The human PKD1 variant IVS45-14T > C decreases miR-1225 abundance and biogenesis without altering host gene (PKD1) splicing. (A) Location of the ADPKD variants in intron 45 of PKD1. Capitalized nucleotides are exonic, lower case nucleotides are intron 45. The red line represents the mature 5p miRNA sequence. (B) Radiolabeled RT-PCR (left) and stem–loop RT-PCR (right) of RNA isolated from HEK-293T cells transiently transfected with WT and mutant minigenes to detect PKD1 splicing and miR-1225 abundance, respectively. Samples were quantitated and normalized to GAPDH, miR-16, and sno65. The quantity of PKD1 is shown relative to expression from the WT plasmid using the equation: (PKD1/GAPDH)mutant/(PKD1/GAPDH)wild-type. The quantity of miR-1225 is shown relative to expression from WT using the equation: (miR-1225/miR-16)mutant/(miR-1225/miR-16)wild-type (top right) or (miR-1225/sno65)mutant/(miR-1225/sno65)wild-type (bottom right). Panel (C) refers to a mock transfection with vehicle. Error bars represent SEM, n = 3 (shown as individual points). (*) P < 0.05, one sample T and Wilcoxon test, (***) P < 0.001, one sample T and Wilcoxon test; all other groups were not significantly different than WT.
