BackgroundRNA editing encompasses a post-transcriptional process in which the genomically templated sequence is enzymatically altered and introduces a modified base into the edited transcript. Mammalian C-to-U RNA editing represents a distinct subtype of base modification, whose prototype is intestinal apolipoprotein B mRNA, mediated by the catalytic deaminase Apobec-1. However, the genome-wide identification, tissue-specificity and functional implications of Apobec-1-mediated C-to-U RNA editing remain incompletely explored.ResultsDeep sequencing, data filtering and Sanger-sequence validation of intestinal and hepatic RNA from wild-type and Apobec-1-deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs, all within 3′ untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites are unique to liver. Changes in RNA editing lead to corresponding changes in intestinal mRNA and protein levels for 11 genes. Analysis of RNA editing in vivo following tissue-specific Apobec-1 adenoviral or transgenic Apobec-1 overexpression reveals that a subset of targets identified in wild-type mice are restored in Apobec-1-deficient mouse intestine and liver following Apobec-1 rescue. We find distinctive polysome profiles for several RNA editing targets and demonstrate novel exonic editing sites in nuclear preparations from intestine but not hepatic apolipoprotein B RNA. RNA editing is validated using cell-free extracts from wild-type but not Apobec-1-deficient mice, demonstrating that Apobec-1 is required.ConclusionsThese studies define selective, tissue-specific targets of Apobec-1-dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific.

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC.

Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membranebound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immunemodulatory and metabolic effects aligning with and possibly "priming" classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein dimerizes with the mGR modulating its function. Molecular & Cellular

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD+-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein β (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model.