Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG)-containing transcripts

Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)(exp)) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5'CAG/3'GAC motif found in r(CAG)(exp) hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)(exp). The compound was identified by first studying the binding of RNA 1 × 1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5'CAG/3'GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate, a small molecule that improves pre-mRNA splicing defects associated with the r(CAG)(exp)-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)(exp) and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)(exp) toxicity. The approach used in these studies, defining the small RNA motifs that bind small molecules with known affinity for nucleic acids and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in the human genomic sequence.     

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Fragile X‐associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′‐UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA‐binding proteins by the expanded CGG repeats. Sam68 is an RNA‐binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA‐binding proteins sequentially, first Sam68, then hnRNP‐G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing‐regulatory function. Consequently, Sam68‐responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain‐of‐function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.     

The four Zn fingers of MBNL1 provide a flexible platform for recognition of its RNA binding elements

Background  

Muscleblind-like 1 (MBNL1) is an alternative splicing factor containing four CCCH Zinc fingers (ZnFs). The sequestration of MBNL1 by expanded CUG and CCUG repeats is a major component in causing myotonic dystrophy. In addition to binding the structured expanded CUG and CCUG repeats; previous results suggested that MBNL1 binds single-stranded RNAs containing GC dinucleotides.     

RNA binding specificity of Drosophila muscleblind

Members of the muscleblind family of RNA binding proteins found in Drosophila and mammals are key players in both the human disease myotonic dystrophy and the regulation of alternative splicing. Recently, the mammalian muscleblind-like protein, MBNL1, has been shown to have interesting RNA binding properties with both endogenous and disease-related RNA targets. Here we report the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Mutagenesis of double-stranded CUG repeats demonstrated that Mbl requires pyrimidine-pyrimidine mismatches for binding and that the identity and location of the C-G and G-C base pairs within the repeats are essential for Mbl binding. Systematic evolution of ligands by exponential enrichment (SELEX) was used to identify RNA sequences that bind Mbl with much higher affinity than CUG repeats. The RNA sequences identified by SELEX are structured and contain a five-nucleotide consensus sequence of 5'-AGUCU-3'. RNase footprinting of one of the SELEX RNA sequences with Mbl showed that Mbl binds both double-stranded and single-stranded regions of the RNA. Three guanosines show the strongest footprint in the presence of Mbl; mutation of any of these three guanosines eliminates Mbl binding. It was also found that Mbl specifically bound a human MBNL1 RNA target, demonstrating the conservation of the muscleblind proteins in recognizing RNA targets. Our results reveal that Mbl recognizes complex RNA secondary structures.     

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.     

Conservation of context-dependent splicing activity in distant Muscleblind homologs

The Muscleblind (MBL) protein family is a deeply conserved family of RNA binding proteins that regulate alternative splicing, alternative polyadenylation, RNA stability and RNA localization. Their inactivation due to sequestration by expanded CUG repeats causes symptoms in the neuromuscular disease myotonic dystrophy. MBL zinc fingers are the most highly conserved portion of these proteins, and directly interact with RNA. We identified putative MBL homologs in Ciona intestinalis and Trichoplax adhaerens, and investigated their ability, as well as that of MBL homologs from human/mouse, fly and worm, to regulate alternative splicing. We found that all homologs can regulate alternative splicing in mouse cells, with some regulating over 100 events. The cis-elements through which each homolog exerts its splicing activities are likely to be highly similar to mammalian Muscleblind-like proteins (MBNLs), as suggested by motif analyses and the ability of expanded CUG repeats to inactivate homolog-mediated splicing. While regulation of specific target exons by MBL/MBNL has not been broadly conserved across these species, genes enriched for MBL/MBNL binding sites in their introns may play roles in cell adhesion, ion transport and axon guidance, among other biological pathways, suggesting a specific, conserved role for these proteins across a broad range of metazoan species.     

Selective inhibition of MBNL1�CCCUG interaction by small molecules toward potential therapeutic agents for myotonic dystrophy type 2 (DM2)

Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disease caused by expanded CCUG repeats that may exhibit toxicity by sequestering the splicing regulator MBNL1. A series of triaminotriazine- and triaminopyrimidine-based small molecules (ligands 1–3) were designed, synthesized and tested as inhibitors of the MBNL1–CCUG interaction. Despite the structural similarities of the triaminotriazine and triaminopyrimidine units, the triaminopyrimidine-based ligands bind with low micromolar affinity to CCUG repeats (K_d ∼ 0.1–3.6 µM) whereas the triaminotriazine ligands do not bind CCUG repeats. Importantly, these simple and small triaminopyrimidine ligands exhibit both strong inhibition (K_i ∼ 2 µM) of the MBNL1–CCUG interaction and high selectivity for CCUG repeats over other RNA targets. These experiments suggest these compounds are potential lead agents for the treatment of DM2.     

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.     

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.     

From dynamic combinatorial 'hit' to lead: in vitro and in vivo activity of compounds targeting the pathogenic RNAs that cause myotonic dystrophy

The myotonic dystrophies (DM) are human diseases in which the accumulation of toxic RNA (CUG or CCUG) repeats in the cell causes sequestration of splicing factors, including MBNL1, leading to clinical symptoms such as muscle wasting and myotonia. We previously used Dynamic Combinatorial Chemistry to identify the first compounds known to inhibit (CUG)-MBNL1 binding in vitro. We now report transformation of those compounds into structures with activity in vivo. Introduction of a benzo[g]quinoline substructure previously unknown in the context of RNA recognition, as well as other modifications, provided several molecules with enhanced binding properties, including compounds with strong selectivity for CUG repeats over CAG repeats or CAG-CUG duplex RNA. Compounds readily penetrate cells, and improve luciferase activity in a mouse myoblast assay in which enzyme function is coupled to a release of nuclear CUG-RNA retention. Most importantly, two compounds are able to partially restore splicing in a mouse model of DM1.     

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.     

Structure of 2′,5′-Linked Tetraribonucleotide Loops: A Novel RNA Motif

Abstract

We report on the three dimensional structure of an RNA hairpin containing a 2′,5′-linked tetraribonucleotide loop, namely, 5′-rGGAC(UUCG)GUCC-3′ (where UUCG = U_2′p5′U_2′p5′C_2′p5′G_2′p5′). We show that the 2′,5′-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3′,5′-linked RNA loop. The 2′,5′- RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar–base contacts, all of which contribute to the extra stability of this hairpin structure.     

Single-molecule study of the CUG repeat–MBNL1 interaction and its inhibition by small molecules

Effective drug discovery and optimization can be accelerated by techniques capable of deconvoluting the complexities often present in targeted biological systems. We report a single-molecule approach to study the binding of an alternative splicing regulator, muscleblind-like 1 protein (MBNL1), to (CUG)_{n = 4,6} and the effect of small molecules on this interaction. Expanded CUG repeats (CUG^exp) are the causative agent of myotonic dystrophy type 1 by sequestering MBNL1. MBNL1 is able to bind to the (CUG)_n–inhibitor complex, indicating that the inhibition is not a straightforward competitive process. A simple ligand, highly selective for CUG^exp, was used to design a new dimeric ligand that binds to (CUG)_n almost 50-fold more tightly and is more effective in destabilizing MBNL1–(CUG)₄. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions.     

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.