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. 2012 Dec 10;7(12):e51393. doi: 10.1371/journal.pone.0051393

Figure 2. Analysis of Rev-dependent export on RNAs with mutations in splice donor site.

Figure 2

(A) A schematic of the psiCHECK-2 vector (psiCHECK) in which the locations of primers to amplify the intron region or exon region of the Rluc are illustrated. The splice donor and the juxtaposed exon sequences (SD) are shown. The sequence mutated from the psiCHECK vector is shown in red. The p5SD vector was exchanged with the fifth SD (6,722–6,730) of pNL4-3. The pmSD has mutations in the highly conserved SD sequences. (B) The nuclear and cytoplasmic level of G3PDH RNA in HeLa cells was analyzed by RT-qPCR. The cytoplasmic level was set to 100. (C) The nuclear and cytoplasmic level of U1 snRNA. The nuclear level was set to 100. (D) The nuclear and cytoplasmic level of firefly luciferase RNA in which the cytoplasmic level was set to 100. (E) The levels of Rluc RNAs transported into the cytoplasm were analyzed by RT-qPCR. The intron region and exon region were amplified using the primers shown in (A). The RNA levels amplified by primers in the exon region were set to 100. The gray arrowhead and arrow point to the intron-containing Rluc RNA levels in each transfected cell. (F) The let-7-mediated silencing of Rev-HA-exported RNAs from p5SD. The red arrow points to the vectors that presented altered Rluc activity in the presence of Rev-HA. The blue arrow points to the Bulge-containing constructs that carry a correctly oriented RRE and were not silenced in the presence of Rev-HA. (G) The let-7-mediated silencing of Rev-HA-exported RNAs from pmSD. The Renilla/firefly luciferase value was assessed, and the data presented are the mean ± S.D. of the percentage normalized to the empty vector C.