyclin E1 mRNA compared to GAPDH mRNA, with values of each transcript set to 1 for stage I. Representative of 5 progesterone maturation experiments. B. Cyclin E1 protein is not expressed until late in oocyte maturation following the polyadenylation of its mRNA. Stage VI oocytes were incubated with progesterone for the indicated times and the corresponding lysates were analysed by Western blot using cyclin E1 and CPEB antibodies (upper panels). RNA extracted from these oocytes was subjected to RNA-ligation-coupled PCR poly(A) analysis (lower panel). *—non-specific band. C. The cyclin E1 3’UTR contains a cluster of three CPE sequences and an additional one overlapping the hexanucleotide as well as two putative miR-15/16 target sites as indicated. Not to scale. D. CPE sequences repress translation in immature oocytes, and activate translation in eggs. Schematic representation of reporters used. Firefly luciferase reporters containing a control 3’UTR (luc400), the wild-type cyclin E1 3’UTR (WT) or the 3’UTR with mutations in the CPEs (CPE mut) were injected into stage VI oocytes. Renilla luciferase reporter mRNA was co-injected as an internal control. Oocytes were incubated for 24h with or without progesterone and both sets were assayed for luciferase expression. Firefly luciferase levels are expressed as a ratio to Renilla internal control. E. CPE sequences in the cyclin E1 3’UTR direct polyadenylation during oocyte maturation. Radiolabelled RNAs representing the WT 3’-terminal 180 nt of the cyclin E1 3’UTR and the same fragment with mutated CPE sequences were injected into oocytes, and maturation was induced by progesterone. RNA extracted from untreated oocytes and progesterone-matured eggs was analysed by denaturing gel electrophoresis and autoradiography. Image published in: Wilczynska A et al. (2016) © 2016 Wilczynska et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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