Figure 6
Interchromosomal region of human ACAT1 chimeric mRNA is required for the exo-endo trans-splicing. (A) Schematic representation of bicistrons with or without a stable hairpin. Rluc cistron and Fluc cistron are linked by the ACAT1 interchromosomal region (ACAT11243-1396, derived from both chromosomes 7 and 1). A stable hairpin (ΔG = −57 kcal/mol) is included upstream or downstream of the Rluc cistron. Mutated nucleotides at the 3′ SS are underlined. (B) Western blot of bicistronic proteins using anti-Fluc or anti-Rluc antibodies. (C) RT-PCR analysis of bicistronic RNAs. Specific primer sets A1-F2/FL-R and asAmp-F/FL-R were used in the RT-PCR analysis. All the RT-PCR products were further analyzed by DNA sequencing. Mutated nucleotides at the 3′ SS are indicated by underlines (right and top panel). The trans-splicing junction (dashed line), partial sequences of exon Xa, mini-exon Xb and exon 1 are shown (right and bottom panel). (D) The human ACAT1 interchromosomal region is required for the exo-endo trans-splicing event. Number 69 construct contains the ACAT1 interchromosomal region (ACAT11243-1396). Constructs of number 70 (1-1279+d790) and 71 (u45+1290-1396) contain partial regions of human ACAT1cDNA exclusively derived from chromosome 7 and 1, respectively. RT-PCR assays were performed by using specific primer sets asAmp-F/A1-R2 and asAmp-F/A1-R3 with amplification cycles 25, 29 and 33, synchronously. Reverse primer A1-R3 is complementary to d790. d790 or u45, 790 bp downstream or 45 bp upstream of the exonic sequences. (E) Splicing efficiency of the trans-splicing between asAmp and ACAT1 RNAs in transfected cells was determined by qRT-PCR assays. Spliced and unspliced RNAs were determined with specific primer sets asAmp-F/A1-R2 and A1-F2/A1-R2 as those used in Figure 4G. The percentage of spliced RNAs was calculated by dividing the amount of spliced RNA products by the total RNAs. Means and SD are shown (n = 3). (F) Model of the exo-endo trans-splicing (bold arrow) between the recombinant plasmid-derived asAmp transcript (donor) and the interchromosomal trans-spliced human ACAT1 mRNA (acceptor). The pre-mRNA from either chromosome 7 or 1 is not acceptor of this exo-endo trans-splicing (dashed arrow). The region and orientation of the recombinant plasmid-derived fragment and recombined cryptic promoter are diagramed. Bold characters, AGA and GGC codons at junction site; italic characters, splice-site signals.
