FIG 3 .

Characterization of SFV miRNA biogenesis reveals noncanonical mechanisms. (A) Northern blot analysis of total RNA extracted from HEK293T cells transfected with the indicated miRNA expression vectors and either treated or not treated with the RNAP II inhibitor α-amanitin as indicated. Ethidium bromide-stained LMW RNA is provided as a loading control. (B) Northern blot analysis of total RNA extracted from HEK293T cells transfected with the indicated miRNA expression vectors and Drosha siRNA (siDro) or negative-control siRNA (siNC). Numbers below lanes indicate signal intensity of pre-miRNA relative to siNC lane for each expression vector. Ethidium bromide-stained low-molecular-weight RNA is provided as a loading control. (C) Northern blot analysis of total RNA extracted from HEK293T cells cotransfected with the indicated miRNA expression vectors and either Drosha/DGCR8 expression vectors (D/D) or empty vector (VEC). Numbers below lanes indicate signal intensity of pre-miRNA relative to VEC lane for each expression vector. Ethidium bromide-stained LMW RNA is provided as a loading control. (D) Drosha cleavage assay was performed on FV miRNA candidates and control miRNAs cloned into the 3′ UTR of Renilla luciferase vectors. The indicated miRNA 3′ UTR vectors were cotransfected with control firefly luciferase vector and either Drosha/DGCR8 expression vectors (D/D) or empty vector (Vector). The mean relative luciferase activity ratios, normalized to the results for the control vector Renilla luciferase vector 3′ UTR, are graphed. Error bars represent the SD of three replicates, and the P values were calculated using Student’s t test. (E) Northern blot analysis of total RNA extracted from untransfected DLD1 (M), wild-type DLD1 (WT), or DLD1 DicerEx 5^−/− hypomorph (EX5-) cells transfected with the indicated miRNA expression vectors. Numbers below lanes indicate pre-miRNA signal divided by mature miRNA signal. Ethidium bromide-stained LMW RNA is provided as a loading control.