In myotonic dystrophy (DM), expression of RNA containing expanded CUG or CCUG repeats leads to misregulated alternative splicing of pre-mRNA. The repeat-bearing transcripts accumulate in nuclear foci, together with proteins in the muscleblind family, MBNL1 and MBNL2. In transgenic mice that express expanded CUG repeats, we show that the splicing defect selectively targets a group of exons that share a common temporal pattern of developmental regulation. These exons undergo a synchronized splicing switch between post-natal day 2 and 20 in wild-type mice. During this post-natal interval, MBNL1 protein translocates from a predominantly cytoplasmic to nuclear distribution. In the absence of MBNL1, these physiological splicing transitions do not occur. The splicing defect induced by expanded CUG repeats in mature muscle fibers is closely reproduced by deficiency of MBNL1 but not by deficiency of MBNL2. A parallel situation exists in human DM type 1 and type 2. MBNL1 is depleted from the muscle nucleoplasm because of sequestration in nuclear foci, and the associated splicing defects are remarkably similar to those observed in MBNL1 knockout mice. These results indicate that MBNL1 participates in the post-natal remodeling of skeletal muscle by controlling a key set of developmentally regulated splicing switches. Sequestration of MBNL1, and failure to maintain these splicing transitions, has a pivotal role in the pathogenesis of muscle disease in DM.

SUMMARY The RNA-mediated disease model for myotonic dystrophy (DM) proposes that microsatellite C(C)TG expansions express toxic RNAs which disrupt splicing regulation by altering MBNL1 and CELF1 activities. While this model explains DM manifestations in muscle, less is known about the effects of C(C)UG expression on the brain. Here, we report that Mbnl2 knockout mice develop several DM-associated CNS features including abnormal REM sleep propensity and deficits in spatial memory. Mbnl2 is prominently expressed in the hippocampus and Mbnl2 knockouts show a decrease in NMDAR synaptic transmission and impaired hippocampal synaptic plasticity. While Mbnl2 loss did not significantly alter target transcript levels in the hippocampus, mis-regulated splicing of hundreds of exons was detected using splicing microarrays, RNA-seq and HITS-CLIP. Importantly, the majority of the Mbnl2-regulated exons examined were similarly mis-regulated in DM. We propose that major pathological features of the DM brain result from disruption of the MBNL2-mediated developmental splicing program.

RNA-mediated pathogenesis is a recently developed disease model that proposes that certain types of mutant genes produce toxic transcripts that inhibit the activities of specific proteins. This pathogenesis model was proposed first for the neuromuscular disease myotonic dystrophy (DM), which is associated with the expansion of structurally related (CTG) n and (CCTG)n microsatellites in two unrelated genes. At the RNA level, these expansions form stable hairpins that alter the pre-mRNA splicing activities of two antagonistic factor families, the MBNL and CELF proteins. It is unclear which altered activity is primarily responsible for disease pathogenesis and whether other factors and biochemical pathways are involved. Here, we show that overexpression of Mbnl1 in vivo mediated by transduction of skeletal muscle with a recombinant adeno-associated viral vector rescues disease-associated muscle hyperexcitability, or myotonia, in the HSA LR poly(CUG) mouse model for DM. Myotonia reversal occurs concurrently with restoration of the normal adult-splicing patterns of four pre-mRNAs that are misspliced during postnatal development in DM muscle. Our results support the hypothesis that the loss of MBNL1 activity is a primary pathogenic event in the development of RNA missplicing and myotonia in DM and provide a rationale for therapeutic strategies designed either to overexpress MBNL1 or inhibit MBNL1 interactions with CUG and CCUG repeat expansions.microsatellite ͉ muscle ͉ pathogenesis ͉ splicing ͉ transgenic D uring mammalian embryonic and postnatal periods, tissues are initially formed and then extensively remodeled to meet the evolving demands of the developing organism. This postnatal remodeling process requires a dynamic series of biochemical events at both the transcriptional and posttranscriptional levels. For vertebrate skeletal muscle, myogenic precursor cells derived from somites migrate during embryogenesis and, ultimately, fuse to form primary and secondary myofibers (1). After birth, fetal muscle must be modified to perform adult functions, and this transition (e.g., a suckling versus a highly mobile adult mouse) requires the sequential replacement of fetal protein isoforms with their adult counterparts. For some muscle genes, this process involves skipping of fetal exons and͞or the inclusion of adult exons during pre-mRNA splicing. Remarkably, this normal splicing transition is altered specifically in the neuromuscular disease myotonic dystrophy (DM) (2-4).Adult-onset DM is a multisystemic degenerative disease (4). Characteristic features of this disorder include skeletal muscle myotonia (muscle hyperexcitability), weakness͞wasting, heart conduction defects, particulate subcapsular cataracts, and insulin insensitivity. The genetic basis of DM is unusual because it is associated with different unstable microsatellites in two unrelated genes. DM type 1 (DM1) is caused by the expansion of a (CTG) n repeat in the 3Ј UTR of the DMPK (dystrophia myotonica protein kinase) gene (5). Normal DMPK microsatellite lengt...

