Date of Award
Doctor of Philosophy (PhD)
Ectopic lipid deposition in non-adipose tissues is a hallmark of metabolic diseases, such as obesity and diabetes, and associated with cell dysfunction, cell death, and organ dysfunction. However, the molecular mechanisms linking excess free fatty acid to cell death are poorly understood. In order to identify genes critical for lipotoxicity, our lab performed a genetic screen in Chinese hamster ovary cells and isolated mutants that were resistant to lipotoxic death. In one of the lines, mutant 2B1, an allele encoding RNASET2, a ribonuclease of the T2 family, was disrupted by proviral insertion. We found that RNASET2 is induced during lipotoxicity and is required for palmitate-induced oxidative stress and cell death. RNASET2 functions upstream of cytoplasmic accumulation of the non-canonical small nucleolar RNAs (snoRNAs), which have previously been shown to be required for amplification of lipotoxic reactive oxygen species (ROS). These functions are critically dependent on its catalytic activity.
Haploinsufficiency of RNASET2 confers increased antioxidant capacity and generalized resistance to ROS production and oxidative stress-mediated cell death in cultured cells. Furthermore, knockdown of RNASET2 in the Drosophila fat body confers increased survival and protection from oxidative damage in the setting of oxidative stress inducers. Together, these findings demonstrate that RNASET2 regulates antioxidant tone and is required for pathological ROS responses.
Our observation that enzymatic activity of RNASET2 is required for its function in lipotoxic and oxidative stress suggested that RNASET2 functions by degrading RNA in these stress response pathways. Through microarray analysis, we identified XIAP mRNA as a potential substrate of RNASET2. Levels of this mRNA are inversely correlated with RNASET2 levels and dependent on lysosomal function. Both haploinsufficiency for RNASET2 and lysosomal perturbation prolong the half-life of this mRNA. Moreover, XIAP mRNA associates with the lysosomal fraction of wild type cells and increased lysosomal XIAP is found in the RNASET2 haploinsuffient 2B1 cells.
Taken together, our data provide evidence of a previously unsuspected role for RNASET2 in the response to metabolic stress and suggest a novel mechanism for selective degradation of an RNA transcript in the lysosome.
Chair and Committee
Jean E Schaffer
Jennifer Duncan, Eric Galburt, Phyllis Hanson, Stephen Kornfeld
Caputa, George, "Lysosomal RNASET2 is Required for Metabolic Stress-Mediated ROS Generation and Cell Death" (2015). Arts & Sciences Electronic Theses and Dissertations. 640.