Extended Data Fig. 4: Microprocessor aggregation reduces the efficiency of pri-miRNA processing and global miRNA dosage, which leads to the de-repression of lipid metabolic genes.
From: Global miRNA dosage control of embryonic germ layer specification
a, Microprocessor in vitro cleavage assay of mouse pri-mir-125b using whole-cell lysate from wild-type and ΔSL1 mES cells. Microprocessor purified by immunoprecipitation from Flag–DROSHA-293T cells was used as a control. The pri-mir125b without lysate was the CK sample. b, Quantification of pri-miRNA cleavage activity calculated based on the density of pre-miRNA bonds in the assays shown in Fig. 2b and Extended Data Fig. 4a. c, Luciferase reporter in vivo cleavage assay of pri-mir-125b in wild-type, DGCR8−/−, and ΔSL1 mES cells. Data are represented as mean ± s.d. (n = 3, technical replicates). ****P < 0.0001, two-sided Student’s t-test. d, Scatter plot of global miRNA expression based small RNA-seq data in wild-type and ΔSL1 mES cells. The small RNA-seq data were normalized on the basis of spike-in RNAs. Differentially expressed miRNAs are represented by coloured circles, and the number of up- and downregulated miRNAs is shown. FC, fold change; two-sided Student’s t-test. e, Heat map of the expression of common up- or downregulated genes in ΔSL1 and DGCR8−/− mES cells compared to wild-type mES cells. The enrichment of Gene Ontology (GO) terms and the number of genes in each group are shown. Two-sided Student’s t-test. f, Venn diagram of mRNAs with expression changes in ΔSL1 and DGCR8−/− cells compared to wild-type mES cells. Number of genes in each group is shown. Two-sided Student’s t-test. g, GSEA analyses of lipid metabolic gene sets by comparing ΔSL1 and DGCR8−/− cells with wild-type mES cells. NES, normalized enrichment score. P values calculated by GSEA software. FDR, false discovery rate. h, Network of miRNAs and lipid metabolic genes. i, miRNA target sites on PDK4, LCLAT1 and GPCPD1 mRNAs, and luciferase miRNA target reporter assay in wild-type, ΔSL1 and DGCR8−/− mES cells. Data are represented as mean ± s.d. (n = 3, technical replicates). ****P < 0.0001, two-sided Student’s t-test. Mutations introduced into the miRNA target sites on PDK4 and LCLAT1, and the mutation sequences are shown in red font. Exact P values are provided in the Source Data (c, i) and Supplementary Table (d–f). Details of statistics replications are given in ‘Statistics and reproducibility’ in Methods.
