Figure 4
Exo-endo trans-splicing of the asAmp and human ACAT1 transcripts. (A) Schematic representation of constructed plasmids (numbers 25-28) containing asAmp or ACAT11-1786 cDNA fused to Flag or Myc tags and empty vectors (numbers 24 and 29) with (pcDNA3) or without (pVk) the Ampr. (B, C) Analysis of trans-spliced RNA products and their translated proteins in intact cells. (B) AC29 cells were transfected with the indicated plasmids. RT-PCR was performed with primer sets asAmp-F/asAmp-R, asAmp-F/A1-R1, A1-F1/A1-R1 to detect the expression of asAmp, trans-spliced asAmp-ACAT1 and ACAT1, respectively. (C) Anti-Flag or anti-Myc antibodies were used in western blot analyses. Calculated MWs of various fusion proteins are shown. Arrows indicate fusion proteins identified by both anti-Flag and anti-Myc antibodies. (D) Schematic representation of in vitro-prepared transcripts as 5′-donor (D1) and 3′-acceptors (A1 and A2). (E) Analysis of trans-spliced RNA in the cell-free system. The cell-free trans-splicing reaction was performed using HeLa cell nuclear extract (NE) and in vitro-prepared 5′-donor and 3′-acceptors depicted in D. Specific primer set asAmp-F/A1-R2 was used in the RT-PCR assay. asAmp-F is the same as in C; A1-R2 is complementary to the sequence of ACAT1 cDNA (upstream of A1-R1); NC, negative-control without templates. (F) DNA sequencing of the RT-PCR product (lane 2) from E. (G) Splicing efficiency of the in vitro trans-splicing between asAmp and ACAT1 RNAs was determined by qRT-PCR assays. The amount of spliced and unspliced RNA products were determined with specific primer sets asAmp-F/A1-R2 (as that used in E) and A1-F2/A1-R2 (complementary to the sequence of ACAT1 cDNA). The percentage of spliced RNAs was calculated by dividing the amount of spliced RNAs by the total RNAs. Means and SD are shown (n = 3). (H) Schematic representation of the exo-endo trans-splicing between asAmp and ACAT1 RNAs and the production of ACAT1 56-kDa/50-kDa isoforms. Human ACAT1 56-kDa isoform is the product of the exo-endo trans-splicing of exogenous asAmp and endogenous ACAT1 transcripts, and the ACAT1 50-kDa isoform is translated by using AUG1397-1399 as the start codon. Underlined characters, AUG start codon; bold characters, AGA and GGC codons at the junction site; italic characters, splice-site signals (5′ SS and 3′ SS).
