Proof of concept of blockmiR induced MBNL rescue in Myotonic dystrophy muscle cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA725240 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14327017_1.fastq.gz | Fastqsanger.gz | |||
| SRR14327017_2.fastq.gz | Fastqsanger.gz | |||
| SRR14327018_1.fastq.gz | Fastqsanger.gz | |||
| SRR14327018_2.fastq.gz | Fastqsanger.gz | |||
| SRR14327019_1.fastq.gz | Fastqsanger.gz |
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Project description: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 DMPK gene that is correctly transcribed and physically sequesters the MBNL family of proteins. The high-affinity binding occurring between the proteins and the repetitions disallows 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 off of previously demonstrated evidence showing that the silencing of miR-23b and miR-218 can increase MBNL1 and 2 protein in DM1 cells. Here we use blockmiR antisense technology to block the binding sites of these miRNAs in order to increase MBNL translation into protein without binding to the microRNAs in DM1 muscle cells. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and transcriptomic expression proving that this novel and highly specific strategy could be a potentially viable therapy in Vivo.
2022-07-31 | GSE173359 | GEO
Project description: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.
| S-EPMC8888893 | biostudies-literature
Project description:For some neurological disorders, disease is primarily RNA-mediated due to expression of non-coding microsatellite expansion RNAs (RNAexp). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNAexp in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases.
2015-07-30 | GSE68890 | GEO
Project description:For some neurological disorders, disease is primarily RNA-mediated due to expression of non-coding microsatellite expansion RNAs (RNAexp). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNAexp in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases. HITS-CLIP analysis was performed to identify RNA binding sites of MBNL2 in control, DM type 1 (DM1), and DM type 2 (DM2) autopsy-derived brain (n=3). Two regions of the brain selected for the study included the frontal cortex and hippocampus. Libraries were sequenced and wiggle files were generated for each biological replicate as well as three pooled biological replicates (3BRs) per group (control, DM1, and DM2). In addition, differential CLIP analysis (dCLIP) was performed to normalize binding data between groups and identify statistically significant changes in binding. The dCLIP analysis generated bedgraph files representing normalized binding profiles of MBNL2 in each group (control, DM1, and DM2) for visualization and comparative analysis. PolyA-seq was performed on control, DM1, and DM2 autopsy-derived brain samples (hippocampus and frontal cortex, n=3) as well as wild-type (WT) and Mbnl1; Mbnl2 conditional double knockout (Mbnl1-/-; Mbnl2c/c; Nestin-Cre+/- or DKO) brain (n=3). Libraries were sequenced and the resultant files were processed and aligned to the reference genomes (hg19 and mm10). Further computational processing was performed to remove internal oligo(dT) mis-priming events, identify valid polyA sites, and trim to the exact polyA sites. BedGraph files were generated for each group in human (control, DM1, and DM2) and mouse (WT and Mbnl DKO) for comparative visualization.
2015-07-30 | E-GEOD-68890 | biostudies-arrayexpress
Project description:Myotonic dystrophy (DM) is a multi-systemic disease that severely impacts cardiac and skeletal muscle functions as well as the central nervous system. DM is unusual because it is RNA-mediated disease due to the expression of C(C)UG expansion RNAs that inhibit the activities of the muscleblind-like (MBNL) proteins. In mice, studies using Mbnl1 and Mbnl2 single knockouts have revealed that Mbnl1 plays a predominant role in skeletal and heart muscle alternative splicing regulation while Mbnl2 performs an analogous splicing function in the brain. However, Mbnl single knockout models fail to recapitulate the full-range of adult-onset DM muscle symptoms. Here, we report that Mbnl1; Mbnl2 double knockouts are embryonic lethal while Mbnl1-/-; Mbnl2+/- mice, which express no Mbnl1 and reduced levels of Mbnl2, are viable but develop cardinal features of adult-onset DM cardiac and skeletal muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in both Mbnl1-/- and Mbnl1-/-; Mbnl2+/- knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the MBNL loss-of-function model for DM and provide novel Mbnl compound knockout models to investigate the molecular pathways disrupted by RNA-mediated disease. Mbnl2 protein-RNA interactions were assessed in 4-month-old WT and Mbnl1-/- quadriceps muscles in triplicates by HITS-CLIP.
