Figure 5

Promoter reporter assay result and two validated miRNA promoters. (a) Luciferase assay results on six identified miRNA promoters plus a control. The y-axis is the ratio between firefly luciferase and Renilla reniformis luciferase activity. Transfection efficiencies are normalized to the expression of a co-transfecting plasmid expressing Renilla reniformis luciferase. Experiments are shown as average plus/minus standard deviation of three independent transfections of three independent experiments. The promoter of miR-122 shows significantly higher activity with respect to the control (fold change approximately 5, P < 0.01). The miR-130a intronic promoter shows an activity about tenfold higher than the control (P < 0.0001). (b) Genomic characteristics of the region up to 5,000 bp upstream of pre-miR-122, including the validated promoter location (CAGE tracks), the RNA-seq read coverage and the conservation score from the UCSC Genome Browser. (c) Genomic characteristics of the 2,500 bp long region upstream of pre-miR-130a including the annotated transcripts of the host gene, the DNase clusters corresponding to open chromatin regions, the H3K4me3 and H3K27Ac histone marks (generally associated with active promoters), the PolII ChIP-seq peaks in different cell lines, the conservation score, the location of both host gene and intronic promoters (CAGE tracks) and the RNA-seq reads. The presence of a DNase cluster, the H3K4me3 and H3K27Ac marks and a second PolII peak in NB4 cells in the region of the identified intronic promoter, are additional evidence for the miR-130a intronic promoter.