Reactive oxygen species (ROS) are natural byproducts of respiration and are damaging in cells that have weakened cellular defense systems or are exposed to environmental stress. While the deleterious effects of DNA,protein, and lipid oxidation are well-known, the impact of oxidatively damaged RNAs has not been investigated. To address this issue, Saccharomyces cerevisiae will be used as a model system to study the mechanisms by which oxidized RNAs are handled in eukaryotic cells. Preliminary studies showed that S. cerevisiae lacking SSD1 was more sensitive to the oxidant diamide than wild-type cells. SSD1 encodes for a protein (Ssd1) that is homologous to exoribonucleases, regulates longevity, and may be potentially conserved in humans. Following treatment of yeast cells with diamide, epitope-tagged Ssd1 localizes to P-bodies, which are cytoplasmic structures that are thought to be translationally repressed mRNAs undergoing decay. These results suggest that SSD1may be involved in the decay or repair of RNAs damaged by oxidants.
The specific aims of this research proposal are to examine if SSD1functions in the handling of oxidatively damaged RNAs, to elucidate how SSD1mediates oxidative stress resistance, and to identify other components involved in handling of oxidized RNAs. Since little is known about the fate of oxidized RNAs in cells, P-bodies will be analyzed to determine if they are the sites of localization for oxidatively damaged RNAs using immunofluorescence and confocal microscopy. SSD1 will also be examined to determineif it functions in repressing accumulation of oxidatively damaged RNAs in vivo, P- body formation, binding of oxidized RNAs in vivo and in vitro, translational repression, decapping, and degradation of RNAs damaged by oxidants. Results of this study may elucidate how oxidatively damaged RNAs contribute to aging in eukaryotic cells and may give insight into how neurodegenerative diseases that are associated with the cellular accumulation of oxidized RNAs develop. Relevance: The goal of this research is to better understand how our cells detect and repair RNAs damaged by oxidants, which all humans are exposed to when food is converted into energy that our bodies use. Findings from this study will greatly enhance our knowledge of how RNA damage contributes to cellular aging and may suggest ways of medically treating neurodegenerative diseases brought on by old age.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F08-G (20))
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Humble, Michael C
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Yale University
Anatomy/Cell Biology
Schools of Medicine
New Haven
United States
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Rojas, Marta; Farr, George W; Fernandez, Cesar F et al. (2012) Yeast Gis2 and its human ortholog CNBP are novel components of stress-induced RNP granules. PLoS One 7:e52824