5'-UTR and ORF elements, as well as the 3'-UTR regulate the translation of Cyclin

Mammalian oocyte maturation is wholly dependent on the translation of accumulated maternal transcripts. Therefore, measuring the translation of specific genes, especially Ccnb1 and Ccnb2, which are key regulators of the oocyte cell cycle in mice, is essential to monitor oocyte cell cycle progression. For this purpose, almost all previous research has used a reporter construct containing the 3'-untranslated region (UTR) of Ccnb. It is based on the concept that the 3'-UTR is the main modulator of translation. Here, we investigated the expression pattern of Renilla luciferase (RL) reporters combining the 5'-UTR and/or open reading frame (ORF) as well as the 3'-UTR (RL-3', 5'-RL-3', RL-ORF-3', and 5'-RL-ORF-3') of Ccnb1 and Ccnb2 in somatic cells and mouse oocytes. The addition of the 5'-UTR and/or ORF of Ccnb altered the expression of the RL-3' reporter in HEK293T cells and mouse oocytes. The ORF tended to suppress RL expression, whereas the 5'-UTR enhanced the expression in most cases. The increased rate in expression was the highest when only the 3'-UTR of Ccnb1 (RL-3') was used, whereas the 5'-RL-ORF-3' reporter showed a relatively lower increase during oocyte maturation. For Ccnb2, the RL-ORF-3' reporter showed the largest increase, and other reporters exhibited a similar increase in expression during oocyte maturation. Results show that the expression of these genes is modulated not only by the 3'-UTR but also by the 5'-UTR and ORF. Therefore, special caution should be taken when using only the 3'-UTR to monitor the expression of specific genes.