FIG 2.
miRNA-repressed mRNAs are targeted to endosome/MVB postrepression. (A) Experimental setup. (Left) Relative level of RL-3xbulge-miR-122 was quantified in isolated MVB and ER fractions after 24 and 48 h of doxycycline treatment in TET-OFF stable HEK293 cells. (Right) Mean threshold cycle (CT) values of RL-3xbulge-miR-122 quantification have also been plotted to show absolute distribution of mRNA between the two fractions. (B) MVB targeting of an mRNA is dependent on the presence of its cognate miRNA. Relative quantification of RL-3xbulge-miR-122 in the MVB fraction in the presence of miR-122-expressing plasmids is presented. Cells not expressing miR-122 served as a control. (C) MVB targeting of mRNA occurs postrepression. A model of the experiments has been outlined. Relative luciferase expression of RL-3xbulge-miR-122 was plotted after 0, 6, 12, 16, 20, and 24 h of induction. MVB association of RL-3xbulge-miR-122 was measured quantitatively and plotted. Experiments were done in TET-ON stable HEK293 cells. In both cases, 0 h of doxycycline treatment was taken as 1. In all reverse transcriptase qPCR (RT-qPCR) experiments, 18S rRNA served as the endogenous control. For estimation of RNA and proteins, we used cell equivalent amounts in individual reactions. RT-qPCR results from three independent experiments ± standard deviations (SD) are shown, and the values of the control are normalized to 1 (*, P < 0.05; **, P < 0.01; ***, P < 0.001).