Cytoplasmic CUG RNA Foci Are Insufficient to Elicit Key DM1 Features

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)₄₀₀ repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology.     

Effect of the [CCTG]n repeat expansion on ZNF9 expression in myotonic dystrophy type II (DM2)

Myotonic dystrophy is caused by two different mutations: a (CTG)n expansion in 3' UTR region of the DMPK gene (DM1) and a (CCTG)n expansion in intron 1 of the ZNF9 gene (DM2). The most accredited mechanism for DM pathogenesis is an RNA gain-of-function. Other findings suggest a contributory role of DMPK-insufficiency in DM1. To address the issue of ZNF9 role in DM2, we have analyzed the effects of (CCTG)n expansion on ZNF9 expression in lymphoblastoid cell lines (n=4) from DM2 patients. We did not observe any significant alteration in ZNF9 mRNA and protein levels, as shown by QRT-PCR and Western blot analyses. Additional RT-PCR experiments demonstrated that ZNF9 pre-mRNA splicing pattern, which includes two isoforms, is unmodified in DM2 cells. Our results indicate that the (CCTG)n expansion in the ZNF9 intron does not appear to have a direct consequence on the expression of the gene itself.     

Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive

RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence.     

Expanded CUG repeats Dysregulate RNA splicing by altering the stoichiometry of the muscleblind 1 complex

To understand the role of the splice regulator muscleblind 1 (MBNL1) in the development of RNA splice defects in myotonic dystrophy I (DM1), we purified RNA-independent MBNL1 complexes from normal human myoblasts and examined the behavior of these complexes in DM1 myoblasts. Antibodies recognizing MBNL1 variants (MBNL1(CUG)), which can sequester in the toxic CUG RNA foci that develop in DM1 nuclei, were used to purify MBNL1(CUG) complexes from normal myoblasts. In normal myoblasts, MBNL1(CUG) bind 10 proteins involved in remodeling ribonucleoprotein complexes including hnRNP H, H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9. Of these proteins, only MBNL1(CUG) colocalizes extensively with DM1 CUG foci (>80% of foci) with its partners being present in <10% of foci. Importantly, the stoichiometry of MBNL1(CUG) complexes is altered in DM1 myoblasts, demonstrating an increase in the steady state levels of nine of its partner proteins. These changes are recapitulated by the expression of expanded CUG repeat RNA in Cos7 cells. Altered stoichiometry of MBNL1(CUG) complexes results from aberrant protein synthesis or stability and is unlinked to PKCα function. Modeling these changes in normal myoblasts demonstrates that increased levels of hnRNP H, H2, H3, F, and DDX5 independently dysregulate splicing in overlapping RNA subsets. Thus expression of expanded CUG repeats alters the stoichiometry of MBNL1(CUG) complexes to allow both the reinforcement and expansion of RNA processing defects.     

Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios

The mechanism by which (CTG)n expansion in the 3' UTR of the DMPK gene causes myotonic dystrophy (DM) is unknown. We identified four RNA splicing factors--hnRNP C, U2AF (U2 auxiliary factor), PTB (polypyrimidine tract binding protein), and PSF (PTB associated splicing factor)--that bind to two short regions 3' of the (CUG)n, and found a novel 3' DMPK exon resulting in an mRNA lacking the repeats. We propose that the (CUG)n is an essential cis acting element for this splicing event. In contrast to (CUG)n containing mRNAs, the novel isoform is not retained in the nucleus in DM cells, resulting in imbalances in relative levels of cytoplasmic DMPK mRNA isoforms and a new dominant effect of the mutation on DMPK.     

Modifications to toxic CUG RNAs induce structural stability,rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model

CUG repeat expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) cause myotonic dystrophy type 1 (DM1). As RNA, these repeats elicit toxicity by sequestering splicing proteins, such as MBNL1, into protein–RNA aggregates. Structural studies demonstrate that CUG repeats can form A-form helices, suggesting that repeat secondary structure could be important in pathogenicity. To evaluate this hypothesis, we utilized structure-stabilizing RNA modifications pseudouridine (Ψ) and 2′-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with Ψ or 2′-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Ψ modification of CUG repeats rescued mis-splicing in a DM1 cell model and prevented CUG repeat toxicity in zebrafish embryos. This study indicates that the structure of toxic RNAs has a significant role in controlling the onset of neuromuscular diseases.     

Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a clinically and genetically heterogeneous neuromuscular disorder. DM is characterized by autosomal dominant inheritance, muscular dystrophy, myotonia, and multisystem involvement. Type 1 DM (DM1) is caused by a (CTG)(n) expansion in the 3' untranslated region of DMPK in 19q13.3. Multiple families, predominantly of German descent and with clinically variable presentation that included proximal myotonic myopathy (PROMM) and type 2 DM (DM2) but without the DM1 mutation, showed linkage to the 3q21 region and were recently shown to segregate a (CCTG)(n) expansion mutation in intron 1 of ZNF9. Here, we present linkage to 3q21 and mutational confirmation in 17 kindreds of European origin with PROMM and proximal myotonic dystrophy, from geographically distinct populations. All patients have the DM2 (CCTG)(n) expansion. To study the evolution of this mutation, we constructed a comprehensive physical map of the DM2 region around ZNF9. High-resolution haplotype analysis of disease chromosomes with five microsatellite and 22 single-nucleotide polymorphism markers around the DM2 mutation identified extensive linkage disequilibrium and a single shared haplotype of at least 132 kb among patients from the different populations. With the exception of the (CCTG)(n) expansion, the available markers indicate that the DM2 haplotype is identical to the most common haplotype in normal individuals. This situation is reminiscent of that seen in DM1. Taken together, these data suggest a single founding mutation in DM2 patients of European origin. We estimate the age of the founding haplotype and of the DM2 (CCTG) expansion mutation to be approximately 200-540 generations.     

Myotonic dystrophies 1 and 2: complex diseases with complex mechanisms

Two multi-system disorders, Myotonic Dystrophies type 1 and type 2 (DM1 and DM2), are complex neuromuscular diseases caused by an accumulation of expanded, non-coding RNAs, containing repetitive CUG and CCUG elements. Similarities of these mutations suggest similar mechanisms for both diseases. The expanded CUGn and CCUGn RNAs mainly target two RNA binding proteins, MBNL1 and CUGBP1, elevating levels of CUGBP1 and reducing levels of MBNL1. These alterations change processing of RNAs that are regulated by these proteins. Whereas overall toxicity of CUGn/CCUGn RNAs on RNA homeostasis in DM cells has been proven, the mechanisms which make these RNAs toxic remain illusive. A current view is that the toxicity of RNA CUGn and CCUGn is associated exclusively with global mis-splicing in DM patients. However, a growing number of new findings show that the expansion of CUGn and CCUGn RNAs mis-regulates several additional pathways in nuclei and cytoplasm of cells from patients with DM1 and DM2. The purpose of this review is to discuss the similarities and differences in the clinical presentation and molecular genetics of both diseases. We will also discuss the complexity of the molecular abnormalities in DM1 and DM2 caused by CUG and CCUG repeats and will summarize the outcomes of the toxicity of CUG and CCUG repeats.