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. 2011 Jan 28;286(4):2853-63.
doi: 10.1074/jbc.M110.155523. Epub 2010 Nov 23.

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes

Ryoma Ota  1 , Tomoya Kotani, Masakane Yamashita
Affiliations

Biochemical characterization of Pumilio1 and Pumilio2 in Xenopus oocytes

Ryoma Ota et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Characterization of antibodies and detection of Pum1 and Pum2 in oocytes. A, E. coli-produced Pum1-His (10 ng) (lanes 1, 3, and 5) and Pum2-His (10 ng) (lanes 2, 4, and 6) were probed with anti-His antibody (lanes 1 and 2), anti-Pum1N antibody (lanes 3 and 4), and anti-Pum2N antibody (lanes 5 and 6). B, anti-Pum1N and anti-Pum2N immunoblots of crude extracts from immature (Im) and mature (M) oocytes. C, phosphorylation of Pum1 and Pum2 accompanied by changes in electrophoretic mobility. Extracts from [γ-32P]ATP-injected immature (Im) and mature (M) oocytes were immunoprecipitated with anti-Pum1 or anti-Pum2 antibody, treated with (+) or without (−) λ-phosphatase (λ-PPase), immunoblotted (IB) with the same antibodies as those used for IP, and subjected to autoradiography (1-month exposure for Pum1 and 1-week exposure for Pum2) to detect the label of 32P (32P). Extracts before IP were also immunoblotted (Initial). D and E, quantification of the protein contents of endogenous Pum1 (D) and Pum2 (E) in one oocyte. One microliter of immature oocyte extracts (Im), which is equivalent to one oocyte, was immunoblotted with anti-Pum1N and anti-Pum2N antibodies with quantified recombinant Pum1-His and Pum2-His. The signal intensity of individual bands of Pum1 and Pum2 on the immunoblots is expressed as arbitrary units. WB, Western blot.
FIGURE 2.
FIGURE 2.
Pum1, Pum2, CPEB, cyclin B1, and MAPK during oocyte maturation. A, immature oocytes were treated with progesterone, collected at intervals of 2 h, extracted, and immunoblotted with antibodies against Pum1, Pum2, CPEB, cyclin B1, cdc2, phospho-MAPK (P-MAPK) and MAPK. Asterisks indicate the time points at which phosphorylation of Pum1 and Pum2 occurs, and an arrowhead indicates the time point at which synthesis of cyclin B1 protein occurs. The progression of oocyte maturation is indicated by the percentage of GVBD. B, oocyte extracts used in A were immunoprecipitated by anti-Pum1N (α-Pum1N-IP) or anti-Pum2N antibody (α-Pum2N-IP) and immunoblotted with antibodies against CPEB, Pum1, and Pum2. Asterisks indicate phosphorylated Pum1 and Pum2. Immunoglobulins (Ig) of the antibody used for IP are also indicated. Three independent experiments were performed using different batches of oocytes, and representative results are shown (for results of the remaining two experiments, see supplemental Fig. S3). C, quantification of the contents of CPEB in crude oocyte extracts (Total CPEB), anti-Pum1N immunoprecipitates (Pum1-associated CPEB), and anti-Pum2N immunoprecipitates (Pum2-associated CPEB). The signal intensity of individual bands of CPEB, Pum1, and Pum2 on immunoblots was measured by using ImageJ software. Because the efficiency of IP was not constant, the value of Pum1- or Pum2-associated CPEB was divided by that of Pum1 or Pum2 in each immunoprecipitate to standardize the values. The content of CPEB at each time point is expressed as a value relative to that at time 0. Data are shown as means ± S.E. for three independent experiments using different batches of oocytes (A and B and supplemental Fig. S3). The timings of Pum1 phosphorylation, Pum2 phosphorylation, and cyclin B1 protein synthesis are also indicated in the figure.
FIGURE 3.
FIGURE 3.
Acceleration of oocyte maturation by anti-Pumilio antibodies. Oocytes were injected with buffer, anti-GST, anti-Pum1N, or anti-Pum2N antibody. The injected oocytes were incubated overnight and induced to mature with progesterone. A and D, time course of GVBD in oocytes injected with buffer, anti-GST, anti-Pum1N (A), or anti-Pum2N antibody (D). Representative results are shown. B and E, percentage of GVBD in the control buffer- or GST-injected oocytes and in the anti-Pum1N (B) or anti-Pum2N (E) antibody-injected oocytes. Values are means ± S.E. of three independent experiments. To clearly show the difference in the kinetics of GVBD, the experiments were stopped when the control oocytes (GST-injected oocytes) had undergone ∼25% GVBD. Both Pum1N antibody and Pum2N antibody accelerated progesterone-induced GVBD significantly (p < 0.05, Student's t test). C and F, immunoblots of extracts from oocytes injected with buffer, anti-GST, anti-Pum1N (C), or anti-Pum2N (F) antibody, indicating expression patterns of cyclin B1, cdc2, and CPEB during oocyte maturation. Note that the synthesis of cyclin B1 and the phosphorylation and destruction of CPEB are initiated at earlier timing in oocytes injected with anti-Pum1 or anti-Pum2 antibody than in oocytes injected with buffer or anti-GST antibody.
FIGURE 4.
FIGURE 4.
