Tau exon 2 responsive elements deregulated in myotonic dystrophy type I are proximal to exon 2 and synergistically regulated by MBNL1 and MBNL2

The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n > 50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscleblind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.     

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.     

RNA Splicing Is Responsive to MBNL1 Dose

Myotonic dystrophy (DM1) is a highly variable, multi-system disorder resulting from the expansion of an untranslated CTG tract in DMPK. In DM1 expanded CUG repeat RNAs form hairpin secondary structures that bind and aberrantly sequester the RNA splice regulator, MBNL1. RNA splice defects resulting as a consequence of MBNL1 depletion have been shown to play a key role in the development of DM1 pathology. In patient populations, both the number and severity of DM1 symptoms increase broadly as a function of CTG tract length. However significant variability in the DM1 phenotype is observed in patients encoding similar CTG repeat numbers. Here we demonstrate that a gradual decrease in MBNL1 levels results both in the expansion of the repertoire of splice defects and an increase in the severity of the splice alterations. Thus, MBNL1 loss does not have an all or none outcome but rather shows a graded effect on the number and severity of the ensuing splice defects. Our results suggest that once a critical threshold is reached, relatively small dose variations of free MBNL1 levels, which may reflect modest changes in the size of the CUG tract or the extent of hairpin secondary structure formation, can significantly alter the number and severity of splice abnormalities and thus contribute to the phenotype variability observed in DM1 patients.     

Mechanistic determinants of MBNL activity

Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUG^exp) RNAs. Pathogenic interactions between MBNL and CUG^exp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To address this question, we coexpressed three MBNL paralogs in cells at equivalent levels and characterized both specific and redundant roles of these proteins in alternative splicing and RNA foci dynamics. When coexpressed in the same cells, MBNL1, MBNL2 and MBNL3 bind the same RNA motifs with different affinities. While MBNL1 demonstrated the highest splicing activity, MBNL3 showed the lowest. When forming RNA foci, MBNL1 is the most mobile paralog, while MBNL3 is rather static and the most densely packed on CUG^exp RNA. Therefore, our results demonstrate that MBNL paralogs and gene-specific isoforms possess inherent functional differences, an outcome that could be enlisted to improve therapeutic strategies for DM1.     

DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1

Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to ‘sponge’ splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG–RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.     

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.     

Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.     

Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs

The MBNL and CELF proteins act antagonistically to control the alternative splicing of specific exons during mammalian postnatal development. This process is dysregulated in myotonic dystrophy because MBNL proteins are sequestered by (CUG)_n and (CCUG)_n RNAs expressed from mutant DMPK and ZNF9 genes, respectively. While these observations predict that MBNL proteins have a higher affinity for these pathogenic RNAs versus their normal splicing targets, we demonstrate that MBNL1 possesses comparably high affinities for (CUG)_n and (CAG)_n RNAs as well as a splicing target, Tnnt3. Mapping of a MBNL1-binding site upstream of the Tnnt3 fetal exon indicates that a preferred binding site for this protein is a GC-rich RNA hairpin containing a pyrimidine mismatch. To investigate how pathogenic RNAs sequester MBNL1 in DM1 cells, we used a combination of chemical/enzymatic structure probing and electron microscopy to determine that MBNL1 forms a ring-like structure which binds to the dsCUG helix. While the MBNL1 N-terminal region is required for RNA binding, the C-terminal region mediates homotypic interactions which may stabilize intra- and/or inter-ring interactions. Our results provide a mechanistic basis for dsCUG-induced MBNL1 sequestration and highlight a striking similarity in the binding sites for MBNL proteins on splicing precursor and pathogenic RNAs.     

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T

Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. We demonstrate that MBNL can bind short structured CUG and CCUG repeats with high affinity and specificity. Only 6 base pairs are necessary for MBNL binding: two pyrimidine mismatches and four guanosine-cytosine base pairs in a stem. MBNL also has a preference for pyrimidine mismatches, but many other mismatches are tolerated with decreased affinity. We also demonstrate that MBNL binds the helical region of a stem-loop in the endogenous pre-mRNA target, the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. In vivo splicing results indicate that MBNL-regulated splicing is dependent upon the formation of stem-loops recognized by MBNL. These results suggest that MBNL may bind all of its RNA substrates, both normal and pathogenic, as structured stem-loops containing pyrimidine mismatches.     

MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families—muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins—are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5′ end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.