FIGURE 3.

General tests of the dual regulatory capacity of miRNA genes. (A) Renilla luciferase sensors containing four antisense matches to either the mature miRNA or star species of a given miRNA gene were tested for their response to a cognate pri-miRNA expression plasmid in HeLa cells. Sensor values were normalized to an internal firefly luciferase transfection control and then represented as the fold repression relative to the sensor level in the presence of a non-cognate miRNA expression plasmid (usually mir-1-2; the control for mir-1-2 was mir-199a-2). We deemed a miRNA expression plasmid to be “dual function” if the mean repression value was at least two standard deviations above 1. Values are derived from two independent sets of quadruplicate transfection experiments performed on different batches of cells; standard deviations are shown. Some miRNA constructs were only capable of repressing the mature strand sensor, but a majority of constructs could repress both mature strand and star sensors. Three genes for which the inferred miRNA* species (based on meta-analysis of published library data) yielded slightly higher repression than their partner miRNA species are segregated to the right. (B) Correlation of ectopic miRNA sensor tests and endogenous cloning ratios. We collected small RNA reads from the 22 loci tested in Figure 2 and this figure and compared their miRNA:miRNA* cloning ratios with their miRNA:miRNA* sensor repression ratios. A rank analysis was performed to group genes with higher cloning ratio (i.e., more asymmetric accumulation of the duplex strands) or lower cloning ratio (i.e., more balanced accumulation of the two strands). The correlation with higher versus lower sensor repression ratios was statistically significant. (C) A linear regression was performed between the miRNA:miRNA* sensor repression ratio and the log₂(miRNA:miRNA*) cloning ratio. The correlation was significant according to both Pearson's tests.