SUMMARY Inhibition of muscleblind-like (MBNL) activity due to sequestration by microsatellite expansion RNAs is a major pathogenic event in the RNA-mediated disease myotonic dystrophy (DM). Although MBNL1 and MBNL2 bind to nascent transcripts to regulate alternative splicing during muscle and brain development, another major binding site for the MBNL protein family is the 3′ untranslated region of target RNAs. Here, we report that depletion of Mbnl proteins in mouse embryo fibroblasts leads to mis-regulation of thousands of alternative polyadenylation events. HITS-CLIP and minigene reporter analyses indicate that these polyadenylation switches are a direct consequence of MBNL binding to target RNAs. Mis-regulated alternative polyadenylation also occurs in skeletal muscle in a mouse polyCUG model and human DM resulting in the persistence of neonatal polyadenylation patterns. These findings reveal a novel developmental function for MBNL proteins and demonstrate that DM is characterized by mis-regulation of pre-mRNA processing at multiple levels.

Myotonic dystrophy (DM) is a multi-systemic disease that impacts cardiac and skeletal muscle as well as the central nervous system (CNS). DM is unusual because it is an RNA-mediated disorder due to the expression of toxic microsatellite expansion RNAs that alter the activities of RNA processing factors, including the muscleblind-like (MBNL) proteins. While these mutant RNAs inhibit MBNL1 splicing activity in heart and skeletal muscles, Mbnl1 knockout mice fail to recapitulate the full-range of DM symptoms in these tissues. Here, we generate mouse Mbnl compound knockouts to test the hypothesis that Mbnl2 functionally compensates for Mbnl1 loss. Although Mbnl1−/−; Mbnl2−/− double knockouts (DKOs) are embryonic lethal, Mbnl1−/−; Mbnl2+/− mice are viable but develop cardinal features of DM muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in Mbnl1−/− knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the hypothesis that compound loss of MBNL function is a critical event in DM pathogenesis and provide novel mouse models to investigate additional pathways disrupted in this RNA-mediated disease.

SUMMARY 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.