Cytoplasmic Polyadenylation Element Binding Protein Deficiency Stimulates PTEN and Stat3 mRNA Translation and Induces Hepatic Insulin Resistance

The cytoplasmic polyadenylation element binding protein CPEB1 (CPEB) regulates germ cell development, synaptic plasticity, and cellular senescence. A microarray analysis of mRNAs regulated by CPEB unexpectedly showed that several encoded proteins are involved in insulin signaling. An investigation of Cpeb1 knockout mice revealed that the expression of two particular negative regulators of insulin action, PTEN and Stat3, were aberrantly increased. Insulin signaling to Akt was attenuated in livers of CPEB–deficient mice, suggesting that they might be defective in regulating glucose homeostasis. Indeed, when the Cpeb1 knockout mice were fed a high-fat diet, their livers became insulin-resistant. Analysis of HepG2 cells, a human liver cell line, depleted of CPEB demonstrated that this protein directly regulates the translation of PTEN and Stat3 mRNAs. Our results show that CPEB regulated translation is a key process involved in insulin signaling

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

CPEB controls the synthesis of Stat3 and PTEN.

<p>(A and B) 3′ UTR sequences of Stat3 and PTEN from human, mouse and cow. The nucleotides in bold represent putative CPEs. (C and D) Western blots of Stat3 and PTEN following CPEB depletion in HepG2 cells. In panels C-H, tubulin served as a negative or input control. (E and F) Quasi-quantitative RT-PCR for Stat3 and PTEN RNAs following CPEB depletion. (G and H) HepG2 cells depleted of CPEB were pulse labeled with ³⁵S-methionine for 15 min followed by Stat3, PTEN and tubulin (as a control) immunoprecipitation and SDS-PAGE analysis. These same proteins were also analyzed by western blots. (I) Representation of Renilla and firefly luciferase RNAs that were electroporated into HepG2 cells. Renilla luciferase RNA, which contained an irrelevant 3′ UTR, served as a normalization control. Firefly luciferase contained the Stat3 or PTEN 3′ UTRs as noted in panels A and B; in some cases, the CPEs in these 3′ UTRs were mutated. (J and K) The firefly and Renilla RNAs noted above were electroporated into HepG2 cells, some of which were depleted of CPEB. Firefly luciferase was normalized to the Renilla luciferase transfection control; luciferase activity derived from all RNAs was then made relative to the control shRNA. The Stat3 and PTEN data were analyzed with ANOVA; p values were 0.009 and 0.005, respectively. The asterisk refers to statistical significance (p<0.05). Data are represented as mean +/− SEM. The firefly and Renilla luciferase RNAs were also analyzed for relative stability by quasi-quantitative RT-PCR; all the RNAs had similar stabilities. At least 3 animals were used for each experiment.</p

Dramatic and widespread changes in insulin signaling molecules in CPEB knockout mice.

<p>(A) Western blot analysis and quantification of IRS1, IRS2, PTEN, PDK1, phospho-Stat3 (S727), total Stat3, Socs3, and tubulin as a loading control from WT and CPEB knockout liver, fat, and muscle. (B) Quantitative RT-PCR analysis of mRNAs encoding IRS1, IRS2, PTEN, PDK1, Stat3, and Socs3 mRNAs from WT and <i>Cpeb1</i> KO liver. Data are represented as mean +/− SEM. At least 3 animals per group were used for the experiment. Asterisks refer to statistical significance at the p<0.05 (*) or p<0.01 (**) levels (Student's t test).</p