DAZL and CPEB1 regulate mRNA translation synergistically during oocyte maturation

Abstract

Meiotic progression requires exquisitely coordinated translation of maternal messenger (m)RNA that has accumulated during oocyte growth. A major regulator of this program is the cytoplasmic polyadenylation element binding protein 1 (CPEB1). However, the temporal pattern of translation at different meiotic stages indicates the function of additional RNA binding proteins (RBPs). Here, we report that deleted in azoospermia-like (DAZL) cooperates with CPEB1 to regulate maternal mRNA translation. Using a strategy that monitors ribosome loading onto endogenous mRNAs and a prototypic translation target, we show that ribosome loading is induced in a DAZL- and CPEB1-dependent manner, as the oocyte reenters meiosis. Depletion of the two RBPs from oocytes and mutagenesis of the 3' untranslated regions (UTRs) demonstrate that both RBPs interact with the Tex19.1 3' UTR and cooperate in translation activation of this mRNA. We observed a synergism between DAZL and cytoplasmic polyadenylation elements (CPEs) in the translation pattern of maternal mRNAs when using a genome-wide analysis. Mechanistically, the number of DAZL proteins loaded onto the mRNA and the characteristics of the CPE might define the degree of cooperation between the two RBPs in activating translation and meiotic progression.

Keywords: CPEB; DAZL; Oocyte maturation; RBPs; Translation control.

Fig. 1.

Immunoprecipitation of ribosome-associated maternal mRNAs predicts translation. (A,B) Expression of the HA tag in oocytes. Ovaries expressing the RiboTag under the control of the Zp3 promoter (Zp3-RiboTag; A) or the control construct (B) were stained for HA and VASA (also known as DDX4). Scale bars: 50 µm (A); 200 µm (B). (C) Polysome array signals of transcripts representative of the three different classes of mRNAs during oocyte maturation. Gdf9 (class I, constitutively expressed), Paip2 (class II, repressed) and Tex19.1 (class III, activated). Mean±s.e.m., n=3 independent biological replicates. (D) qPCR analysis of mRNAs recovered using RiboTag RIP for the three transcripts reported in C. Data are expressed as a ratio between the qPCR signal from immunoprecipitations from control (WT) and Zp3-RiboTag oocytes. Mean±range of two independent experiments. (E) Time course of RiboTag RIP of Tex19.1 and Dppa3 during oocyte maturation. Fold enrichment was calculated as 2^−ΔΔCT over the nonspecific (NS) control IgG. Data were normalized against input values. Mean±s.e.m. (n=5 independent experiments and 3 for the controls). (F) Expression of a luciferase reporter under the control of the Tex19.1 3′ UTR throughout oocyte maturation. The reporter luciferase signal (Renilla luciferase, RLuc) was normalized to that of the firefly luciferase (FFLuc) injection control. Mean±s.e.m., n=15 independent experiments. Inset shows immunodetection of TEX19.1 protein during oocyte maturation; the bar graph represents the average intensity±s.e.m. of three different biological replicates. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase of meiosis I; MII, metaphase of meiosis II; TUB, tubulin.

Fig. 2.

Ribosome loading onto Tex19.1 is under CPEB1 and DAZL control. (A) Knockdown of CPEB1 in mouse oocytes decreases ribosome recruitment onto Tex19.1 and CcnB1 mRNAs. Germinal vesicle Zp3-RiboTag oocytes were injected with a morpholino against Cpeb1 (CPEB1-MO), or a control MO (Ctr MO), incubated overnight, matured for 5 h and then analyzed using RiboTag RIP. qPCR analysis was performed for Tex19.1, CcnB1 and Rpl19 as a control. (B) Knockdown of DAZL significantly reduced ribosome loading onto Tex19.1 but showed no effect in CcnB1 or Rpl19 levels. DAZL^+/−-Zp3-RiboTag (heterozygous, Het) and DAZL^+/+-Zp3-RiboTag (wild type, WT) oocytes were injected with control or DAZL-MO. RiboTag RIP precipitates from oocytes collected at 5-h post meiotic re-entry were used for qPCR analysis for Tex19.1, CcnB1 and Rpl19. Fold enrichment in A and B was calculated as 2^−ΔΔCT against the RNAs from RIP using the non-specific IgG control and normalized against input values. *P<0.05, **P<0.01, ***P<0.001 (two-tailed paired t-test). The data are reported as individual experiments (circles) or as mean±s.e.m. of three or four independent experiments (bars).

Fig. 3.

Translation of the Tex19.1 reporter requires both CPEB1 and DAZL. Germinal vesicle oocytes were co-injected with RBP-specific MOs (CPEB1, A; DAZL, B) or control MO (Cont MO), and a luciferase reporter with the 3′ UTR of Tex19.1. After release from the germinal vesicle stage, injected oocytes were collected at different times of maturation, and luciferase activity was measured. Significance was assessed by using two-way ANOVA with Bonferroni correction comparing the reporter accumulation in MO-knockdown and control MO-injected oocytes. (C,D) Time 0 of oocyte maturation from the time courses shown in A and B of groups injected with control MO and MOs specific to each of the two RBPs. Significance was determined by a two-tailed paired t-test. Renilla Luciferase (RLuc) units were normalized to firefly luciferase (FFLuc) activity injected as a control. Each point is the mean±s.e.m. of five or six independent pools of injected oocytes. *P<0.05; ***P<0.001; ns, not statistically significant.

