Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

Winter 1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Jean E Schaffer

Abstract

Accumulation of excess lipid in non-adipose tissues is associated with oxidative stress and organ dysfunction and plays an important role in diabetic complications. While a number of stress responses have been implicated, the precise molecular mechanisms linking lipid accumulation and cellular dysfunction are not fully understood. To elucidate molecular events critical for lipotoxicity, we used retroviral promoter trap mutagenesis to generate mutant Chinese hamster ovary cell lines resistant to lipotoxic and oxidative stress. This approach uncovered a previously unsuspected role for small nucleolar RNAs: snoRNAs) as critical mediators of lipotoxic cell death.

Herein we show that under lipotoxic conditions, intronic snoRNAs in the rpL13a gene accumulate in the cytosol during metabolic stress, suggesting that these non-coding RNAs function non-canonically to target cytosolic RNAs. Moreover, we demonstrate that the rpL13a snoRNAs play a critical role in vivo in amplification of reactive oxygen species and downstream oxidative stress-mediated tissue injury.

Study of independent mutants from our genetic screen not only identified a role for snoRNAs in lipotoxicity, but also provided new insights into the cellular machinery for production of these non-coding RNAs. We demonstrate that the spliceosomal protein SmD3 plays a critical role in expression of intronic non-coding RNAs, including the rpL13a snoRNAs, by maintaining the abundance of snoRNA-containing intron lariats from which they are processed. Our findings indicate that this function may involve effects of SmD3 on small nuclear RNAs: snRNAs) U4 and U5.

Finally, we demonstrate a link between superoxide induction and cytosolic snoRNA accumulation. Under lipotoxic conditions, superoxide induction precedes cytosolic snoRNA accumulation. Other chemical inducers of superoxide also cause the rpL13a snoRNAs to localize to the cytosol, and manipulation of superoxide dismutase demonstrates that superoxide levels directly correlate with cytosolic snoRNA expression.

Together, our studies identify an important role for snoRNAs in metabolic stress responses. The rpL13a snoRNAs are essential for cell death in response to lipid overload through functions that are distinct from their canonical role in ribosomal RNA modification in the nucleolus. Moreover, these non-coding RNAs respond to chemical inducers of reactive oxygen species and thereby may contribute more broadly to disease pathogenesis that involves oxidative stress.

Comments

Permanent URL: http://dx.doi.org/10.7936/K72N508W

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