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Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy

ABSTRACT: 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.

OTHER RELATED OMICS DATASETS IN: PRJNA382960

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE97806 | GEO | 2017/07/12

SECONDARY ACCESSION(S): PRJNA382960

REPOSITORIES: GEO

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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.

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Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy

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