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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: J Cell Physiol. 2015 Apr;230(4):791–801. doi: 10.1002/jcp.24804

Figure 2. The cooperation between RA and Acinus isoforms in the splicing of a weak 5′ splice site is promoter specific.

Figure 2

A-B. Acinus activates the splicing of CMV-tubg1E8-E9 pre-mRNA (A) and Sp1RE-tubg1E8-E9 pre-mRNA (B) containing a weak 5′splice site without cooperation with RA. C. Acinus activates the splicing of PPRE-tubg1E8-E9 pre-mRNA containing a weak 5′ splice site without cooperation with rosiglitazone. D. The relative level of exogenous Acinus-L mRNA and Acinus-S’ mRNA in cells transfected with pRARE-tubg1E8-E9 and pPPRE-tub1E8-E9. E. The relative level of RARE-tubg1E8-E9 mRNA and PPRE-tubg1E8-E9 mRNA in transfected cells. 293A cells were transfected with pCMV-tubg1E8-E9 (A), pSp1RE- tubg1E8-E9 DNA (B), pPPRE- tubg1E8-E9 (C-E) or pRARE-tubg1E8-E9 (D-E) splicing reporter DNA along with the expression vector DNA for RARβ (A, B, D and E) or PPARγ (C-E) and the expression vector DNA for either V5-Acinus-L or V5-Acinus-S’ (A-E). The V5 empty vector DNA (pcDNA3.1nV5DEST) was transfected as a control for pV5-Acinus-L or pV5-Acinus-S’ (A-E). In addition, the internal control Renilla vector DNA (pRL-CMV) was transfected as a normalizer for transfection efficiency (A-E). Twenty-four hr following transfection, cells in Panels A, B, D, and E were treated with ethanol (Eth) or RA (10−6 M) for 24 hr, and cells in Panels C-E were treated with DMSO or 50 μM Rosiglitazone for 24 hr. Total RNA was prepared and used to analyze splicing as described in the legend to Figure 1. In Panel D, RT-qPCR was performed to quantitate the relative mRNA levels of exogenous Acinus-L and Acinus-S’ as described in Figure 1. In Panel E, schematic diagram of RARE-tubg1E8-E9/PPRE-tubg1E8-E9 is shown in the upper panel. Arrows indicate the positions of the primer pairs for RT-qPCR analysis. All mRNA levels are normalized to Renilla mRNA levels. The normalized relative mRNA level of RARE-tubg1E8-E9 in the empty vector control cells treated with ethanol was set to 1. V, V5 empty vector; L, V5-Acinus-L; S’, V5-Acinus-S’; Eth, ethanol; RA, retinoic acid; DMSO, dimethyl sulfoxide; ROS, rosiglitazone. Values represent mean ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t test.