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.     

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing

In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.     

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.     

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.     

Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.     

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.     

The CUG-binding protein binds specifically to UG dinucleotide repeats in a yeast three-hybrid system

The CUG-binding protein (CUG-BP) has been reported to be involved in the pathogenesis of myotonic dystrophy (DM) through binding to a CUG trinucleotide repeat located in the 3' untranslated region (3'UTR) of the DM protein kinase (DMPK) gene. We found that CUG-BP associates with long CUG trinucleotide repeats ((CUG)(11)(CUG)(12)), but not with short repeats ((CUG)(12)) in a yeast three-hybrid system. On the other hand, CUG-BP+LYLQ, an alternatively spliced isoform of CUG-BP, does not associate with CUG trinucleotide repeats regardless of the repeat length. In addition to these findings, we found that CUG-BP and CUG-BP+LYLQ strongly and specifically associate with UG dinucleotide repeats. Deletion analyse of CUG-BP revealed that the absence of the first or third RNA-binding domain (RBD I and RBD III, respectively) does not affect the interaction between CUG-BP and UG dinucleotide repeats. Loss of the second RNA-binding domain (RBD II) decreases the affinity of CUG-BP for UG dinucleotide repeats by about 40%. Unexpectedly, deletion of the linker domain most severely reduces the interaction, although this region does not contain a known RNA-binding motif. Our results suggest the possibility that both CUG-BP and CUG-BP+LYLQ associate with UG repeat-containing mRNAs and regulate such metabolic properties as mRNA localization, stability, and translation, and provide new insights into the pathogenesis of DM.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.