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.     

MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1

In myotonic dystrophy 1 (DM1), aggregation of the mutant DMPK RNA into RNA-protein complexes containing MBNL1 and MBNL2 has been linked to aberrant splicing of the insulin receptor (IR) RNA. In a parallel line of investigation, elevated levels of CUG-binding protein (CUG-BP) have been shown to result in altered IR splicing in DM1. The relative importance of MBNL1, MBNL2, and CUG-BP in DM1 pathogenesis is, however, unclear. Here we have demonstrated that either small interfering RNA-mediated down-regulation of MBNL1 and MBNL2 or the overexpression of CUG-BP in normal myoblasts results in abnormal IR splicing. Our results suggest that CUG-BP regulates the equilibrium of splice site selection by antagonizing the facilitatory activity of MBNL1 and MBNL2 on IR exon 11 splicing in a dose-dependent manner. We have shown that CUG-BP levels are elevated in DM1 cells by mechanisms that are independent of MBNL1 and MBNL2 loss. Importantly, rescue experiments in DM1 myoblasts demonstrated that loss of MBNL1 function is the key event, whereas the overexpression of CUG-BP plays a secondary role in the aberrant alternative splicing of IR RNA in DM1. Small interfering RNA-mediated down-regulation of MBNL1, MBNL2, and CUG-BP in DM1 myoblasts demonstrated that MBNL1 plays a critical role in the maintenance of DM1 focus integrity. Thus, these experiments demonstrate that sequestration of MBNL1 by the expanded CUG repeats is the primary determinant of both DM1 focus formation and the abnormal splicing of the IR RNA in DM1 myoblasts. The data therefore support MBNL1-mediated therapy for DM1.     

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.     

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.     

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.     

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 proteins regulate alternative splicing

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.     

Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing

In myotonic dystrophy (DM1), both inactivation of muscleblind proteins and increased levels of CUG-BP1 are reported. These events have been shown to contribute independently to aberrant splicing of a subset RNAs. We demonstrate that steady-state levels of the splice regulator, hnRNP H, are elevated in DM1 myoblasts and that increased hnRNP H levels in normal myoblasts results in the inhibition of insulin receptor (IR) exon 11 splicing in a manner similar to that observed in DM1. In normal myoblasts, overexpression of either hnRNP H or CUG-BP1 results in the formation of an RNA-dependent suppressor complex consisting of both hnRNP H and CUG-BP1, which is required to maximally inhibit IR exon 11 inclusion. Elevated levels of MBNL1 show RNA-independent interaction with hnRNP H and dampen the inhibitory activity of increased hnRNP H levels on IR splicing in normal myoblasts. In DM1 myoblasts, overexpression of MBNL1 in conjunction with si-RNA mediated depletion of hnRNP H contributes to partial rescue of the IR splicing defect. These data demonstrate that coordinated physical and functional interactions between hnRNP H, CUG-BP1 and MBNL1 dictate IR splicing in normal and DM1 myoblasts.     

Identification of MBNL1 and MBNL3 domains required for splicing activation and repression

Muscleblind-like 1 (MBNL1) is a splicing regulator that controls developmentally regulated alternative splicing of a large number of exons including exon 11 of the Insulin Receptor (IR) gene and exon 5 of the cardiac Troponin T (cTNT) gene. There are three paralogs of MBNL in humans, all of which promote IR exon 11 inclusion and cTNT exon 5 skipping. Here, we identify a cluster of three binding sequences located downstream of IR exon 11 that constitute the MBNL1 response element and a weaker response element in the upstream intron. In addition, we used sequential deletions to define the functional domains of MBNL1 and MBNL3. We demonstrate that the regions required for splicing regulation are separate from the two pairs of zinc-finger RNA-binding domains. MBNL1 and MBNL3 contain core regulatory regions for both activation and repression located within an 80-amino-acid segment located downstream of the N-terminal zinc-finger pair. Deletions of these regions abolished regulation without preventing RNA binding. These domains have common features with the CUG-BP and ETR3-like Factor (CELF) family of splicing regulators. These results have identified protein domains required for splicing repression and activation and provide insight into the mechanism of splicing regulation by MBNL proteins.     

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.     

The hallmarks of myotonic dystrophy type 1 muscle dysfunction

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3′ untranslated region (3′UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin–proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.     

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.