ADAR2-mediated editing on the RNA transcript complementary to pri-miR-214 decreases the levels of pri-miR-214 and the miR-214.

<p>(<b>A</b>) The RNA extracted from Huh-7 cells infected with individual lenti-ADARs as indicated was used for assessment of the levels of <b>endogenous</b> miR-214 (left), pri-miR-214 (middle), and complementary antisense transcript of miR-214 (comp-AS-RNA) (right). (<b>B</b>) The individual lenti-ADARs were infected to Huh-7 cells, which were already transfected with the plasmids <b>exogenously</b> expressing either the pri-miR-214 or the complementary antisense transcript (comp-AS-RNA) as indicated. The RNA was extracted for quantification of the levels of both transcripts accordingly. (<b>C</b>) The Huh-7 cells were transfected with the plasmid expressing the wild-type comp-AS-RNA, or the ones introducing either the single mutation as A(−37)G or double mutations as A(−37/−28)G. The levels of antisense transcript were then determined by qRT–PCR. (<b>D</b>) The Huh-7 cells were not infected (mock), infected with control lenti-si-GFP (si-GFP), or infected with lenti-si-AS-214, which targets the RNA transcript complementary to pri-miR-214, for evaluating the effects on the expression levels of comp-AS-RNA (left), pri-miR-214 (middle), and miR-214 (right). (<b>E</b>) The Huh-7 cells were transfected with the control vector or the plasmid expressing the comp-AS-RNA, for evaluating the effect on the levels of comp-AS-RNA (left), pri-miR-214 (middle), and mature miR-214 (right). (<b>F</b>) The protein lysates extracted from Huh-7 cells uninfected or infected with lenti-si-Luc or lenti-pri-miR214 were analyzed by Western blotting analysis (left). The Huh-7 cells transfected with pGL3-vector or pGL3-Rab15-3′UTR reporter constructs were infected with lenti-si-GFP or lenti-pri-miR214. The effect on reporter activity was illustrated relative to that of pGL3-vector-transfected cells infected with lenti-si-GFP (middle). The protein lysates extracted from Huh-7 cells either uninfected or infected with various lentiviruses as described were processed for Western blotting, probing with antibodies against Rab15, ADAR1, ADAR2, and β-actin (right).