The MBNL and CELF proteins act antagonistically to control the alternative splicing of specific exons during mammalian postnatal development. This process is dysregulated in myotonic dystrophy because MBNL proteins are sequestered by (CUG)n and (CCUG)n RNAs expressed from mutant DMPK and ZNF9 genes, respectively. While these observations predict that MBNL proteins have a higher affinity for these pathogenic RNAs versus their normal splicing targets, we demonstrate that MBNL1 possesses comparably high affinities for (CUG)n and (CAG)n RNAs as well as a splicing target, Tnnt3. Mapping of a MBNL1-binding site upstream of the Tnnt3 fetal exon indicates that a preferred binding site for this protein is a GC-rich RNA hairpin containing a pyrimidine mismatch. To investigate how pathogenic RNAs sequester MBNL1 in DM1 cells, we used a combination of chemical/enzymatic structure probing and electron microscopy to determine that MBNL1 forms a ring-like structure which binds to the dsCUG helix. While the MBNL1 N-terminal region is required for RNA binding, the C-terminal region mediates homotypic interactions which may stabilize intra- and/or inter-ring interactions. Our results provide a mechanistic basis for dsCUG-induced MBNL1 sequestration and highlight a striking similarity in the binding sites for MBNL proteins on splicing precursor and pathogenic RNAs.

We address the challenge of detecting the contribution of noncoding mutations to disease with a deep-learning-based framework that predicts specific regulatory effects and the deleterious impact of genetic variants. Applying this framework to 1,790 Autism Spectrum Disorder (ASD) simplex families reveals disease causality of noncoding mutations: ASD probands harbor both transcriptional and post-transcriptional regulation-disrupting de novo mutations of significantly higher functional impact than unaffected siblings. Further analysis suggests involvement of noncoding mutations in synaptic transmission and neuronal development, and taken together with prior studies reveal a convergent genetic landscape of coding and noncoding mutations in ASD. We demonstrate that sequences carrying prioritized proband mutations possess allele-specific regulatory activity, and highlight a link between noncoding mutations and IQ heterogeneity in ASD probands. Our predictive genomics framework illuminates the role of noncoding mutations in ASD, prioritizes high impact mutations for further study, and is broadly applicable to complex human diseases.

Efficient delivery of gene therapy vectors across the blood-brain barrier (BBB) is the holy grail of neurological disease therapies. A variant of the neurotropic vector adeno-associated virus (AAV) serotype 9, called AAV-PHP.B, was shown to very efficiently deliver transgenes across the BBB in C57BL/6J mice. Based on our recent observation that this phenotype is mouse strain dependent, we used whole-exome sequencing-based genetics to map this phenotype to a specific haplotype of lymphocyte antigen 6 complex, locus A (Ly6a) (stem cell antigen-1 [Sca-1]), which encodes a glycosylphosphatidylinositol (GPI)anchored protein whose function had been thought to be limited to the biology of hematopoiesis. Additional biochemical and genetic studies definitively linked high BBB transport to the binding of AAV-PHP.B with LY6A (SCA-1). These studies identify, for the first time, a ligand for this GPIanchored protein and suggest a role for it in BBB transport that could be hijacked by viruses in natural infections or by gene therapy vectors to treat neurological diseases.

The muscleblind-like (MBNL) genes encode alternative splicing factors that are essential for the postnatal development of multiple tissues, and the inhibition of MBNL activity by toxic C(C)UG repeat RNAs is a major pathogenic feature of the neuromuscular disease myotonic dystrophy. While MBNL1 controls fetal-to-adult splicing transitions in muscle and MBNL2 serves a similar role in the brain, the function of MBNL3 in vivo is unknown. Here, we report that mouse Mbnl3, which encodes protein isoforms that differ in the number of tandem zinc-finger RNA-binding motifs and subcellular localization, is expressed primarily during embryonic development but also transiently during injury-induced adult skeletal muscle regeneration. Mbnl3 expression is required for normal C2C12 myogenic differentiation and high-throughput sequencing combined with cross-linking/immunoprecipitation analysis indicates that Mbnl3 binds preferentially to the 3' untranslated regions of genes implicated in cell growth and proliferation. In addition, Mbnl3ΔE2 isoform knockout mice, which fail to express the major Mbnl3 nuclear isoform, show age-dependent delays in injury-induced muscle regeneration and impaired muscle function. These results suggest that Mbnl3 inhibition by toxic RNA expression may be a contributing factor to the progressive skeletal muscle weakness and wasting characteristic of myotonic dystrophy.

Highlights d cTag-CLIP provides a strategy to study cell-specific RNA regulation in vivo d NOVA2 controls unique RNA splicing programs in inhibitory and excitatory neurons d NOVA2 cTag-CLIP reveals a new mechanism of cell-specific AS regulation d NOVA2 regulates intron retention as a cis-acting scaffold for AS factor PTBP2