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Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes
Ryoma Ota1, Tomoya Kotani, Masakane Yamashita
1Laboratory of Reproductive and Developmental Biology, Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
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.
Insights
Precise control of mRNA translation during oocyte maturation involves Pumilio1 (Pum1) and Pumilio2 (Pum2). Inhibiting Pum1 or Pum2 accelerates key maturation events, revealing their regulatory roles in dormant mRNA translation.
Area of Science:
- Developmental Biology
- Molecular Biology
- Cell Biology
Background:
- Oocyte maturation relies on precise translational control of stored mRNAs.
- Pumilio1 (Pum1) and Pumilio2 (Pum2) are known regulators of mRNA translation in Xenopus oocytes.
- Pum1 interacts with CPEB for cyclin B1 mRNA control, while Pum2 interacts with DAZL for RINGO mRNA control.
Purpose of the Study:
- To biochemically characterize Pum1 and Pum2 in Xenopus oocytes.
- To investigate the interactions of Pum1 and Pum2 with other translational regulators.
- To elucidate the roles of Pum1 and Pum2 in controlling dormant mRNA translation during oocyte maturation.
Main Methods:
- Production of specific antibodies for Pum1 and Pum2.
- Co-immunoprecipitation assays to study protein interactions.
- Antibody injection experiments to assess functional impact on oocyte maturation.
- Analysis of protein phosphorylation and mRNA-protein interactions.
Main Results:
- Pum1 and Pum2 bind to CPEB and DAZL, respectively, in immature oocytes.
- Pum1 and Pum2 do not directly interact with each other.
- Antibodies against Pum1 or Pum2 accelerated oocyte maturation, CPEB phosphorylation, and cyclin B1 translation.
- Pum1 phosphorylation correlates with CPEB dissociation and cyclin B1 mRNA translational activation.
- Pum2 phosphorylation precedes Pum1 phosphorylation.
Conclusions:
- Pum1 and Pum2 play distinct but coordinated roles in regulating dormant mRNA translation during oocyte maturation.
- The phosphorylation status of Pum1 and Pum2 is critical for translational control and oocyte maturation.
- Pum1 remains associated with cyclin B1 mRNA even after translational activation.
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