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1.
FIGURE 3.

FIGURE 3. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

Restoration of myotube formation by Mef2D(+)β expression in C2C12-MBNL3 cells. C2C12-MBNL3 cells (A–C), clonal pools of C2C12-MBNL3 cells expressing the indicated Mef2D isoform (D–I), and control C2C12 cells (J–L) were maintained in differentiation media for 0 or 5 days. The cells were fixed and analyzed by immunocytochemistry using an anti-myosin heavy chain mAb MF20 (green). The nuclei were visualized by DAPI staining (blue). Scale bars, 50 μm.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.
2.
FIGURE 1.

FIGURE 1. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

MBNL3 promotes exclusion of β-exon from Mef2A and Mef2D transcript during muscle differentiation. A, a schematic diagram of Mef2 alternative splicing and the relative positions of primers used for RT-PCR are provided. The MADS box and the Mef2 domain comprise the highly conserved DNA-binding and dimerization domains. The remainder of the protein represents the transactivation domain. The alternatively spliced β-exon and two forms of the α-exon are shown. B, total RNA was isolated from control (C2C12-control) and MBNL3 (C2C12-MBNL3) expressing C2C12 cells maintained for 0 or 2 days in differentiation media (DM). Splicing pattern of Mef2A and Mef2D was examined by RT-PCR using primers that can distinguish between the (+)β and (−)β isoforms, as diagrammed in A. The levels of Timm17b were measured to control for variations in input RNA. The data shown are representative of four independent experiments.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.
3.
FIGURE 5.

FIGURE 5. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

MBNL3 directly binds to intron 7 of Mef2D pre-mRNA in vitro. A, the relative positions of uniformly radiolabeled RNA segments of Mef2D pre-mRNA transcribed in vitro for UV cross-linking experiments are shown. MBNL3 bound efficiently to the region indicated by the thick black line in vivo. B, bacterially expressed His-tagged MBNL3 was purified by nickel-agarose affinity chromatography. Purified MBNL3 was incubated with the indicated radiolabeled RNA fragments. Protein-RNA interactions were covalently cross-linked. The resulting stable complexes were separated on SDS-polyacrylamide, and protein bound to the radiolabeled probe was detected by autoradiography (lower panel). An aliquot of each RNA probe is shown (upper panel). The data presented are representative of four independent experiments. C, comparable amounts of purified WT MBNL3 and ZFmt were subjected to SDS-PAGE followed by Western blotting using the anti-MBNL3 mAb P1E7. The positions of molecular mass markers (in kDa) are shown on the left. D, RNA binding of WT and ZFmt MBNL3 proteins to the T5 fragment of Mef2D pre-mRNA was examined by UV cross-linking (n = 7) as described in B.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.
4.
FIGURE 4.

FIGURE 4. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

MBNL3 associates with intron sequences downstream of Mef2D β-exon in vivo. A, schematic diagram of introns and exons surrounding the alternatively spliced Mef2D β-exon is provided. The relative positions of RT-PCR products for different intron primer sets are shown. B, C2C12 cells transiently transfected with TAP-tag MBNL3 expression plasmid (lane 3, 6, 9, and 12) were cross-linked with formaldehyde. Whole cell lysates were prepared, sonicated, and precipitated with streptavidin-agarose. The presence of Mef2D intron sequences in TAP-MBNL3-bound RNAs was determined by RT-PCR. The cells transfected with empty control vector (lanes 2, 5, 8, and 11) were subjected to the same protocol in parallel. Results representative of three independent experiments are shown. Lanes 1, 4, 7, and 12 (Input), RT-PCR signal for each primer set using C2C12 total RNA as template.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.
5.
FIGURE 8.

FIGURE 8. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

MBNL3 expression and Mef2D β-exon splicing in muscle of DM patients. A, MBNL3 transcript levels. Total RNA was isolated from psoas (skeletal) and heart (cardiac) muscle tissue obtained from two normal and two age/gender-matched DM patients. The level of MBNL3 expression was determined by quantitative RT-PCR. GAPDH transcript levels were measured to control for RNA input. Relative MBNL3 mRNA levels were calculated using the conventional 2−ΔΔCt method (29). The p values for significant differences are provided and indicated by the asterisks. B, Mef2D β-exon splicing. Mef2D(−)β and (+)β transcript levels in total RNA prepared from normal and DM patient tissues were determined by RT-PCR using human specific primers. Amplified products were separated on polyacrylamide and visualized by ethidium bromide staining. Relative intensity of (+)β and (−)β splice products was determined using National Institutes of Health Image J software and used to calculate the percentage of total Mef2D transcripts for each isoform (n = 3). Significant differences are indicated by the p value.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.
6.
FIGURE 2.

