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Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model.

ABSTRACT: 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 (?) and 2'-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with ? 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 ?-mediated CUG repeat stabilization. ? 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.

SUBMITTER: deLorimier E

PROVIDER: S-EPMC4227782 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model.

deLorimier Elaine E Coonrod Leslie A LA Copperman Jeremy J Taber Alex A Reister Emily E EE Sharma Kush K Todd Peter K PK Guenza Marina G MG Berglund J Andrew JA

Nucleic acids research 20141010 20

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-m ...[more]

PMID: 25303993

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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.

Dataset Information

Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model.

Publications

Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model.

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