Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded non-coding CTG/CCTG repeats, and these CUG/CCUG repeats sequester the Muscleblind-like (MBNL) family of RNA binding proteins. The MBNL proteins are regulators of alternative splicing, and their sequestration has been linked with mis-splicing events in DM. A previously reported screen for small molecules found that pentamidine was able to improve splicing defects associated with DM. Biochemical experiments and cell and mouse model studies of the disease indicate that pentamidine and related compounds may work through binding the CTG*CAG repeat DNA to inhibit transcription. Analysis of a series of methylene linker analogs of pentamidine revealed that heptamidine reverses splicing defects and rescues myotonia in a DM1 mouse model.

Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy. In myotonic dystrophy, the lack of properly localized MBNL1 leads to missplicing of many pre-mRNAs. One of these events is the aberrant inclusion of exon 5 within the MBNL1 pre-mRNA. The region of the MBNL1 gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3 -end of intron 4 is noncanonical in that it contains a predicted branch point that is 141 nucleotides from the 3 -splice site and an AAG 3 -splice site. Using a minigene that includes exon 4, intron 4, exon 5, intron 5, and exon 6 of MBNL1, we showed that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branch point confirmed that branching occurs primarily at the predicted distant branch point. Structure probing and footprinting revealed that the highly conserved region between the branch point and 3 -splice site is primarily unstructured and that MBNL1 binds within this region of the pre-mRNA. Deletion of the MBNL1 response element eliminated MBNL1 splicing regulation and led to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing.Splicing of pre-mRNAs is an important event that contributes to a diverse proteome as well as the regulation of gene expression. It is estimated that more than 90% of human genes undergo alternative splicing (1, 2). To produce a functional mRNA, non-coding regions must be accurately removed, and the coding regions must be ligated together. Splicing occurs via two transesterification reactions that result in removal of the intron and ligation of the exons. This splicing mechanism relies on pre-mRNA sequences, proteins, and small nuclear RNAs (snRNAs) that are necessary for intron and exon definition and the two transesterification reactions. Cis-sequences that are important for splicing include the 5Ј-splice site (ss), 2 the branch point sequence, the polypyrimidine (PY) tract, and the 3Ј-ss. These canonical intronic motifs, plus additional regulatory splicing motifs found in exons and introns, are recognized by splicing factors and small nuclear ribonucleoproteins (U1, U2, U4, U5, and U6) to form the spliceosome, which catalyzes intron removal (for a review, see Ref.3).There are many splicing factors that are only involved in a subset of splicing decisions. These include the human muscleblind-like family of RNA-binding proteins: MBNL1/2/3 also known as MBNL/EXP, MBLL/MPL1, and MBXL/CHCR, respectively. The founding member of this family, muscleblind (Mbl), was discovered in Drosophila and was shown to be important for photoreceptor differentiation and terminal differentiation of muscles (4, 5). Subsequently, MBNL proteins were found to associate with expanded CUG repeats (located in the 3Ј-untranslated region of the DMPK gene) that have been shown to act as a toxic RNA and are at least partially responsible for causing myotonic dystrophy (DM) type 1 (for reviews, see Refs. 6 -8). The expanded...

Summary Myotonic Dystrophy type 1 (DM1) is an inherited disease characterized by the inability to relax contracted muscles. Affected individuals carry large CTG expansions that are toxic when transcribed. One possible treatment approach is to reduce or eliminate transcription of CTG repeats. Actinomycin D (ActD) is a potent transcription inhibitor and FDA-approved chemotherapeutic that binds GC-rich DNA with high affinity. Here, we report that ActD decreased CUG transcript levels in a dose-dependent manner in DM1 cell and mouse models at significantly lower concentrations (nanomolar) compared to its use as a general transcription inhibitor or chemotherapeutic. ActD also significantly reversed DM1-associated splicing defects in a DM1 mouse model, and did so within the currently approved human treatment range. RNA-seq analyses showed that low concentrations of ActD did not globally inhibit transcription in a DM1 mouse model. These results indicate that transcription inhibition of CTG expansions is a promising treatment approach for DM1.

Myotonic dystrophy type 1 (DM1) is a microsatellite expansion disorder caused by the aberrant expansion of CTG repeats in the 3’ untranslated region of the DMPK gene. When transcribed, the toxic RNA CUG repeats sequester RNA binding proteins, which leads to disease symptoms. The expanded CUG repeats can adopt a double-stranded structure, and targeting this helix is a therapeutic strategy for DM1. In order to better understand the 5’CUG/3’GUC motif, and how it may interact with proteins and small molecules, we designed a short CUG helix attached to a GAAA tetraloop/receptor in order to facilitate crystal packing. Here we report the highest resolution structure (1.95 Å) to date of a GAAA tetraloop/receptor and the CUG helix it was used to crystallize. Within the CUG helix, we identify two different forms of non-canonical U-U pairs and reconfirm that CUG repeats are essentially A-form. An analysis of all non-canonical U-U pairs in the context of CUG repeats revealed six different classes of conformations that the non-canonical U-U pairs are able to adopt.

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.

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