Fig 2: Pharmacological manipulation of CPEB1 phosphorylation regulates SC activation, muscle regeneration and Pax7+ SC number after regeneration.a, b FACS-isolated SCs were plated down and treated with MK5108 and/or insulin for 36 hours. Cells were also continuously supplied with EdU for EdU incorporation analysis. a 36 hours after MK5108 and/or insulin treatment, SCs were harvested for EdU detection and pCPEB1 immunostaining. Nuclei were stained with DAPI. b Quantification of EdU+ SCs after MK5108 and/or insulin treatment. (n = 4 independent experiments). c Timeline of muscle injury, intramuscular injection of MK5108 and/or insulin, and muscle regeneration study for d–i. d–f Tibialis anterior (TA) muscles were injured and injected with insulin and/or MK5108, then allowed to regenerate. d Histological analysis of the cross-sectioned TA muscles using hematoxylin and eosin (H&E) staining 7 days and 14-days post-injury (DPI). e, f Quantification of the size (in cross-sectional area) of regenerated fibers in d. (n = 4 independent experiments). g–i TA muscles were injured and injected with insulin and/or MK5108, then allowed to regenerate. g Immunostaining for Pax7 and laminin of cross-sectioned muscle fibers 7DPI and 14DPI. h, i Quantification of Pax7+ SCs in g. (n = 3 independent experiments). Data are presented as mean ± SD in panels b, e, f, h, i. The p values calculated by two-tailed unpaired t-test are used for comparing two groups in b, e, f, h, and i, ns not significant. Source data are provided as a Source Data file.

Fig 3: CPEB1 regulates transcripts encoding for proteins involved in transcriptional, translational, and energy metabolism during SC quiescence-to-activation transition.a–d CPEB1-associated genes analysis using RNA immunoprecipitation sequencing (RIP-seq). a Schematic illustration of the workflow of the CPEB1 RIP-seq. b Scatter plot of the genes detected in two independent CPEB1 RIP experiments. Each dot represents one gene. The X- and Y-axis represent the fold change of each CPEB1 RIP experiment by normalizing to IgG controls. (n = 2 independent experiments). c Venn diagram showing the genes significantly enriched after CPEB1 RIP when compared with IgG controls (Fold change (CPEB1-Ab-IP/IgG) > 2, DEseq2, P-adj < 0.05). DESeq294 is a method to perform differential analysis of count data, using shrinkage estimation for dispersions and fold changes. d Bar graph showing percentage of CPE-containing genes in CPEB1-associated genes and IgG-associated genes. e Functional analysis of CPEB1-associated genes in KEGG pathways using g: Profiler99. The g:Profiler uses a hypergeometric test to measure the significance of functional terms in the input gene list. f–h Heatmaps of the percentile rank of representative CPEB1-associated genes involved in f spliceosome, g energy metabolism, and h translational regulation.