Fig. 4.

Meiotic re-entry is dependent on cytoplasmic polyadenylation. (A) Tex19.1 is polyadenylated upon meiotic re-entry. PAT assay of Tex19.1 from mouse oocytes at different stages of maturation. n=6 independent experiments. (B) CcnB1 is polyadenylated upon meiotic re-entry. PAT assay of CcnB1 from mouse oocytes at different stages of maturation. The experiment was repeated three times, and the mean and range is reported. GV, germinal vesicle; MI, metaphase of meiosis I; MII, metaphase of meiosis II; bp, base pairs. Average±range of three independent experiments. **P<0.01; ns, not statistically significant (one-way ANOVA).

Fig. 5.

CPEB1 has a direct effect on Tex19.1 translation. (A) CPEB1 is bound to Tex19.1. Oocytes were matured in vitro and used for a RIP with CPEB1; qPCR analysis was performed on the immunoprecipitated pellets. Fold enrichment was calculated as 2^−ΔΔCT against the RNAs from RIP with the non-specific IgG control. Data are represented as mean±s.e.m. of three independent experiments, or reporting biological replicates as individual points. (B,C) Germinal vesicle (GV) oocytes were injected with a luciferase reporter containing the wild-type 3′ UTR of Tex19.1 with a non-consensus CPE (WT-ncCPE) or a mutated CPE (ΔCPE; B), or with a mutant 3′ UTR modified to a consensus CPE (cCPE; C) and matured in vitro. Luciferase activity was measured between times 0 and 17 h of oocyte maturation. ****P<0.0001; ns, not statistically significant. Significance was assessed with a two-way ANOVA with Bonferroni correction comparing the progression of the WT-ncCPE with the mutated reporters (cCPE or ΔCPE) during oocyte maturation.

Fig. 6.

DAZL regulates translation of Tex19.1. (A) Multiple DAZL proteins bind to the Tex19.1 3′ UTR simultaneously. HEK293T cells were co-transfected with DAZL–Flag and one of three Tex19.1 luciferase reporters [wild type (WT), mutant of site 1 (Δ1) and mutant of site 2 (Δ2)]. qPCR analysis for Renilla luciferase was performed on the FLAG RIP (α-FLAG) samples. FLAG RIP data were normalized to the negative control IgG RIP. (B) Germinal vesicle oocytes were injected with a luciferase reporter with the 3′ UTR of Tex19.1 containing three putative DAZL-binding sites, or reporters with mutations in the putative DAZL-binding sites. The reporter luciferase signal (Renilla Luciferase, RLuc) was normalized to the firefly luciferase (FFLuc) injection control. (C) Deletion of putative DAZL-binding sites caused a decrease in ribosome loading into a Tex19.1 reporter. Zp3-RiboTag-expressing oocytes were injected with a luciferase reporter containing the endogenous 3′ UTR of Tex19.1 or a reporter with two putative DAZL-binding sites mutated (Δ1+3); oocytes were then matured for 5 h. A RiboTag RIP was performed followed by qPCR analysis for Renilla luciferase. Fold enrichment was calculated as 2^−ΔΔCT against those RNAs from RIP with the non-specific IgG control. Data are reported as mean±s.e.m., and independent biological replicates are also reported as single points. Statistical significances for A and C were calculated with a two-tailed paired t-test, and for B by a two-way ANOVA with Bonferroni correction comparing the progression of the wild type to the mutated reporters during oocyte maturation. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not statistically significant.

Fig. 7.

Synergistic effect between CPEB1 and DAZL. (A) Deletion of one or two DAZL-binding sites (Δ3 or Δ1+3) results in a decrease in CPEB1 binding to a common mRNA target. RIP was performed in germinal vesicle oocytes, followed by a qPCR analysis for Renilla luciferase. Fold enrichment was calculated as 2^−ΔΔCT against those RNAs from RIP with the non-specific IgG control; data are expressed as percentages of the results obtained using the wild-type Tex19.1 3ʹ UTR (WT). Data are reported as mean±s.e.m. with individual points representing each biological replicate. (B) The properties of the CPE determines the effect of DAZL on translation. Oocytes were co-injected with a control MO or a Dazl MO, and a Tex19.1 reporter construct with the endogenous CPE (WT) or a CPE mutated to consensus (cCPE). At the times indicated in the abscissa, oocytes were harvested and luciferase activity measured. Each bar represents the mean±s.e.m. of six to eight independent experiments. **P<0.01 (two-tailed paired t-test). IP, immunoprecipitation.

Fig. 8.

Bioinformatic analysis of the relationship between the number of DAZL-binding and CPE elements and the recruitment of oocyte transcripts to the polysomes. (A) Computational analysis of the polysome array dataset for CPE and DAZL-binding site interactions. The 3′ UTRs of all the present transcripts were scanned for CPE or DAZL-binding putative sites. Transcripts were then subdivided into groups according to the presence and number of putative elements. Samples were binned according to the Log2 fold-change in polysome loading during maturation as derived from the array. Note the progressive shift to the right of transcripts containing DAZL-binding and CPE sites. (B) The cumulative fold-change for transcripts containing no DAZL-binding sites (No Dazl) or one (1 Dazl) or more (>1 Dazl) DAZL-binding elements was related to the presence of non-consensus or consensus CPEs.