Unstructured HSUR1 down-regulates miR-27a levels most effectively. (A) Partial structure models for HSUR1 and its mutants. The colored residues on WT HSUR1 indicate the location of the mutated residues in the mutants. Watson–Crick base pairs are represented by solid lines and wobble base pairs by dotted lines. Predicted base-pairing interactions with miR-27a and the folding energies of predicted hairpins are indicated (units are kcal/mol [k/m]). (B) Northern blot comparing the levels of HSUR1 and miR-27a in marmoset (marm.) T cells transformed with HVS (either WT or HSUR1-deleted [Δ2A]), and in BJAB cells transduced with either WT HSUR1 or BS HSUR1, in which the miR-27 binding site is disrupted. Each lane contained 10 µg total RNA. For quantification, in vitro transcribed HSUR1 and synthetic miR-27a were used in the amounts indicated at the top of the blot (fmol). (C) Northern blot detecting the levels of three miRNAs, HSUR1 and U6 in BJAB cells stably expressing various HSUR1 mutants (at the same levels as shown in B). EV, empty vector. (D) Levels of miR-27a in the BJAB cells expressing the HSUR1 mutants were normalized to the geometric means of miR-16 and miR-20a levels. Averages from four independent experiments with standard deviations are shown. P-values between WT HSUR1 and its mutants were calculated using a Student's t-test; (*), (**), and (***) indicate P-values <0.05, <0.01, and <0.001, respectively.