FIGURE 2. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

Mef2D(+)β expression overcomes muscle differentiation defect of C2C12-MBNL3 cells. A, C2C12-MBNL3 cells that constitutively express MBNL3 were infected with no virus (C2C12), the empty control retrovirus (pclBabe), or retroviruses that express either the (−)β or (+)β isoforms of Mef2D. After puromycin selection for virally infected cells, drug-resistant cells were pooled, and whole cell lysates were prepared. Mef2D protein level was determined by Western blotting. GAPDH levels were used to control for variations in protein load. The positions of molecular mass markers (in kDa) are shown on the left. B, total RNA was isolated for RT-PCR analysis from C2C12 and different virally infected C2C12-MBNL3 cells. Positions of PCR products for (+)β and (−)β isoforms of Mef2D are indicated. Timm17b message levels were measured to control for input RNA. C, control C2C12 cells and virally infected drug-resistant cell pools were maintained in differentiation media (DM) for the indicated number of days. Whole cell lysates were prepared, and the ability of cells to execute the muscle differentiation program was determined by following two myogenic markers, myogenin (Myog) and MHC, by Western blotting. Tubulin protein levels were used to control for total protein load. The protein standards are indicated on the left. The data presented was observed in three independent experiments.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.
7.
FIGURE 6.

FIGURE 6. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

β-Exon splicing pattern of Mef2D minigene constructs in C2C12 cells. A, schematic diagrams of WT and mutant (ΔT5) Mef2D minigene constructs consisting of exons 6, β, and 7 and the intervening introns are shown. B, WT Mef2D minigene construct was cotransfected into C2C12 cells with either pcDNA3.0FLAG vector (−), an expression plasmid for the ZFmt, or the WT-MBNL3 expression plasmid (WT). Approximately 24 h post-transfection, total RNA was isolated, and the β-exon splicing pattern was determined by RT-PCR. Amplified products were loaded onto native polyacrylamide gels and detected by ethidium bromide staining. The intensity of (+)β and (−)β splice products was determined using National Institutes of Health Image J software and used to calculate the percentage of total Mef2D transcript for each isoform. Percentages from three independent experiments (n = 5) are provided. C, level of WT and ZFmt MBNL3 protein expressed from the transfected expression vector was determined by Western blotting. Tubulin protein levels were measured to control for variations in protein load. D, WT or ΔT5 Mef2D minigene was transfected into C2C12 cells in the absence (−) or presence (+) of WT-MBNL3 expression plasmid. The samples and resulting data were analyzed as described in B. E, expression levels of MBNL3 protein were determined by Western blotting. Tubulin protein levels were used to monitor for differences in protein loading. Statistically significant differences are indicated by the asterisks, and the p values are provided.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.
8.
FIGURE 7.

FIGURE 7. From: RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) ?-Exon Splicing.

Increased MBNL3 protein levels accompany defects in Mef2D β-exon splicing and muscle gene expression in a cell culture model for myotonic dystrophy. A, C2C12 clonal cell pools that express GFP fused to the full-length DMPK 3′-UTR containing either 5 (CTG5) or 200 (CTG200) CTG repeats were kindly provided by Dr. Mani Mahadevan. These cells and control C2C12 cells were exposed to differentiation conditions, and the splicing pattern of Mef2D was examined by RT-PCR and polyacrylamide gel electrophoresis. B, expression of the muscle differentiation markers MHC and myogenin (Myog) were determined by Western blotting of whole cell lysates prepared from the indicated cell lines. GAPDH protein levels were monitored to control for total protein load. The positions of molecular mass markers (in kDa) are shown on the left. C, elevated levels of MBNL3 but not MBNL1 in C2C12 cells expressing CUG200 repeat transcripts. Whole cell lysates were prepared from C2C12 cells that expressed DMPK 3′-UTR containing either 5 (CTG5) or 200 (CTG200) CTG repeats. The cells were maintained in either growth (GM) or differentiation (DM) medium before the extracts were prepared. Approximately 100 μg of lysate was loaded onto SDS-polyacrylamide gels, transferred to nitrocellulose, and subjected to Western blotting with the MBNL3 mAb P1E7 and MBNL1 mAb MB1a. Tubulin levels were used to control for protein loading. The protein levels of MBNL3 and MBNL1 were determined using National Institutes of Health Image J software and used to calculate the relative levels, shown below each lane, under growth and differentiation medim conditions. The data presented were reproducibly observed in three independent experiments.

Kyung-Soon Lee, et al. J Biol Chem. 2010 October 29;285(44):33779-33787.

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