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...

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.