Fragile X-associated Tremor Ataxia Syndrome (FXTAS) results from a CGG repeat expansion in the 5’UTR of FMR1. This repeat is thought to elicit toxicity as RNA yet disease brains contain ubiquitin-positive neuronal inclusions, a pathologic hallmark of protein-mediated neurodegeneration. We explain this paradox by demonstrating that CGG repeats trigger repeat associated non-AUG initiated (RAN) translation of a cryptic polyglycine-containing protein, FMRpolyG. FMRpolyG accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, mouse disease models and FXTAS patient brains. CGG RAN translation occurs in at least two of three possible reading frames at repeat sizes ranging from normal (25) to pathogenic (90), but inclusion formation only occurs with expanded repeats. In Drosophila, CGG repeat toxicity is suppressed by eliminating RAN translation and enhanced by increased polyglycine protein production. These studies expand the growing list of nucleotide repeat disorders where RAN translation occurs and provide evidence that RAN translation contributes to neurodegeneration.

SUMMARY Repeat associated non-AUG (RAN) translation produces toxic polypeptides from nucleotide repeat expansions in the absence of an AUG start codon and contributes to neurodegenerative disorders such as ALS and Fragile X Tremor/Ataxia Syndrome (FXTAS). How RAN translation occurs is unknown. Here we define the critical sequence and initiation factors that mediate CGG repeat RAN translation in the 5′ leader of Fragile X mRNA, FMR1. Our results reveal that CGG RAN translation is 30–40% as efficient as AUG initiated translation, is m7G-cap and eIF4E-dependent, requires the eIF4A helicase, and is strongly influenced by repeat length. However, it displays a dichotomous requirement for initiation site selection between reading frames, with initiation in the +1 frame, but not the +2 frame, occurring at near-cognate start codons upstream of the repeat. These data support a model where RAN translation at CGG repeats utilizes cap-dependent ribosomal scanning, yet bypasses normal requirements for start codon selection.

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Fragile X-associated tremor ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG trinucleotide repeat expansion in the 5' UTR of the Fragile X gene, FMR1. FXTAS is thought to arise primarily from an RNA gain-of-function toxicity mechanism. However, recent studies demonstrate that the repeat also elicits production of a toxic polyglycine protein, FMRpolyG, via repeat-associated non-AUG (RAN)-initiated translation. Pathologically, FXTAS is characterized by ubiquitin-positive intranuclear neuronal inclusions, raising the possibility that failure of protein quality control pathways could contribute to disease pathogenesis. To test this hypothesis, we used Drosophila- and cell-based models of CGG-repeat-associated toxicity. In Drosophila, ubiquitin proteasome system (UPS) impairment led to enhancement of CGG-repeat-induced degeneration, whereas overexpression of the chaperone protein HSP70 suppressed this toxicity. In transfected mammalian cells, CGG repeat expression triggered accumulation of a UPS reporter in a length-dependent fashion. To delineate the contributions from CGG repeats as RNA from RAN translation-associated toxicity, we enhanced or impaired the production of FMRpolyG in these models. Driving expression of FMRpolyG enhanced induction of UPS impairment in cell models, while prevention of RAN translation attenuated UPS impairment in cells and suppressed the genetic interaction with UPS manipulation in Drosophila. Taken together, these findings suggest that CGG repeats induce UPS impairment at least in part through activation of RAN translation.

Fragile X Tremor Ataxia Syndrome (FXTAS) is a common inherited neurodegenerative disorder caused by expansion of a CGG trinucleotide repeat in the 5′UTR of the fragile X syndrome (FXS) gene, FMR1. The expanded CGG repeat is thought to induce toxicity as RNA, and in FXTAS patients mRNA levels for FMR1 are markedly increased. Despite the critical role of FMR1 mRNA in disease pathogenesis, the basis for the increase in FMR1 mRNA expression is unknown. Here we show that overexpressing any of three histone deacetylases (HDACs 3, 6, or 11) suppresses CGG repeat–induced neurodegeneration in a Drosophila model of FXTAS. This suppression results from selective transcriptional repression of the CGG repeat–containing transgene. These findings led us to evaluate the acetylation state of histones at the human FMR1 locus. In patient-derived lymphoblasts and fibroblasts, we determined by chromatin immunoprecipitation that there is increased acetylation of histones at the FMR1 locus in pre-mutation carriers compared to control or FXS derived cell lines. These epigenetic changes correlate with elevated FMR1 mRNA expression in pre-mutation cell lines. Consistent with this finding, histone acetyltransferase (HAT) inhibitors repress FMR1 mRNA expression to control levels in pre-mutation carrier cell lines and extend lifespan in CGG repeat–expressing Drosophila. These findings support a disease model whereby the CGG repeat expansion in FXTAS promotes chromatin remodeling in cis, which in turn increases expression of the toxic FMR1 mRNA. Moreover, these results provide proof of principle that HAT inhibitors or HDAC activators might be used to selectively repress transcription at the FMR1 locus.

