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First published online 16 June 2005
doi: 10.1242/jcs.02404

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Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
⁴ Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
² Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
³ Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of Florida, College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610-0266, USA

View larger version (43K): [in a new window]   Fig. 1. GFP-MBNL1 colocalizes with CUG RNA foci and CAG RNA foci in COSM6 cells. (A) Diagram of repeat-expressing construct. Interrupted 960 CUG or CAG repeats are expressed in a human DMPK minigene containing the genomic segment with exons 11-15 (see Materials and Methods). The DMPK minigene without repeats expresses mRNA identical to the truncated DMPK mRNA, but without repeats in the 3'UTR. (B) CUG and CAG RNA foci detected by FISH. COSM6 cells were transfected with DMPK minigenes containing 960 CTG repeats or 960 CAG repeats. Cy3-labeled (CAG)5 and (CTG)5 antisense PNA probes were used to detect CUG and CAG repeats, respectively. Nuclei were counterstained with DAPI. (C) Expression of GFP-MBNL1 (71 kDa), was detected by western blot analysis using an anti-GFP monoclonal antibody. (D) CUG and CAG foci colocalize with GFP-MBNL1. Cells were co-transfected with a GFP-MBNL1 expression plasmid and DMPK minigenes containing 960 CTG, 960 CAG or 0 repeats. (E) CUG and CAG foci are RNase-sensitive and DNase-insensitive. GFP-MBNL1 was coexpressed with DMPK minigenes containing 960 CTG or 960 CAG repeats. (F) CUG and CAG foci are not detectable using PNA sense probes (CTG)5 and (CAG)5, consistent with the detection of RNA by FISH rather than plasmid DNA. DMPK minigenes containing 960 CTG or 960 CAG repeats were co-transfected with the GFP-MBNL1 expression plasmid. Bars, 10 µM.

View larger version (51K): [in a new window]   Fig. 2. Endogenous MBNL1 colocalizes with CUG and CAG foci in COSM6 cells. (A) Combined FISH/immunofluorescence using Cy5-labeled antisense oligonucleotide probes (Cy5-Oligo), MBNL1 mAb 3A4 (MBNL1) (secondary antibody is Alexa Fluor488) and DAPI staining (DAPI) demonstrating the colocalization of endogenous MBNL1 with CUG or CAG RNA. Magnification x 40. (B) Same as A with magnification x 100. Bars, 10 µM.

View larger version (32K): [in a new window]   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.

View larger version (45K): [in a new window]   Fig. 4. FRAP analysis reveals an immobile fraction of GFP-MBNL1 molecules in both CUG and CAG foci. COSM6 cells were transfected with GFP-MBNL1 alone or with DMPK minigenes containing 960 CTG or CAG repeats. (A) Photobleaching of foci and nucleoplasm in 960 CUG repeat and CAG repeat or 0 repeat plasmids. White circles denote the photobleached regions in the foci and nucleoplasm. Bars, 10 µM. (B) Recovery curves of photobleached foci and nucleoplasm in cells expressing CUG or CAG repeats. Five images were taken pre-bleach, followed by bleaching pulses as indicated on the graph. The half-time of recovery was calculated from at least three independent experiments. The duration of the experiment was 100 seconds. The graph represents the data collected from 42, 34 and 45 cells for the expression 960 CUG repeats, 960 CAG repeats and GFP-MBNL1, respectively. RFI, recovery of fluorescence intensity.

View larger version (36K): [in a new window]   Fig. 5. FRAP analysis of GFP-MBNL1 mobility in DM1 fibroblasts. DM cells (GM3987, GM3132) with CTG repeats of different lengths (500 and 2000 repeats, respectively) were transfected with GFP-MBNL1. (A) GFP-MBNL1 colocalizes with nuclear foci of endogenously expressed CUG repeat RNA. Arrows indicate the location of the foci. CUG-containing RNA was detected using Cy3-labeled (CAG)5 antisense PNA probe, nuclei were counterstained with DAPI. Bar, 10 µm. (B) Photobleaching of foci and nucleoplasm in DM1 fibroblasts (2000 CTG repeats) transfected with GFP-MBNL1. Circles denote the photobleached regions in the foci and nucleoplasm. Bar, 10 µM. (C) Recovery curves of photobleached foci and nucleoplasm in DM1 fibroblasts. GM3987 and GM3132 fibroblasts were transfected with GFP-MBNL1. The graph represents data collected for 37 GM3132 fibroblasts and 31 GM3987 fibroblasts over three independent experiments.

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