Figure 4. Target mRNA drives increased biogenesis of mature miRNA from pre-miRNA.

(a) De novo synthesis of mature miR-122 in the presence of target mRNA is accompanied by a simultaneous drop in pre-miR-122 level. Experimental format is illustrated in the left panel. Tet-ON HEK293 cells were induced for specific time points with doxycycline to synthesize pre-miR-122 from a plasmid with Tet-response element. Cells were harvested after 14 and 24 h, and mature and pre-miR-122 levels quantified. To measure the relative changes at 24 h, values at 14 h are taken as the unit. (b) Target mRNA-induced increase of miRNA levels does not occur due to enhanced stability of a preformed miRNP in the presence of target mRNA. Cells were transfected with 1 μM synthetic pre-miR-122. After 48 h, cells were again transfected with RL-con or RL-3 × bulge-miR-122 plasmids. This was followed by RNA isolation after 24, 48 and 72 h, and mature miR-122 levels quantified to plot the decay rate of mature miR-122. Relative changes in levels of target RNAs over time have been plotted. (c) FH-AGO2 was immunoprecipitated from FH-AGO2-stable HEK293 cells transfected with pre-miR-122 plasmid and FH-AGO2 beads corresponding to ∼2 × 10⁶ cells were incubated with 500 ng in vitro-transcribed RL-con or RL-3 × bulge-miR-122 mRNA in a 20 μl reaction for increasing time. The supernatant was removed and on-bead RISC cleavage assay was subsequently performed to quantify the amount of miR-122 retained with AGO2 post interaction with target mRNA. Paired two-tailed Student's t-tests were used for all comparisons. *P<0.05, **P<0.01 and ***P<0.001. Values plotted are means from at least three biological replicates for a and two for b and c. Error bars represent s.d.