Date of Award

Summer 8-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Metabolic diseases, such as obesity and diabetes, are associated with excess levels of lipids, which can lead to organelle dysfunction, cell death and eventually organ dysfunction. This process, termed lipotoxicity, is still not completely understood. In a genetic screen used to identify genes critical for lipotoxicity, the Schaffer lab has identified small nucleolar RNAs (snoRNAs) within the ribosomal protein L13a (Rpl13a) locus that mediate the cellular response to lipotoxic and general metabolic stress. These snoRNAs are non-canonical in that they accumulate in the cytosol after metabolic stresses like lipotoxicity and oxidative stress, suggesting that cells have specific mechanisms for regulating snoRNA distribution within the cell.

To begin elucidating the underlying mechanism of snoRNA transport from the nucleus to the cytoplasm during metabolic stress, we modeled oxidative stress by treating cells with the chemotherapeutic drug doxorubicin (DOX). DOX is a potent inducer of superoxide through the activation of NADPH oxidase splice isoform 4D (NOX4D) and results in the cytosolic accumulation of Rpl13a snoRNAs. NOX inhibitors and genetic knockdown of NOX4D led to a decrease in the accumulation of Rpl13a snoRNAs in the cytosol after DOX treatment. Furthermore, RNA-sequencing studies demonstrated that snoRNAs as a class accumulated in the cytosol after DOX treatment, while simultaneous treatment with NOX inhibitors blunted the increase in cytosolic levels of snoRNAs. Together, these data indicate that snoRNAs as a class are present in the cytoplasm, where their levels are dynamically regulated by NOX4D during oxidative stress.

The finding that loss-of-function of NXF3, a member of the nuclear export family of RNA transporters, protects cells from lipotoxic cell death suggested that this protein may function as a regulator of snoRNA distribution. In transient transfection assays, NXF3 knockdown increased abundance of cytosolic Rpl13a snoRNAs, whereas NXF3 overexpression led to decreased cytosolic levels. These observations suggested that snoRNAs traffic constitutively between the nucleus and cytoplasm, and NXF3 functions to efficiently concentrate snoRNAs in the nucleus. Consistent with a role for NXF3 as a snoRNA transporter, we found the Rpl13a snoRNAs co-immunoprecipitated with NXF3. Treatment of cells with forskolin caused a rapid decrease in cytosolic snoRNAs and increased both nuclear localization of NXF3 and association of NXF3 with Rpl13a snoRNAs. These effects of forskolin were abrogated by NXF3 knockdown.

Our findings identify a novel trafficking pathway for snoRNAs that cycle between the nucleus and the cytosol. This pathway is regulated by oxidative stress and levels of the RNA transporter NXF3.

Language

English (en)

Chair and Committee

Jean E. Schaffer

Committee Members

Thomas Baranski, Joseph Dougherty, Scot Matkovich, Jeanne Nerbonne,

Comments

Permanent URL: https://doi.org/10.7936/K7GM86P8

Download

Share

COinS