Biochemical Characterization of Pumilio1 and Pumilio2 in Xenopus Oocytes

Ryoma Ota,Tomoya Kotani,Masakane Yamashita

doi:10.1074/jbc.m110.155523

Abstract

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.

Highlights

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Co-IP experiments revealed that both Pum1 and Pum2 interact with other key proteins responsible for cytoplasmic polyadenylation and translational control, including cytoplasmic polyadenylation element-binding protein (CPEB), Maskin, Symplekin, CPSF, PARN, GLD2, and Deleted in Azoospermia-like protein (DAZL), in immature oocytes in an RNAindependent manner (Figs. 4, D–I, and 5, A–H) but that Pum1 and Pum2 do not form a complex (Fig. 4, C–E)
These findings indicate that oocytes have at least two types of protein complex that act as a machinery for controlling translation of mRNAs in oocytes; one type contains Pum1 and the other contains Pum2, both of which include CPEB, Maskin, Symplekin, CPSF, PARN, GLD2, and DAZL as common components

Summary

Introduction

Society for the Promotion of Science for Young Scientists Injection of anti-Pum1 antibody or overexpression of Pum1 protein has a significant impact on the timing of oocyte maturation and cyclin B1 synthesis suggesting the involvement of Pum1 in translational control of cyclin B1 mRNA [10].

Results

Conclusion