Document Type

PhD diss.

Date of Degree

2010

Degree Name

PhD (Doctor of Philosophy)

Department

Free Radical and Radiation Biology

First Advisor

Prabhat C. Goswami

Abstract

Eukaryotic gene expression is a complex process and can be controlled at the level of transcription, post-transcription or translation and post-translation. In recent years, there is a growing interest in understanding the role of 3'-untranslated region (UTR) in regulating mRNA turnover and translation. The 3'-UTR harbors the poly(A) signal and post-transcriptional regulatory sequences like miRNA and AU-rich elements (AREs). The presence of multiple poly(A) sites often results in multiple transcripts; shorter transcripts correlating with more protein abundance. Manganese superoxide dismutase (MnSOD) is a nuclear encoded and mitochondrial matrix localized antioxidant enzyme that converts mitochondrial generated superoxide to hydrogen peroxide. Human MnSOD has two poly(A) sites resulting in two transcripts: 1.5 and 4.2 kb. We hypothesize that the 3'-UTR of MnSOD regulates its mRNA and protein levels as well as activity in response to growth states and environmental stress. Results from a Q-RT-PCR assay showed a preferential accumulation of the shorter MnSOD transcript during quiescence, which correlated with an increase in MnSOD activity. The accumulation of the longer MnSOD transcript during proliferation was associated with a decrease in MnSOD activity. Log transformed expression ratio of the longer to shorter transcript was also higher in proliferating epithelial non-cancerous (mammary: MCF-10A) and cancer cells (mammary: MB-231, SUM 159; oral squamous: SQ20B, FaDu, Cal27; and lung: A549, H292), suggesting that the abundance of the longer transcript is independent of cellular transformation status, instead it is dependent on cellular growth state. Interestingly, the abundance of the longer transcript directly correlated with percent S-phase (R2=0.86). The shorter transcript was enriched in irradiated MB-231 cells. MCF-10A cells exposed to 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of the environmental pollutant polychlorinated biphenyl 3, showed a significant decrease in the abundance of the 4.2 kb transcript due to a faster mRNA turnover, 14 h compared to 20 h in untreated control cells. The decrease in the 4.2 kb transcript levels was associated with a corresponding decrease in MnSOD protein levels and activity, which resulted in a significant inhibition of quiescent cells entry into the proliferative cycle. Deletion and reporter assays showed: (a) a significant decrease in reporter activity in constructs carrying multiple AREs that are present in the 3'-UTR of the longer MnSOD transcript; (b) irradiation increased the reporter activity of the constructs carrying the 3'-UTR sequence of the shorter MnSOD transcript and (c) N-acetyl-cysteine increased the reporter activity of constructs carrying multiple AREs. Because the longer transcript carries AREs, our results identified redox sensitive AREs as novel regulators of MnSOD transcript levels. We conclude that MnSOD 3'-UTR is a novel molecular sensor regulating MnSOD mRNA levels in response to different growth states and environmental stress. A better understanding of the 3'-UTR regulating gene expression could lead to the development of new molecular biology-based redox therapy designed to treat proliferative disorders.

Pages

xiii, 128

Bibliography

118-128

Copyright

Copyright 2010 Leena Chaudhuri