Trinucleotide expansions cause disease by both protein-and RNAmediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.T ranslation of mRNA into protein is an exquisitely regulated, almost error-free process. Well-established rules of translational initiation have been used as a cornerstone in biology to understand gene expression and to predict the consequences of disease-causing mutations (1). For microsatellite expansion disorders, mutations within or outside ATG-initiated ORFs are thought to cause disease either by protein gain-of-function, protein loss-of-function, or RNA gain-of-function mechanisms (2, 3).Microsatellite expansion mutations that express polyglutamine (polyGln) expansion proteins include Huntington disease (Huntingtin, HD), spinal bulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17. Since the discovery of these CAG·CTG expansion mutations, efforts to understand disease mechanisms have focused on elucidating the molecular effects of the polyGln proteins expressed from these loci. Although these polyGln expansion proteins bear no similarity to each other apart from the polyGln tract, a hallmark of these diseases is protein accumulation and aggregation in nuclear or cytoplasmic inclusions. Surprisingly, although the polyGln expansion proteins are widely expressed in the CNS and other tissues, only restricted populations of neurons are vulnerable in each disease (3).Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are the best-characterized examples of RNA-mediated expansion disorders (2). The mutation causing DM1 is a CTG-repeat expansion located in the 3′ UTR of the dystrophia myotonica-protein kinase (DMPK) gene. Although DM1 can be clinically more severe than DM2, the discovery of the DM2 mutation and several mouse models provide strong support that many features of these diseases result from RNA gain-of-function effects in which the dysregulation of RNA-binding proteins is mediated by the expression of CUG and CCUG transcripts (4). Additionally, RNA gain-of-function effects have been reported for CGG and CAG expansion RNAs (5, 6).Both RNA and protein mechanisms appear to operate...

The human transcriptome is composed of a vast RNA population that undergoes further diversification by splicing. Detecting specific splice sites in this large sequence pool is the responsibility of the major and minor spliceosomes in collaboration with numerous splicing factors. This complexity makes splicing susceptible to sequence polymorphisms and deleterious mutations. Indeed, RNA mis-splicing underlies a growing number of human diseases with substantial societal consequences. Here, we provide an overview of RNA splicing mechanisms followed by a discussion of disease-associated errors, with an emphasis on recently described mutations that have provided new insights into splicing regulation. We also discuss emerging strategies for splicing-modulating therapy.

The neuromuscular disease myotonic dystrophy (DM) is caused by microsatellite repeat expansions at two different genomic loci. Mutant DM transcripts are retained in the nucleus together with the muscleblind (Mbnl) proteins, and these abnormal RNAs somehow interfere with pre-mRNA splicing regulation. Here, we show that disruption of the mouse Mbnl1 gene leads to muscle, eye, and RNA splicing abnormalities that are characteristic of DM disease. Our results support the hypothesis that manifestations of DM can result from sequestration of specific RNA binding proteins by a repetitive element expansion in a mutant RNA.

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.

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Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene. In skeletal muscles, DM1 may involve a novel, RNA-dominant disease mechanism in which transcripts from the mutant DMPK allele accumulate in the nucleus and compromise the regulation of alternative splicing. Here we show evidence for a similar disease mechanism in brain. Examination of post-mortem DM1 tissue by fluorescence in situ hybridization indicates that the mutant DMPK mRNA, with its expanded CUG repeat in the 3'-untranslated region, is widely expressed in cortical and subcortical neurons. The mutant transcripts accumulate in discrete foci within neuronal nuclei. Proteins in the muscleblind family are recruited into the RNA foci and depleted elsewhere in the nucleoplasm. In parallel, a subset of neuronal pre-mRNAs show abnormal regulation of alternative splicing. These observations suggest that CNS impairment in DM1 may result from a deleterious gain-of-function by mutant DMPK mRNA.

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF-and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.

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.