Our long-term goal is to alleviate the frataxin insufficiency that causes Friedreich ataxia (FRDA), the most common inherited ataxia. FRDA is a relentlessly progressive neurodegenerative disease with associated hypertrophic cardiomyopathy, diabetes and skeletal deformities. The ataxia is debilitating and the cardiomyopathy is often fatal. FRDA is caused by triplet repeat expansion within the first intron of the frataxin gene. Expanded GAA-TTC repeats reduce frataxin mRNA expression, but the mechanism by which this occurs is poorly understood. Currently FRDA has no effective treatment. Most FRDA patients have intact frataxin coding sequences, so the mRNA deficiency is a logical therapeutic target. To design effective therapies, we must first extend and refine our understanding of the cause for this deficiency. Our hypothesis is that transcription instigates dynamic structure formation within the GAA-TTC repeat that leads to reduced mRNA expression through multiple pathways. We hypothesize that this structure includes an extensive and persistent RNA-DNA hybrid that contributes to decreased transcription elongation efficiency and compromises the integrity of the primary transcript. To characterize the mechanism(s) leading to reduced mRNA expression in FRDA, we will: 1) Isolate transcription elongation within defined lengths of GAA-TTC repeats and measure the impediment presented by the repeats using a novel tandem reporter construct in human cell lines. 2) Determine to what extent intronic GAA repeats disrupt downstream transcription elongation or primary transcript integrity in human cells using a combination of nuclear run-on assays and oligonucleotide-directed interference with structure formation. 3) Determine the extent of transcription-dependent RNA'DNA hybrid formation within GAA-TTC repeats in human cells. The novel reporter constructs we developed to dissect the underlying causes of frataxin insufficiency will also serve as high throughput cell-based assays for candidate therapies.