2013-12-01 | E-GEOD-47794 | biostudies-arrayexpress
Project description:Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. Myotonic dystrophy is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of myotonic dystrophy in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2 which is characterized by nucleotide repeat expansions often greater than 5000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this cell model, we found impaired dystrophin expression, MBNL foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls, DM1 and DM2 subjects and differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. IPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing and MBNL sequestration. High resolution imaging revealed tight association between MBNL clusters and RNA FISH foci in DM1. Ca2+ transients differed between DM1 and DM2 IPSC-derived cardiomyocytes and from healthy control cells. RNA-sequencing from DM1 and DM2 iPSC-derived cardiomyocytes both altered gene expression as well as distinct splicing patterns as differential between DM1 and DM2. Together these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.
2019-02-12 | GSE125638 | GEO
Project description:In this Study, we used RNA-targeting Cas9 (RCas9) to reverse characteristic Myotonic Dystrophy (DM1) cellular phenotypes such as elimination of RNA foci, MBNL relocalization, and reversal of transcriptome-wide splicing in a mouse model of myotonic Dystrophy (DM1). Furthermore we show that gene expression is not altered with RCas9 treatment in WT mice with or without treatment with immunosuppression
2020-07-01 | GSE152033 | GEO
Project description:Myotonic dystrophy (DM) is a multi-systemic disease that severely impacts cardiac and skeletal muscle functions as well as the central nervous system. DM is unusual because it is RNA-mediated disease due to the expression of C(C)UG expansion RNAs that inhibit the activities of the muscleblind-like (MBNL) proteins. In mice, studies using Mbnl1 and Mbnl2 single knockouts have revealed that Mbnl1 plays a predominant role in skeletal and heart muscle alternative splicing regulation while Mbnl2 performs an analogous splicing function in the brain. However, Mbnl single knockout models fail to recapitulate the full-range of adult-onset DM muscle symptoms. Here, we report that Mbnl1; Mbnl2 double knockouts are embryonic lethal while Mbnl1-/-; Mbnl2+/- mice, which express no Mbnl1 and reduced levels of Mbnl2, are viable but develop cardinal features of adult-onset DM cardiac and skeletal muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in both Mbnl1-/- and Mbnl1-/-; Mbnl2+/- knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the MBNL loss-of-function model for DM and provide novel Mbnl compound knockout models to investigate the molecular pathways disrupted by RNA-mediated disease.
2013-12-01 | GSE47794 | GEO
Project description:MBNL Sequestration by Toxic RNAs and RNA Mis-Processing in the Myotonic Dystrophy Brain
| PRJNA283977 | ENA
Project description:Myotonic dystrophy type 1 (DM1) is a CTG microsatellite expansion (CTGexp) disorder caused by expression of CUGexp RNAs. These mutant RNAs alter the activities of RNA processing factors, including MBNL proteins, leading to reversion to specific fetal isoforms in adult tissues and DM1 pathology. While this pathogenesis model accounts for adult-onset disease, the molecular basis of congenital DM (CDM) is unknown. Here, we test the hypothesis that disruption of developmentally regulated RNA alternative processing pathways contributes to CDM disease. Analysis of alternative splicing (AS) transitions during human myogenesis reveals hundreds of AS events that undergo prenatal isoform transitions and both AS and alternative polyadenylation abnormalities are prominently detectable in infant CDM muscle biopsies. While the majority of RNA targets are also mis-regulated in adult-onset DM1, splicing dysregulation is significantly more severe in CDM. Since many of these mis-processing events are MBNL-regulated, we generated mouse Mbnl double (Mbnl1; Mbnl2) and triple (Mbnl1; Mbnl2; Mbnl3) muscle-specific knockout models that recapitulate the congenital myopathy, gene expression and spliceopathy defects characteristic of CDM. This study demonstrates RNA mis-processing is a major pathogenic factor in CDM and provides novel mouse models to further examine roles for co/post-transcriptional gene regulation during tissue development.
2017-07-12 | GSE97806 | GEO