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. ? ?

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Molecular Genetics B Study Section (MGB)
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Gwinn, Katrina
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Louisiana State Univ Hsc New Orleans
Schools of Medicine
New Orleans
United States
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Halabi, Anasheh; Ditch, Scott; Wang, Jeffrey et al. (2012) DNA mismatch repair complex MutS? promotes GAA·TTC repeat expansion in human cells. J Biol Chem 287:29958-67
Mancuso, Miriam; Sammarco, Mimi C; Grabczyk, Ed (2010) Transposon Tn7 preferentially inserts into GAA*TTC triplet repeats under conditions conducive to Y*R*Y triplex formation. PLoS One 5:e11121
Banerjee, Ayan; Sammarco, Mimi C; Ditch, Scott et al. (2009) A dual reporter approach to quantify defects in messenger RNA processing. Anal Biochem 395:237-43
Ditch, Scott; Sammarco, Mimi C; Banerjee, Ayan et al. (2009) Progressive GAA.TTC repeat expansion in human cell lines. PLoS Genet 5:e1000704
Banerjee, Ayan; Sammarco, Mimi C; Ditch, Scott et al. (2009) A novel tandem reporter quantifies RNA polymerase II termination in mammalian cells. PLoS One 4:e6193
Grabczyk, Ed; Mancuso, Miriam; Sammarco, Mimi C (2007) A persistent RNA.DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro. Nucleic Acids Res 35:5351-9