Figure 1. The role of splicing and Rev-dependent export on miRNA silencing.

(A) A schematic of the SV40 promoter, intron and 3′ UTR from which the Renilla luciferase (Rluc) was transcribed in the psiCHECK-2 vector (psiCHECK). “SD” and “SA” indicate the splice donor and splice acceptor sites, respectively. The 3′ UTR has three restriction sites (XhoΙ, PmeΙ and NotΙ). Each let-7 targeting sequence was inserted into the PmeΙ site, and the “SD” or Rev response element (RRE) was inserted into the XhoΙ or NotΙ sites in various combinations. (B) The silencing of the RNA and mutated sequences cloned into the PmeΙ restriction site in the Rluc 3′ UTR in the presence of Rev-HA was assessed. The Rluc activity was normalized to the firefly luciferase activity, and an empty vector C was used as a control. (C) The miRNA-mediated silencing of the spliced Rev-HA-exported RNAs was assessed. “RRE” and “SD” were inserted into the restriction sites (XhoΙ or NotΙ) in the Rluc 3′ UTR. The red arrow points to the vectors that presented altered Rluc activity in the presence of Rev-HA. The red arrowhead points to the Bulge-, 3×Bulge- or Perfect-containing constructs that carry a correctly oriented RRE and were silenced in the presence of Rev-HA. (D) A schematic of the SV40 promoter and intron region and the truncated promoters without an intron. (E) The effects of splicing and the presence of enhancers on miRNA-mediated silencing were analyzed using plasmids containing the SV40 promoter or its truncated versions. The blue arrow points to the Bulge-containing constructs without an intron that carry a correctly oriented RRE and were not silenced in the presence of Rev-HA. For each promoter tested, the empty vector C was used as a control. The Renilla/firefly luciferase value was assessed, and six independent experiments were performed and expressed as the mean ± S.D. as a percentage of the control. ***P<0.001.