Fig 2: Compound ISOX and vorinostat increase MBNL1 expression in DM1-patient-derived fibroblasts. (A) Immunoblot images of MBNL1 levels in DM1 patient-derived (top) and normal (bottom) fibroblasts after 5 µM ISOX or 5 µM vorinostat treatment for 2 days. Three biological replicates were generated for each treatment. Tubulin was used as a loading control. (B) MBNL1 levels from A were normalized to tubulin signal and plotted as a bar graph. Error bars show the standard deviation (SD) of the biological triplicates and statistic differences were characterized by a t test. *P = 0.05; **P = 0.01; ***P = 0.001.

Fig 3: MBNL-like proteins repress Tpm1 exon 3. (A) Schematic representation of the mutually exclusive splicing of Tpm1 exons 2 and 3. The essential negative regulatory elements flanking exon 3 are indicated. The P3 and DY elements bind PTB and are denoted by black and white rectangles, respectively. The U and D elements are indicated by black and white diamonds, respectively, and their sequences are indicated. Matches to MBNL1 consensus sequences are underlined (YGCU(U/G)Y) and overlined (YGCY). (B) Western blot of siRNA knockdown, probed with a-MBNL1, a-MBNL2 or a-actin antibodies with a titration of control (C2) knockdown sample at 20, 40, 60, 80 and 100% (lanes 1–5) compared with knockdown of MBNL1, MBNL2 or MBNL1 plus MBNL2 (lanes 6–8). (C) qRT–PCR analysis of endogenous Tpm1 on knockdown of MBNL1 and MBNL2. The histogram shows the fold change of exon 2 or exon 3 products comparing control siRNA (C2) with knockdown of MBNL1 (1), MBNL2 (2) or MBNL1 plus MBNL2 (1 + 2). (D) Schematic representation of Tpm1 minigene reporter containing exons 1, 3 and 4 on the left, and Venus-tagged MBNL1 on the right. RT–PCR analysis of RNA isolated from PAC-1 cells (left panel) or HeLa cells (right panel) transfected with the Tpm1 minigene reporter (lanes 1 and 11). In HeLa cells, the Tpm1 mingene has a branch point mutation changing the wild-type sequence from GGCUAAC to GGCUGGC. Lanes 2–10, siRNA knockdown of MBNL1 and MBNL2 in PAC-1 cells together with overexpressed Venus–MBNL1 with the siRNA site mutated (lane 3), or various C-terminal truncations of Venus–MBNL1 (lanes 4–9); the yellow dots indicate Venus and the adjacent number represents the position of the C-terminal deletion. Lane 10 is transfected with an N-terminal MBNL1 deletion containing amino acids 239–382. Lanes 12 and 13, siRNA knockdown of MBNL1 in HeLa cells together with overexpressed GFP–MBNL1 with the siRNA site mutated, MBNL1m (lane 13). Right panel: anti-GFP (detects Venus) western blot; lane numbers correspond to those in the left panel.

Fig 4: Site-specific integration of ZsGreen into endogenous MBNL1 locus generates ZsGreen-MBNL1 cells expressing green fluorescent fusion protein. (A) Schematic diagram of the strategy to insert a ZsGreen cassette into the MBNL1 locus (not to scale). The asterisks indicate the position of the single-strand breaks generated by Cas9 nickase/sgRNAs. The middle diagram shows the donor vector that contains the left and right homologous MBNL1 arms and the ZsGreen reporter. (B) ZsGreen integration in MBNL1 locus is confirmed by PCR followed by agarose gel analysis. Primer sets and PCR products are indicated in the upper diagram. (C) Droplet digital PCR (ddPCR) quantifying MBNL1 and ZsGreen copy number in no-template control (NTC), parental HeLa and ZsGreen-MBNL1 genomic DNA and plotted on the bar graph. (D) Immunoblotting shows MBNL1 and ZsGreen-MBNL1 protein expression in parental HeLa and ZsGreen-MBNL1 cells. MBNL1-silencing siRNA (siMBNL1) and non-silencing siRNA (siNS) control were used to demonstrate MBNL1 antibody specificity. Tubulin was used as a loading control. (E) Fluorescent microscopy images of parental HeLa and clonal ZsGreen-MBNL1 #27 using the same exposure. The green fluorescent images are on the left; the merged images of green and blue (DAPI staining) signal are on the right. Scale bar is 20 µm. (F) Flow cytometry quantification of green fluorescent signal in parental HeLa cells and ZsGreen-MBNL1 clone #27.

Fig 5: Interaction of MBNL truncations with PTB. (A) Schematic representation of Venus-tagged MBNL1 fusion proteins with the amino acid boundaries indicated (upper). Black boxes represent the four ZF domains, the grey shaded box the C-terminus and the yellow dot the Venus tag. Schematic representation of mCherry-tagged PTB (lower); white numbered boxes represent the RRM domains and ‘L’ denotes inter-RRM linkers. (B) Western blot of input (left panel, 10% of immunoprecipitation) and anti-flag immunoprecipitation (right panel) of Venus–MBNL1 co-expressed with FLAG–PTB4 in formaldehyde cross-linked 293 T extracts. All lanes had FLAG–PTB4 co-expressed with Venus-tagged proteins; vector (lanes 1 and 10), MBNL1-2–382 (lanes 2 and 11), MBNL1-2–253 (lanes 3 and 12), MBNL1-2–183 (lanes 4 and 13), MBNL1-2–115 (lanes 5 and 14), MBNL1-2–88 (lanes 6 and 15), MBNL1-2–104 (lanes 7 and 16), MBNL1-2–72 (lanes 8 and 17) and MBNL1-239–382 (lanes 9 and 18). Top panel, anti-PTB western, lower panel, anti-GFP western. (C) FLIM–FRET analysis of MBNL1 interaction with PTB. MBNL1 and PTB were fused to Venus as the FRET donor or mCherry as the acceptor and expressed in HeLa cells as indicated. MBNL fusion constructs are indicated with the N-terminal ZFs shown in black, and the C-terminal region shaded. First two rows show the Venus fluorescence and the corresponding fluorescence lifetime images of the indicated MBNL1 constructs in absence or presence of full-length mCherry–PTB, respectively. Third row shows the corresponding histograms (red line = donor only control; blue line = double transfection FRET). An energy transfer and, therefore, a reduction of the fluorescence lifetime was observed for N- (a) and C-terminal (b) labelled full-length MBNL1 (Venus–MBNL-2–382 and MBNL-2–382–Venus, respectively) in presence of mCherry-tagged PTB. Furthermore, the N-terminus of MBNL1 was sufficient to establish this interaction (c), whereas the C-terminal end alone did not show a FRET (d). (D) Statistical analysis of (C). Error bars are SEM of at least five independent fields of view with approximately four cells per image. Scale bar = 10 µm.