Figure 3. Effect of target mRNA concentration on substrate-dependent miRNA increase in human cells.

(a) Amount of mature miR-122 formed per unit of target mRNA in HEK293 cells transfected with pmiR-122 and respective reporter plasmids. Values were calculated by normalizing the amount of mature miR-122 against the amount of respective target mRNA level and plotted. (b) Effect of increasing concentration of target mRNA on mature miRNA levels. HEK293 cells expressing pre-miR-122 were transfected with increasing amounts of in vitro-transcribed mRNA (RL-con or RL-3 × bulge-miR-122) and mature miR-122 and pre-miR-122 levels were quantified 6 h post transfection. In the left panel, changes in relative level of mature miR-122 has been plotted for experiments done with RL-con or RL-3 × bulge-miR-122. Relative change of mature and pre-miR-122 in the presence of different amounts of RL-3 × bulge-miR-122 was plotted (right panel). Values obtained with 100 ng of transcript to transfect 2 × 10⁵ cells were considered as 1. (c) IRE-RL-3 × bulge-miR-122 mRNA with Ferritin IRE element in 5′-UTR is schematically depicted. (d) Cells transfected with pre-miR-122 and IRE-RL-3 × bulge-miR-122 were split 24 h post transfection and iron chelator DFMO (100 μM) or Fe²⁺ source Hemin (50 μM) was added after an additional 6 h. Cells were harvested after 16 h post Hemin or DFMO addition for analysis. Polysomal enrichment of IRE-RL-3 × bulge-miR-122 was estimated by normalizing polysomal mRNA content by total mRNA level. (e) Target mRNA and mature miR-122 level were measured in cells treated with either Hemin or DFMO. Paired two-tailed Student's t-tests were used for all comparisons. *P<0.05, **P<0.01 and ***P<0.001. In a–e, error bars represent s.d. (n⩾3).