Fig 2: CPEs containing 3'-UTR confer in vitro oncoselectivity to engineered transgenes.(a) The upper panel shows representative western blots showing CPEB1 and CPEB4 expression in pancreatic primary fibroblasts, normal cells (HPDE) and tumour cells (RWP-1, MIA PaCa-2 and PANC-1). The lower panel shows quantification of CPEB1 and CPEB4 signals normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (b) Schematic representation of the assessed 3'-UTR. Regulatory sequences are indicated. (c) Quantification of relative d2EGFP/dRFP fluorescence intensity levels in cell lines transduced with the indicated lentiviruses and relative to the mean intensity/content of the d2EGFP/dRFP from cells transduced with Lv-WT 3'-UTR. Data are shown as mean±s.e.m. from three independent biological replicates and were analysed by a linear mixed model fit by REML and a Tukey's contrast test to assess the significance of the differences. **P<0.01, ***P<0.001. cCPE, consensus CPE; ncPCE, nonconsensus CPE.

Fig 3: CPEB1, CPEB2 and CPEB4 are required to coordinate proper polyadenylation dynamics during the cell cycle.HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down and cells carrying a GFP-3'UTR with mutated CPE (CPE-) were induced or not with IPTG. Two days after induction cells were synchronized through double thymidine blockade (DTB). Samples were collected at the indicated time points after DTB release. Total RNA was extracted in order to analyze GFP and RFP poly(A) tail length. Digestion with RNAse H (lane 13) indicates the deadenylated GFP and RFP mRNAs. The positions of the deadenylated and polyadenylated RNAs are indicated. Schematic representation of the transfected constructs is shown. GFP open reading frame in green, RFP open reading frame in red. 3’UTRs, 3’-untranslated region; CPE, cytoplasmic polyadenylation element; CTRL, control.

Fig 4: CPEB1 is required for prophase entry; CPEB2 for metaphase-to-anaphase transition and CPEB4 for cytokinesis.(A) HEK-293 cells stably expressing an IPTG-inducible system for each CPEB knock-down were induced or not with IPTG. Two days after induction, cells were transfected with a plasmid encoding fluorescent histon H2B. After one additional day cells were recorded by live imaging experiments. Images were acquired every 10 minutes and analyzed for mitotic progression. Representative images are shown for individual cells in interphase or during specific phases of mitosis that are readily identified by chromosome condensation state and organisation. (B) Mitotic-stage analysis of 50 cells from S1–S5 Videos as in Fig 2A. On Y axis, each lane represents one cell. On X axis, time is represented as minutes. Colors represent the indicated mitotic phases. Mitotic entry was determined by analyzing the first signs of DNA condensation cross-reinforced with cell-rounding. Mitotic exit was scored based on chromosome segregation at anaphase and DNA decondensation. Sh, short-hairpin; CTRL, control.

Fig 5: Supplemental characterization of TgCPEB4?4 mice.a, Breeding protocol to obtain TgCPEB4?4 mice. Number of mice and percentages of births observed and expected for the four experimental genotypes. b, Kaplan-Meier curve for cumulative survival (continuous line) and probability of developing cranial dysmorphology (dashed line), (n = 44 for control, n = 39 for TgCPEB4?4 mice). c, Evolution of mice body weight (grams). Males (continuous line), n = 25 controls, n = 9 TgCPEB4?4 mice. Females (dashed line), n = 26 control, n = 7 TgCPEB4?4 mice. d, ß-GAL nuclear staining in forebrain neurons from 1.5-month-old controls (n = 6) and TgCPEB4?4 mice (n = 4). Cx, cortex; St, striatum; Hipp, hippocampus; LV, lateral ventricle. Scale bars represent 250 µm. e, St CPEB4 immunohistochemistry shows cytoplasm pattern in control (n = 6), no staining in CPEB4 KO (n = 2) and overexpressing neurons in TgCPEB4?4 mice (n = 4). Scale bars represent 50 µm. f, Protein and g, mRNA expression levels of CPEB1-4 in forebrain at embryonic day 18 (n = 3) and Cx at 1.5 months (n = 6), 1 year (n = 4) and 2 years (n = 5) of control and TgCPEB4?4 mice. For gel source data, see Supplementary Figure 1. a, Pearson’s chi-squared test. c, f, g, Two-sided unpaired t-test. Data are mean ± s.e.m. 95% CIs. *P < 0.05, **P < 0.01, ***P < 0.001.