Fig. 7
a, b RT-PCR analysis of mRNA levels of pluripotency (a) or early neural commitment (b) markers in Dicer flox/- conditional ES cells [33] 8 days after transduction with a CRE-GFP carrying lentiviral vector. Values are relative to β-actin mRNA expression. Expression levels were normalized to GFP-transduced cells. c Log2 fold change (FC; y-axis) and mean value of miRNA expression (x-axis) between ES and ELA cells. The blue dashed box indicates miRNAs significantly down-regulated (set “D”; mean log2 RPM ≥ 5 and log2 fold change ≤ −1) during priming. d Z-scores of the expression of miRNAs predicted to bind top Ago-released (ΔE < −2; left panel) or top Ago-loaded (ΔE > 2; right panel) mRNAs during the ES–ELA transition. Global miRNA/mRNA binding prediction was performed by the miRVestigator framework, which is designed to take as input a list of co-expressed genes and return the miRNA most likely regulating these genes [82]. miRNA expression was evaluated by small RNA-seq of ES cells cultured in 2i medium and LIF (ES#), ES cells cultured in LIF/serum (ES), ELA cells obtained from ES in 2iL (ELA#), and ELA obtained from ES. Ago enrichment variation provides a good estimation of miRNAs changing at the ES–ELA transition. e Z-scores of expression (RPM) of miRNAs selected in the blue box in c (set “D”), in ES cells cultured in 2i medium and LIF (ES#), ES cells cultured in LIF/serum (ES), ELA cells obtained from ES in 2iL (ELA#), and ELA obtained from ES (ELA). f The distribution of predicted binding affinity, calculated as cumulative Miranda scores of set “D” miRNAs in the 3′ untranslated region (UTR) of genes from the indicated families. The dashed red line marks the median score of a random gene set. g–i Normalized enhanced GFP (EGFP)/DsRed fluorescence ratios as obtained by co-transfection of plasmids and miRNA mimics/controls in ES and ELA cells (see “Methods”). All values, normalized on a DsRed plasmid as an internal control for transfection efficiency, are relative to the ratio displayed by ES cells transfected with an EGFP plasmid devoid of any 3′ UTR. AU arbitrary units.*p = 0.05, **p = 0.01, ***p = 0.001; a, b, REST randomization test; d–f, Wilcoxon test between pairs of conditions (d, e) or between each family and a randomized set of 3′ UTRs (f; see “Methods”); g–i, Student’s t-test