FIGURE 2

Effects of shRNA- and miRNA-based vectors on artificial miRNA biogenesis and function. (a) Cartoon of RNAi and RNAi luciferase reporter vectors. "TTTTT" designates the Pol-III termination signal. (b) HEK293 cells were transfected with miGFP and GFP RNAi luciferase reporter expression plasmids to establish baseline silencing levels (dotted line). To assess for disruptions in miGFP biogenesis and function, shSCA1 or miSCA1 competitors were added to the transfections at varying doses of RNAi:target. U6 serves as the promoter-only control. (c) Gene silencing assays were performed by co-transfecting HEK293 cells with SCA1 RNAi and RNAi reporter plasmids at varying doses of RNAi:target. (d,e) Reciprocal experiments evaluating the effects of GFP RNAi competitors (shGFP or miGFP) on miSCA1 activity, in parallel with GFP RNAi efficacy studies. All bar graphs represent mean values SEM (n = 3; ***, ** and NS indicate P < 0.001, P < 0.01, and no significance, P > 0.05, respectively). (f) Northern blot analyses assessing the processing of miGFP in the presence of SCA1 RNAi competitors. Blots were probed for either GFP (top blot-pair) or SCA1 (bottom blot-pair) RNAi transcripts.