Fig. 9: DAZL positive or negative effects on translation depend on the 3′UTR context and involve an interaction with the CPEB1-dependent adenylation.

a Scheme summarizing the domain organization of the two long and short Oosp1 variant 3′UTRs expressed in mouse oocytes. PAS: Cleave and polyadenylation signal; Dazl: consensus DAZL binding sequence; CPE: CPEB1 binding consensus sequence. The mutated element is marked by an X. b, c Comparison of the effect of DAZL element mutagenesis on the translational activation of Oosp1 reporter during maturation. The Oosp1 long and short wild type (WT, no mutation) and mutant (ΔDazl) reporters were generated by fusing the two 3′UTRs to a YFP sequence and were injected in GV oocytes together with the mCherry constitutive reporter. After overninght recovery, oocytes were transferred to maturation medium and fluorescence recorded by time lapse microscopy. Each experiment was repeated three or more times in different days and oocyte recordings were combined. Each point is the Mean ± SEM of single oocyte measurements. The total number of oocytes used for each construct is reported among brackets. d Effect of single or combined CPE and DAZL binding element mutagenesis on polyadenylation and translational rate of the Oosp1-short 3′UTR reporter. Oligo- or poly-adenylated wild type and mutant reporters were generated as described in the Methods and injected in GV oocytes. After 3 h recovery, fluorescent recordings were monitored by time lapse microscopy in oocytes maintained in GV. Rates of reporter translation were calculated for each injected oocyte at the beginning (0–3 h.) and at the end (7–10 h.) of the incubation (see Methods for details). The average ± SEM from two experiments was calculated for each group of measurements and the statistical significance was calculated by T-test with Welch correction. To estimate the adenylation or deadenylation of the reporter, the ratio between 0–3 h and 7–10 h was calculated for each oocyte and the log2 average reported below the measurements (Upper panel: unpaired two tailed t-test, WT p = 0.6985, ΔCPE p < 0.0001, ΔDazl p = 0.3536, ΔCPE + ΔDazl p < 0.0001; Lower panel: unpaired two tailed t-test, WT p < 0.0001, ΔCPE p < 0.0001, ΔDazl p < 0.0001, ΔCPE + ΔDazl p = 0.0912). e Consequences of single or combined mutation of the CPE and DAZL element on the translation of the Oosp1-short reporter during oocyte maturation. The experimental design is as in c. Each point is the mean ± SEM of single oocyte measurements from three independent experiments. The total number of oocyte used in the experiment is reported among brackets.