Objective Repeat associated non-AUG (RAN) translation drives production of toxic proteins from pathogenic repeat sequences in multiple untreatable neurodegenerative disorders. Fragile X-associated tremor/ataxia syndrome (FXTAS) is one such condition, resulting from a CGG trinucleotide repeat expansion in the 5′ leader sequence of the FMR1 gene. RAN proteins from the CGG repeat accumulate in ubiquitinated inclusions in FXTAS patient brains and elicit toxicity. In addition to the CGG repeat, an antisense mRNA containing a CCG repeat is also transcribed from the FMR1 locus. We evaluated whether this antisense CCG repeat supports RAN translation and contributes to pathology in FXTAS patients. Methods We generated a series of CCG RAN translation specific reporters and utilized them to measure RAN translation from CCG repeats in multiple reading frames in transfected cells. We also developed antibodies against predicted CCG RAN proteins and used immunohistochemistry and immunofluorescence on FXTAS patient tissues to measure their accumulation and distribution. Results RAN translation from CCG repeats is supported in all three potential reading frames, generating polyproline, polyarginine, and polyalanine proteins, respectively. Their production occurs whether or not the natural AUG start upstream of the repeat in the proline reading frame is present. All three frames show greater translation at larger repeat sizes. Antibodies targeted to the antisense FMR polyproline and polyalanine proteins selectively stain nuclear and cytoplasmic aggregates in FXTAS patients and colocalize with ubiquitinated neuronal inclusions. Interpretation RAN translation from antisense CCG repeats generates novel proteins that accumulate in ubiquitinated inclusions in FXTAS patients.

Fragile X premutation-associated disorders, including Fragile X-associated Tremor Ataxia Syndrome, result from unmethylated CGG repeat expansions in the 5' untranslated region (UTR) of the FMR1 gene. Premutation-sized repeats increase FMR1 transcription but impair rapid translation of the Fragile X mental retardation protein (FMRP), which is absent in Fragile X Syndrome (FXS). Normally, FMRP binds to RNA and regulates metabotropic glutamate receptor (mGluR)-mediated synaptic translation, allowing for dendritic synthesis of several proteins. FMRP itself is also synthesized at synapses in response to mGluR activation. However, the role of activity-dependent translation of FMRP in synaptic plasticity and Fragile X-premutation-associated disorders is unknown. To investigate this question, we utilized a CGG knock-in mouse model of the Fragile X premutation with 120-150 CGG repeats in the mouse Fmr1 5' UTR. These mice exhibit increased Fmr1 mRNA production but impaired FMRP translational efficiency, leading to a modest reduction in basal FMRP expression. Cultured hippocampal neurons and synaptoneurosomes derived from CGG KI mice demonstrate impaired FMRP translation in response to the group I mGluR agonist 3,5-dihydroxyphenylglycine. Electrophysiological analysis reveals enhanced mGluR-mediated long-term depression (mGluR-LTD) at CA3-CA1 synapses in acute hippocampal slices prepared from CGG KI mice relative to wild-type littermates, similar to Fmr1 knockout mice. However, unlike mGluR-LTD in mice completely lacking FMRP, mGluR-LTD in CGG knock-in mice remains dependent on new protein synthesis. These studies demonstrate partially overlapping synaptic plasticity phenotypes in mouse models of FXS and Fragile X premutation disorders and support a role for activity-dependent synthesis of FMRP in enduring forms of synaptic plasticity.

Nucleotide repeat expansions can elicit neurodegeneration as RNA by sequestering specific RNA-binding proteins, preventing them from performing their normal functions. Conversely, mutations in RNA-binding proteins can trigger neurodegeneration at least partly by altering RNA metabolism. In Fragile X-associated tremor/ataxia syndrome (FXTAS), a CGG repeat expansion in the 5'UTR of the fragile X gene (FMR1) leads to progressive neurodegeneration in patients and CGG repeats in isolation elicit toxicity in Drosophila and other animal models. Here, we identify the amyotrophic lateral sclerosis (ALS)-associated RNA-binding protein TAR DNA-binding protein (TDP-43) as a suppressor of CGG repeat-induced toxicity in a Drosophila model of FXTAS. The rescue appears specific to TDP-43, as co-expression of another ALS-associated RNA-binding protein, FUS, exacerbates the toxic effects of CGG repeats. Suppression of CGG RNA toxicity was abrogated by disease-associated mutations in TDP-43. TDP-43 does not co-localize with CGG RNA foci and its ability to bind RNA is not required for rescue. TDP-43-dependent rescue does, however, require fly hnRNP A2/B1 homologues Hrb87F and Hrb98DE. Deletions in the C-terminal domain of TDP-43 that preclude interactions with hnRNP A2/B1 abolish TDP-43-dependent rescue of CGG repeat toxicity. In contrast, suppression of CGG repeat toxicity by hnRNP A2/B1 is not affected by RNAi-mediated knockdown of the fly TDP-43 orthologue, TBPH. Lastly, TDP-43 suppresses CGG repeat-triggered mis-splicing of an hnRNP A2/B1-targeted transcript. These data support a model in which TDP-43 suppresses CGG-mediated toxicity through interactions with hnRNP A2/B1 and suggest a convergence of pathogenic cascades between repeat expansion disorders and RNA-binding proteins implicated in neurodegenerative disease.