Dependence on Drosha and Exportin-5 for mirtron-mediated knockdown. (A,B) HEK cells were transfected with 250 ng of psiCheck2.2 and 500 ng of mirtron/variant and assayed 2 d later. Splicing efficiency of NAD, DMPK-Mirt5, and DMPK-Mirt5US using RT-PCR with eGFP primers flanking the intron (A) or GFP fluorescence (B). (C) Knockdown of luciferases target by NAD, DMPK-Mirt5, DMPK-Mirt5US, an unspliceable construct. (**) p < 0.01 versus NAD. (D) Knockdown of putative targets in Renilla luciferases 3′ UTR by an shRNA targeting a HIV transcript (positive control) or by DMPK-Mirt5 in HEK cells stably transfected with Lentivirus (MOI = 1) carrying either a nonspecific (+Drosha) or Drosha shRNA (−Drosha), induced for 8 d with 5 μg/mL doxycycline and transfected on Day 6 with mirtron/amiRNA and targets. (**) p < 0.01 between +Drosha versus −Drosha. (E) HEK cells were cotransfected with 250 ng of target with or without 500 ng of mirtron (Mirt), 500 ng of pAdvantage (Adv), and 500 ng of exportin-5 (XPO5). pEGFP-NAD was used to maintain the total plasmid levels (1750 ng per well). Cells were harvested 2 d after and assayed for luciferase activity. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 versus control.
