rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

Fig. 1. Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP32] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f Quantification of the co-localization of CUG or CCUG RNA foci with candidate proteins in transfected C2C12 cells. Error bars indicate s.e.m. of three…

Fig. 2. rbFOX1 binds to expanded CCUG RNA repeats. a Left panel, gel-shift assays of 0, 0.1, 0.3, 1, 3, 10, 30, and 100 nM of purified recombinant GST-rbFOX1 with 10 pM (3000 CPM) of uniformly [αP32] internally labeled in vitro transcribed RNAs containing either 10 CUG or CCUG repeats. Right panel, gel-shift quantification. b Gel-shift as in a but with purified recombinant GST-MBNL1∆101. c Alignment of the UGCAUGC consensus RNA binding site for rbFOX1 with expanded CCUG repeats that constitute the DM2 mutation. d Model of rbFOX1 RRM bound to UGCCUGC. RNA is shown in stick representation (yellow) and potential hydrogen bonds in dashed lines (purple). e Magnification of d showing that guanine 2 and cytosine 4 form a non-Watson-Crick base pair, in an analogous way to guanine 2 and adenine 4 in rbFOX1 RRM bound to UGCAUGU described previously (pdb 2ERR)49. Error bars indicate s.e.m. of five independent experiments

Fig. 3. rbFOX1 co-localizes with CCUG RNA foci in DM2 patients. a Left panel, representative confocal images of RNA FISH against CCUG repeats coupled to immunofluorescence against MBNL1 and rbFOX1 on four days differentiated muscle cells originating from muscle biopsies of individuals with either DM1 or DM2. Right panel, quantification of the co-localization of MBNL1 and rbFOX1 with CUG or CCUG RNA foci. b Left panel, CUG or CCUG repeats RNA FISH combined with immunofluorescence against MBNL1 and rbFOX1 on skeletal muscle sections of adult individuals with DM1 or DM2. Right panel, quantification of the co-localization of rbFOX1 within a hundred CUG or CCUG RNA foci. c RNA FISH/ immunofluorescence as in b but with an antibody directed against rbFOX2. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. of three independent experiments. Student’s t-test, asterisk (*) indicates p < 0.5, asterisk (***) indicates p < 0.001

Fig. 4. rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1. Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1. Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right…

Fig. 5. rbFOX1 competes with MBNL1 for binding to expanded CCUG repeats. a Upper panel, UV-cross-linking binding of 1 µg of GST-MBNL1∆101 to 10,000 CPM of uniformly [αP32] internally labeled in vitro transcribed RNA containing 30 CCUG repeats was competed by increasing amounts (0.25, 0.5, 1, and 2 µg) of GST-rbFOX1. Middle panel, loading of recombinant MBNL1 and of rbFOX1 proteins was verified by coomassie staining. Lower panel, quantification of the binding of MBNL1 and rbFOX1 to expanded CCUG repeats. b UV-cross-linking as in a but with binding of 0.5 µg of GST-rbFOX1 competed by 0.5, 1, and 2 µg of GST-MBNL1∆101. c Left panel, RNA FISH of expanded CUG or CCUG repeats coupled to immunofluorescence against Flag-rbFOX1 and MBNL1 on primary cultures of myoblasts originating from muscle biopsies of individuals with either DM1 or DM2 and transfected with a plasmid expressing FLAG-tagged rbFOX1. Each image shows at least two different cells, one transfected (T) and one not transfected (U), with insets showing higher magnification focusing on RNA foci. Right panel, quantification of the signal of endogenous MBNL1 localized within the CCUG RNA foci. d As in c but with transfection of…

Fig. 6. rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t-test, asterisk (*) indicates p < 0.5, asterisk (**) indicates p < 0.01, asterisk (***) indicates p < 0.001

Fig. 7. rbFOX1 alleviates the symptoms caused by expanded CCUG repeats. a Left panel, representative dorsoventral sections of resin-embedded adult thoraces showing indirect flight muscles (IFMs) of flies expressing expanded CCUG repeats and GFP-tagged rbFOX1 or MBNL1. Right panel, IFM muscle area quantification. b As in a but with DM1 flies expressing expanded CUG repeats. c Notched box plot showing the median and the distribution of average landing height (cm) data from flight assay of control, expanded CUG and expanded CCUG expressing flies with or without expression of GFP-rbFOX1. The horizontal lines within the boxes represent median values, whereas bottom and top edges of the boxes represent the 25th and 75th percentiles and bottom and top whiskers reach the 10th and 90th percentiles, respectively. d Quantification of climbing speed in mm/s of control, expanded CUG and expanded CCUG expressing flies with or without expression of GFP-rbFOX1. e Upper panel, RT-PCR analysis of endogenous Fhos exon 16 in flies expressing expanded CUG or CCUG repeats and GFP-rbFOX1. Lower panel, quantification of Fhos exon 16 inclusion. f Quantification of Serca exon 13 expression in flies expressing…

Fig. 8. Model of splicing alteration in myotonic dystrophy. a, b Lifespan in days of control and expanded CCUG (a) or CUG (b) expressing flies with or without expression of GFP-tagged rbFOX1. Flies expressing expanded CCUG or CUG repeats showed a median life of 10 and 21 days, respectively, which was significantly reduced in comparison to control flies (n = 50 flies, p < 0.0001, Gehan-Breslow-Wilcoxon test). Expression of rbFOX1 partly corrects median life and lifespan in CCUG expressing flies (n = 50 flies, CCUG rbFOX1 L1 p < 0.0001 and CCUG rbFOX1 L2 p = 0.0002). In contrast, expression of rbFOX1 does not improve lifespan of CUG expressing flies. c In myotonic dystrophy type 1, expanded CUG repeats represent an excess of ligand for MBNL proteins, resulting in their titration and sequestration in discrete nuclear RNA foci, ultimately resulting in alternative splicing changes that are inadequate to adult physiology. d In myotonic dystrophy type 2, expanded CCUG repeats also titrate MBNL proteins, but with a lesser efficiency due to competition with rbFOX proteins, which ultimately results in milder alternative splicing changes and milder symptoms compared to DM1