Interaction of Pumilio with mRNAs and other proteins. A, oocytes overexpressing GST-Pum1 were subjected to GST pulldown (GST-PD) followed by RT-PCR to detect mRNAs indicated in the figure. As controls, oocytes injected with distilled water (DW) or GST mRNA were also examined. RT-PCR was also carried out using total RNAs before GST pulldown (Initial, RNAs corresponding to 5% of those subjected to GST pulldown were used) to confirm the presence of indicated mRNAs in the initial samples. Lower figure shows results of an experiment to verify the specific precipitation of GST and GST-Pum1 by GST pulldown. B, immature (Im) and mature (M) oocyte extracts were immunoprecipitated with anti-CPEB (α-CPEB-IP) or anti-Pum1N (α-Pum1N-IP) antibody or without an antibody (Control IP), and the resulting precipitates were subjected to RT-PCR to detect mRNAs indicated in the figure. RT-PCR was also carried out using samples before IP (Initial) to confirm the presence of indicated mRNAs in the initial samples. C, immature oocyte extracts incubated with anti-Pum1N (α-Pum1N-IP) or anti-Pum2N antibody (α-Pum2N-IP) or without an antibody (Control IP) were precipitated with protein A- or protein G-Sepharose beads and immunoblotted with anti-Pum1N antibody (left) and anti-Pum2N antibody (right). Extracts before IP were also immunoblotted to confirm the presence of Pum1 and Pum2 (Initial). D, immature oocytes extracts were treated with (+) or without (−) RNase A, incubated in the presence (+) or absence (−) of anti-Pum1N antibody (Ab), and subjected to IP followed by immunoblotting with antibodies against Pum1, Pum2, CPSF100, CPEB, and DAZL. E, similar experiments to those in D using anti-Pum2 antibody for IP. Immunoglobulins (Ig) of the antibody used for IP are indicated. F, similar experiments to those in D using anti-Symplekin antibody for IP and Symplekin and Pum1 and Pum2 antibodies for immunoblotting. G, similar experiments to those in F using anti-Maskin antibody for IP. H, similar experiments to those in D using anti-CPEB antibody for IP and CPEB, DAZL, Pum1, and Pum2 antibodies for immunoblotting. I, similar experiments to those in H using anti-DAZL antibody for IP. Indistinct bands found in anti-Pum2 immunoblots of anti-DAZL immunoprecipitates are derived from immunoglobulins of the antibody used for IP but not from Pum2. The absence of Pum1 and Pum2 in the anti-DAZL immunoprecipitates is probably due to the failure of antibody in accessing the epitope that was buried in the Pumilio-DAZL complex.
FIGURE 5.
FIGURE 5.
Interaction of Pumilio with PARN and GLD2. A, oocytes were injected with mRNAs encoding GST-Pum1 or T7-tagged GST and incubated with or without progesterone to induce maturation. Extracts from immature (Im) and mature (M) oocytes were incubated with GSH-Sepharose for GST pulldown assay (GST-PD) or incubated in the presence (+) or absence (−) of anti-PARN antibody for IP (α-PARN-IP). The resulting precipitates were immunoblotted with antibodies (Ab) against Pum1, PARN, and T7 tag (for T7-GST in GST-PD). Extracts before IP were also immunoblotted (Initial). B, similar experiments to those in A using oocytes overexpressing GST-Pum2. C, oocyte extracts used in A were treated with (+) or without (−) RNase A and incubated in the presence (+) or absence (−) of anti-PARN antibody for IP. The resulting precipitates were immunoblotted with antibodies against Pum1 and PARN. Extracts before IP were also immunoblotted (Initial). D, similar experiments to those in C using oocyte extracts used in B. E, oocytes were injected with mRNAs encoding T7-GLD2 and GST-Pum1 and incubated with or without progesterone to induce maturation. Oocytes injected with GST mRNA were used as a control. Extracts from immature (Im) and mature (M) oocytes were subjected to GST pulldown assay (GST-PD) or incubated in the presence (+) or absence (−) of anti-T7 antibody for IP (α-T7-IP). The resulting precipitates were immunoblotted with antibodies against Pum1, T7 tag (for T7-GLD2 and T7-GST in GST-PD), and GLD2 (for T7-GLD2 in α-T7-IP to avoid visualization of the antibody used for IP). F, similar experiments to those in E using oocytes overexpressing GST-Pum2. G, oocyte extracts used in E were treated with (+) or without (−) RNase A and incubated in the presence (+) or absence (−) of anti-T7 antibody for IP. The resulting precipitates were immunoblotted with antibodies against Pum1 and T7 tag (for T7-GLD2). H, similar experiments to those in G using oocyte extracts used in F. I, immature (Im) and mature (M) oocyte extracts were incubated with anti-CPEB (α-CPEB-IP), anti-Pum1N (α-Pum1N-IP), or anti-PARN antibody (α-PARN-IP) or incubated without an antibody (Control IP). Following IP, the samples were subjected to RT-PCR to detect cyclin B1 and β-actin mRNAs. Samples before IP (Initial, RNAs corresponding to 5% of those subjected to IP were used) were also analyzed to confirm the presence of cyclin B1 and β-actin mRNAs. J, oocytes overexpressing T7-GLD2 were treated with or without progesterone to induce maturation. Immature (Im) and mature (M) oocyte extracts were incubated with anti-T7 antibody (α-T7(GLD2)-IP) to precipitate T7-GLD2 or incubated without an antibody as a control (Control IP). Samples were subjected to RT-PCR to detect cyclin B1 and β-actin mRNAs.
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