Loss of function of members of the muscleblind-like (MBNL) family of RNA binding proteins has been shown to play a key role in the spliceopathy of RNA toxicity in myotonic dystrophy type 1 (DM1), the most common muscular dystrophy affecting adults and children. MBNL1 and MBNL2 are the most abundantly expressed members in skeletal muscle. A key aspect of DM1 is poor muscle regeneration and repair, leading to dystrophy. We used a BaCl2-induced damage model of muscle injury to study regeneration and effects on skeletal muscle satellite cells (MuSCs) in Mbnl1∆E3/∆E3 and Mbnl2∆E2/∆E2 knockout mice. Similar experiments have previously shown deleterious effects on these parameters in mouse models of RNA toxicity. Muscle regeneration in Mbnl1 and Mbnl2 knockout mice progressed normally with no obvious deleterious effects on MuSC numbers or increased expression of markers of fibrosis. Skeletal muscles in Mbnl1∆E3/∆E3/ Mbnl2∆E2/+ mice showed increased histopathology but no deleterious reductions in MuSC numbers and only a slight increase in collagen deposition. These results suggest that factors beyond the loss of MBNL1/MBNL2 and the associated spliceopathy are likely to play a key role in the defects in skeletal muscle regeneration and deleterious effects on MuSCs that are seen in mouse models of RNA toxicity due to expanded CUG repeats.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

Muscleblind (mbl) is an essential muscle and neuronal splicing regulator. Mbl hosts multiple circular RNAs (circRNAs), including circMbl, which is conserved from flies to humans. Here, we show that mbl-derived circRNAs are key regulators of MBL by cis- and trans-acting mechanisms. By generating fly lines to specifically modulate the levels of all mbl RNA isoforms, including circMbl, we demonstrate that the two major mbl protein isoforms, MBL-O/P and MBL-C, buffer their own levels by producing different types of circRNA isoforms in the eye and fly brain, respectively. Moreover, we show that circMbl has unique functions in trans, as knockdown of circMbl results in specific morphological and physiological phenotypes. In addition, depletion of MBL-C or circMbl results in opposite behavioral phenotypes, showing that they also regulate each other in trans. Together, our results illuminate key aspects of mbl regulation and uncover cis and trans functions of circMbl in vivo.

Myotonic dystrophy type 1 is a debilitating neuromuscular disease causing muscle weakness, myotonia, and cardiac dysfunction. The phenotypes are caused by muscleblind-like (MBNL) protein sequestration by toxic RNA in the DM1 protein kinase (DMPK) gene. DM1 patients exhibit a pathogenic number of repetitions in DMPK, which leads to downstream symptoms. Another disease characteristic is altered microRNA (miRNA) expression. It was previously shown that miR-23b regulates the translation of MBNL1 into protein. Antisense oligonucleotide (AON) treatment targeting this miRNA can improve disease symptoms. Here, we present a refinement of this strategy targeting a miR-23b binding site on the MBNL1 3\' UTR in DM1 model cells and mice by using AONs called blockmiRs. BlockmiRs linked to novel cell-penetrating peptide chemistry showed an increase in MBNL1 protein in DM1 model cells and HSALR mice. They also showed an increase in muscle strength and significant rescue of downstream splicing and histological phenotypes in mice without disturbing the endogenous levels of other miR-23b target transcripts.

In Huntington\'s disease (HD), the uninterrupted CAG repeat length, but not the polyglutamine length, predicts disease onset. However, the underlying pathobiology remains unclear. Here, we developed bacterial artificial chromosome (BAC) transgenic mice expressing human mutant huntingtin (mHTT) with uninterrupted, and somatically unstable, CAG repeats that exhibit progressive disease-related phenotypes. Unlike prior mHTT transgenic models with stable, CAA-interrupted, polyglutamine-encoding repeats, BAC-CAG mice show robust striatum-selective nuclear inclusions and transcriptional dysregulation resembling those in murine huntingtin knockin models and HD patients. Importantly, the striatal transcriptionopathy in HD models is significantly correlated with their uninterrupted CAG repeat length but not polyglutamine length. Finally, among the pathogenic entities originating from mHTT genomic transgenes and only present or enriched in the uninterrupted CAG repeat model, somatic CAG repeat instability and nuclear mHTT aggregation are best correlated with early-onset striatum-selective molecular pathogenesis and locomotor and sleep deficits, while repeat RNA-associated pathologies and repeat-associated non-AUG (RAN) translation may play less selective or late pathogenic roles, respectively.

In vivo RNA structure analysis has become a powerful tool in molecular biology, largely due to the coupling of an increasingly diverse set of chemical approaches with high-throughput sequencing. This has resulted in a transition from single target to transcriptome-wide approaches. However, these methods require sequencing depths that preclude studying low abundance targets, which are not sufficiently captured in transcriptome-wide approaches. Here we present a ligation-free method to enrich for low abundance RNA sequences, which improves the diversity of molecules analyzed and results in improved analysis. In addition, this method is compatible with any choice of chemical adduct or read-out approach. We utilized this approach to study an autoregulated event in the pre-mRNA of the splicing factor, muscleblind-like splicing regulator 1 (MBNL1).

Unstable CTG expansions in the 3\' UTR of the DMPK gene are responsible for myotonic dystrophy type 1 (DM1) condition. Muscle dysfunction is one of the main contributors to DM1 mortality and morbidity. Pathways by which mutant DMPK trigger muscle defects, however, are not fully understood. We previously reported that miR-7 was downregulated in a DM1 Drosophila model and in biopsies from patients. Here, using DM1 and normal muscle cells, we investigated whether miR-7 contributes to the muscle phenotype by studying the consequences of replenishing or blocking miR-7, respectively. Restoration of miR-7 with agomiR-7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth. Conversely, oligonucleotide-mediated blocking of miR-7 in normal myoblasts led to fusion and myotube growth defects. miR-7 was found to regulate autophagy and the ubiquitin-proteasome system in human muscle cells. Thus, low levels of miR-7 promoted both processes, and high levels of miR-7 repressed them. Furthermore, we uncovered that the mechanism by which miR-7 improves atrophy-related phenotypes is independent of MBNL1, thus suggesting that miR-7 acts downstream or in parallel to MBNL1. Collectively, these results highlight an unknown function for miR-7 in muscle dysfunction through autophagy- and atrophy-related pathways and support that restoration of miR-7 levels is a candidate therapeutic target for counteracting muscle dysfunction in DM1.

Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3\' UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. In this work, we demonstrate that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in Drosophila and mouse (HSALR) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSALR mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

TRIM71/LIN-41, a phylogenetically conserved regulator of development, controls stem cell fates. Mammalian TRIM71 exhibits both RNA-binding and protein ubiquitylation activities, but the functional contribution of either activity and relevant primary targets remain poorly understood. Here, we demonstrate that TRIM71 shapes the transcriptome of mouse embryonic stem cells (mESCs) predominantly through its RNA-binding activity. We reveal that TRIM71 binds targets through 3\' untranslated region (UTR) hairpin motifs and that it acts predominantly by target degradation. TRIM71 mutations implicated in etiogenesis of human congenital hydrocephalus impair target silencing. We identify a set of primary targets consistently regulated in various human and mouse cell lines, including MBNL1 (Muscleblind-like protein 1). MBNL1 promotes cell differentiation through regulation of alternative splicing, and we demonstrate that TRIM71 promotes embryonic splicing patterns through MBNL1 repression. Hence, repression of MBNL1-dependent alternative splicing may contribute to TRIM71\'s function in regulating stem cell fates.