Repeat Associated Non-AUG initiated Translation mediates neurodegeneration in a Drosophila models of Fragile X-associated Tremor Ataxia Syndrome. Michelle A. Frazer¹, Fang He², Peter K. Todd². 1) Cellular & Molecular Biology, University of Michigan, Ann Arbor, MI; 2) Neurology, University of Michigan, Ann Arbor, MI.

   Fragile X-associated Tremor Ataxia Syndrome (FXTAS) is a neurodegenerative disease that results from a CGG repeat expansion in the 5UTR of FMR1. Pathogenesis in FXTAS is thought to involve a dominant RNA gain of function mechanism, whereby the CGG repeat mRNA binds to and sequesters specific RNA binding proteins. However, our group has recently discovered that the repeats are also capable of eliciting aberrant translation initiation in the 5UTR in the absence of an AUG start codon (RAN translation), leading to the production of a polyglycine-containing protein that forms ubiquitinated aggregates in cells and animal models, as occurs in patients. A critical question that emerges from this work is whether this polyglycine protein contributes directly to toxicity, or whether the neurodegeneration is mediated strictly via RNA toxic mechanisms. To investigate this question, we created strains of drosophila that decouple the potential RNA and protein mediated toxic processes. This was achieved by placing the CGG repeat in either the 5UTR or 3UTR of a heterologous gene, eGFP. Placement in the 3UTR precludes RAN translation. To enhance the protein mediated toxic effects, we have inserted an AUG start codon 5 to the repeat, which leads to increased production of the polyglycine protein. As previously reported, expression of a (CGG)₁₀₀ repeat in the 5UTR of eGFP leads to a modest rough eye phenotype with isolated oomatidial expression and a decrease in viability with ubiquitous expression compared to control flies. In lines where the CGG repeat is in the 3UTR of eGFP, there is very little overt oomatidial degeneration and no effect on viability. In contrast, flies with an ATG codon inserted 5 to the repeat, exhibit a dramatic degenerative eye phenotype and further reduced viability compared to flies lacking this ATG. These studies support a model where aberrant translation of a polyglycine protein in FXTAS contributes significantly to disease pathogenesis.