Fig. 5

CPEB1 and the CPEB2-4 subfamily target distinct mRNA subsets. A Western blot detection of HA and DDX6 of HA immunoprecipitates from HA-CPEB1-4 overexpressing oocytes (n = 3). The number of oocytes loaded is indicated in parentheses. I, input; E, eluate; NI: not-injected. B Overlap between the CPEB1-4 targets defined from RIP-Seq experiments. Targets are at least fourfold enriched relative to the input (P-adj ≤ 0.05) and twofold relative to the not-injected background control IP (P-adj ≤ 0.05). C Left: CPEB-mRNA enrichment heatmap for targets of at least one CPEB. The enrichment is expressed as the module of the two centered fold changes. The clustering tree was created with the full-linkage method. Right: CPEB1-CPEB2-4 differential enrichment heatmap. Colored genes are enriched at least twofold in one group versus the other (P-adj ≤ 0.05). D RIP-qPCR enrichment (expressed as delta CT) of indicated candidates in the CPEB1 IP relative to CPEB2/3/4 IPs. The candidates are either CPEB1-preferential targets or CPEB2-4-preferential targets, as indicated with dashed lines. Data points represent the mean and standard deviation (n = 3). E Motifs differentially enriched in the 3′ UTRs of targets of any CPEB-, CPEB1-, or CPEB2-4-preferential targets relative to RIP-Seq input minus targets and each group of preferential targets relative to the other, as determined with HOMER. The background used is indicated for the table rows. F AlphaScreen assay of purified CPEB1 and CPEB4 (50 nM) binding to CPE-A or CPE-G oligonucleotides. Error bars represent the standard deviation of the technical replicates (n = 2). The experiment was performed in triplicate. G Gene set enrichment in CPEB1-preferential targets (234 genes), CPEB2-4-preferential targets (414), or non-preferentially regulated targets (shared, 1148) determined with Enrichr. Only ontologies within “pathways” and “ontologies” with significant gene sets are included. Signaling by NOTCH1 (condensed) includes four redundant Reactome 2016 categories. GO MF, CC, and BP refer to molecular function, cellular component, and biological process, respectively. Significance scale: ****P-adj < 0.0001; ***P-adj < 0.001; **P-adj < 0.01; *P-adj < 0.05