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Fig. 3. CUG repeats, but not CAG repeats alter the splicing of cTNT and IR minigene pre-mRNAs. (A,B) Splicing of cTNT (A) or IR (B) minigene pre-mRNAs coexpressed with DMPK constructs containing 960 CUG repeats, 960 CAG repeats or 0 repeats in COSM6 cells. Exon inclusion was assayed by RT-PCR. The percent exon inclusion was calculated as [(mRNA + exon) ÷ (mRNA – exon + mRNA + exon)] x100. Results are from at least three independent experiments. (C) Quantitation of CUG and CAG nuclear foci by FISH analysis. The number of foci per cell is indicated. The results are the average of three independent transfections. (D) S1 nuclease protection analysis of repeat RNA expression in cells transfected with 0, 100 or 300 ng of repeat-expressing plasmid. Top: diagram of the DMPK S1 probe. The 567-bp probe end-labeled with ³²P hybridizes to the 3' UTR of DMPK to yield a 125 bp protected fragment. The probe detects transcripts expressed from the DMPK minigenes that contain 960 CUG repeats, 960 CAG repeats or 0 repeats. The labeled end is denoted with a *. Bottom: representative results show that cells express equivalent steady-state levels of CUG and CAG repeat RNA. (E) Quantitation of S1 nuclease protection analysis using endogenous ß-actin as an internal standard. S1 probes specific to the 3' UTR of DMPK or ß-actin were quantitated by phosphoimager analysis and the ratio of DMPK to ß-actin signal was calculated. The results of three independent experiments were averaged. P values comparing CUG and CAG repeat expression at 100 ng and 300 ng were 0.3770 and 0.7539, respectively.

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