Fig. 4 |. ATI-mediated miRNA dosage control mechanism is conserved in human cells and tissues.

a, Sequencing reads mapping to the 5’-end of the DGCR8 gene from RNA-seq and ChIP-seq for histone modifications and transcription factors (TFs) in several different cell lines. Transcription start sites (TSSs) predicted by SwitchGear Genomics from UCSC dataset are shown. CHIP-seq signals for various TFs at this region are shown, and representative TFs are listed in red. The number of TFs binding to the two distinct promoters is shown by Histograms. Reads number of exon junctions (above the arcs) and sequencing abundance (in brackets) are indicated. b, 5’RACE for DGCR8 mRNA in H1299 and K562 cells. Positions of 5’RACE ends and the number of colonies localized upstream and downstream of SL1 structure of annotated DGCR8 gene are shown. c, Imaging of tagged mCherry-DGCR8 and eGFP-DROSHA in living RPE1, HepG2, and K562 cells transfected with corresponding plasmids. d, Box plot and heatmap of the expression of global mature miRNAs in U2OS, H1299, HCT116, K562, and HepG2 cells. The small RNA-seq data were normalized based on spike-in RNAs. Number of miRNAs analyzed is shown and representative miRNAs are listed. ****p<0.0001, Two-tailed Student’s t-test. e, Western blot of DGCR8 and DROSHA in WT and ΔSL1 H1299 cells. Density of the DROSHA and DGCR8 bands were normalized to the ACTINB band to calculate the relative protein ratios. f, Imaging of tagged mCherry-DGCR8 and eGFP-DROSHA expressing in living WT and ΔSL1 H1299 cells transfected with corresponding plasmids. g, q.RT-PCR analysis of mature miRNA expression. Data were normalized to U2 snoRNA. Data are represented as mean +/− SD (n=2). h, Box plot shows the ATI of DGCR8 mRNA appearance in different human tissues based on RNA-seq from GTEX dataset. Tissues mainly derived from embryonic endoderm are highlighted in red font.