Fig 4: CPEB binding to mRNAs activated during maturation is necessary, but not sufficient, for full translational activation (A) Pattern of ribosome loading onto UP mRNAs during meiotic maturation. mRNAs whose translation increased by at least 3-fold from Pro I to Met I in our RiboTag/RNA-Seq dataset are shown. Traces of the 149 mRNAs with the highest activation are in grey and transcripts recovered in the pellet of RNA-IP/RT-qPCR with CPEB1 antibody are in black. * denotes transcripts that are also immunoprecipitated by DAZL antibodies (data under review). (B) RiboTag-IP/RT-qPCR validation of ribosome loading for selected UP candidates. Zp3-CreTRiboTagF/F mice were hormone primed and the oocytes isolated. Oocytes were either maintained in Pro I or matured in vitro for 8 h and collected for downstream RiboTag-IP/RT-qPCR analysis. We quantified several candidates with some of the greatest fold changes in ribosome loading from Pro I to Met I. Dppa3 was used as a reference gene as it is known to be constitutively translated during this time. Data are represented as fold changes in message levels as compared to 0 h. Three biological replicates of 200 oocytes per time point were used and RT-qPCR reactions were run in triplicate. The bars represent the mean ± SEM of three experiments. Statistical significance was evaluated by unpaired, two-tailed t-tests; ****P < 0.0001. (C) The effect of CDK1 inhibition on the translation of Ccnb1 mRNA (UP). Pro I-arrested oocytes were collected and microinjected with oligoadenylated YPet-Ccnb1 3′UTR mRNA along with polyadenylated mCherry mRNA. Oocytes were incubated for 16 h then two groups of oocytes were maintained in Pro I with either cilostamide (empty, black circle) or dinaciclib without cilostamide (blue circle). Another two groups of oocytes were either matured without (solid, black circle) or with dinaciclib added at 2 h after release (red circle). Imaging started 2 h after cilostamide release and lasted for 10 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in three separate experiments. The total number of oocytes analyzed is in parentheses. (D) Translation rates of YPet-CcnB1 and YPet-Ewsr1 are affected by CDK1 inhibition during meiotic maturation. The translation rate for each oocyte was calculated by linear regression of the reporter data (C and Supplementary Figure S14C and D) between 8 and 12 h. Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; ns: not significant; ****P < 0.0001. (E) Detailed analysis of the relationship between mRNAs that are translationally activated during meiotic resumption and the presence of CPEs in the 3′UTR. Pie charts report the percentage of UP mRNAs in Pro I-arrested oocytes that have or lack CPEs in the 3′UTR. (F) CPEB1 is required for efficient translational activation of CcnB1. CPEB1+/+ (black), CPEB1+/− (light red) and CPEB1−/− (red) oocytes were collected, maintained in Pro I, and microinjected with oligoadenylated YPet-CcnB1 mRNA along with polyadenylated mCherry mRNA. After 2.5 h incubation, oocytes were matured and imaged for 10 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in two separate experiments. The total number of oocytes analyzed is in parentheses. (G) Translation rates of the YPet-CcnB1 reporter during oocyte maturation in CPEB1+/+, CPEB1+/− and CPEB1−/− oocytes. The translation rate for each oocyte was calculated by linear regression of the reporter data (F) between 0 and 2 h or 6 and 10 h. Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; ns: not significant; ****P < 0.0001. (H) Accumulation of wild type Oosp1 and mutant Oosp1 YPet reporters during meiotic maturation. Pro I-arrested oocytes were collected and microinjected with oligoadenylated YPet-Oosp1 (circle), YPet-Oosp1(ΔCPE1) (square), YPet-Oosp1(ΔCPE2) (triangle) or YPet-Oosp1(ΔCPE1+2) (diamond) mRNA along with polyadenylated mCherry mRNA. After 16 h of recovery after microinjection, oocytes were allowed to mature, and imaged for 10 h with a sampling frequency of 15 min. Each point is the mean ± SEM of individual oocyte traces obtained in two separate experiments. The total number of oocytes analyzed is in parentheses. (I) Translation rates of wild type Oosp1 and mutant Oosp1 YPet reporters during meiotic maturation. The translation rate for each oocyte was calculated by linear regression of the reporter data (H) between 0 and 2 h or 6 and 10 h (post-GVBD). Mean ± SEM is reported. Statistical significance was evaluated by Kruskal–Wallis test; ns: not significant; ****P < 0.0001.

Fig 5: Decreased CPEB1 expression accelerates meiosis reentry.a, b GV oocytes from young and old mice (a), or GV oocytes from Cpeb1+/+;Zp3Cre mice (CPEB1+/+ oocytes) and Cpeb1fl/+;Zp3Cre mice (CPEB1+/− oocytes) (b) were incubated with cilostamide-free medium and brightfield images were captured every 15 min. The cumulative GVBD times were plotted. Experiments were repeated at least three times, and the data are shown as the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document}± SD. Two-tailed multiple unpaired Student’s t tests were used to evaluate statistical significance at each time point. a P = 0.00032 for 30 min, P = 0.0076 for 45 min, P = 0.016 for 60 min. b P = 0.0036 for 45 min, P = 0.0051 for 60 min, P = 0.019 for 75 min. c–f CDK1 activity were measured using Kinase assays. Young and old GV oocytes (c, d), or CPEB1+/+ and CPEB1+/− GV oocytes (e, f) were incubated with cilostamide-free medium for 45 min. After oocytes were lysed by freezing and thawing, oocytes were incubated with GST-PP1 fragment as a substrate for 30 min. Oocytes were lysed in a sample buffer and used for western blot analysis. T320 PP1 phosphorylation was detected by a phospho-specific antibody (pT320-PP1). The level of total substrate loaded was evaluated by Ponceau S staining (Total PP1). Ten oocytes per lane were loaded for the experiment. c, e A representative of experiments is reported. d, f A graph shows the quantification of two or three independent western blot analyses. The data are shown as the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document}± SD. pT320-PP1/ total PP1 ratios were expressed as fold changes over young controls. Two-tailed unpaired Student’s t tests were used to evaluate statistical significance, d *P = 0.025, f *P = 0.033. Source data are provided as a Source Data file.