The differentiating erythroid cell, particularly in thalassemia, is exposed to severe cellular stresses including oxidative and hypoxic stress. Both reactive oxygen species and hypoxia lead to the phosphorylation of eIF2a, a translation factor vital for the initiation of protein synthesis, and genetic studies have demonstrated that the regulation of this phosphorylation plays an important role in erythropoiesis. We have determined that the inhibition of nonsense mediated RNA decay in hypoxic cells is dependent on eIF2a phosphorylation. Nonsense mediated RNA decay (NMD) is a multi-step pathway responsible for the degradation of 30% of all human mutated mRNAs, as well as up to 10% of normal cellular mRNAs. But because NMD has not been thought of as a regulated pathway, its activity in normal or thalassemic erythroid cells has not been closely studied, and its significance in erythropoiesis has not been determined. We hypothesize that NMD is inhibited by cellular hypoxia and reactive oxygen species in differentiating thalassemic erythroid cells. This inhibition, mediated by eIF2a phosphorylation, alters gene expression, augments the stress response, and improves the survival of these cells. We propose to 1) Determine if eIF2a is phosphorylated by reactive oxygen species in human thalassemic erythroid cells, and whether this phosphorylation is sufficient for the inhibition of NMD. Briefly, we will amplify and differentiate erythroid cells from peripheral blood, and also examine bone marrow biopsies, from control and thalassemic volunteers. These samples will be assessed for eIF2a phosphorylation status, induction of stress responsive genes, and reactive oxygen species. We will then use a variety of stresses and engineered cell lines to determine if eIF2a phosphorylation is sufficient to inhibit NMD. 2) Determine the biological significance of eIF2a phosphorylation and NMD inhibition in normal and thalassemic erythroid cells. Briefly, we will determine if eIF2a phosphorylation by reactive oxygen species or cellular hypoxia correlates with apoptosis in differentiating erythroid cells. Using expression arrays we will determine the mRNAs that are stabilized when NMD is genetically deleted in erythroid cells and/or when NMD is inhibited by hypoxia in these cells. NMD targets alternatively spliced mRNAs, and by using expression arrays that identify mRNA splice variants we will determine if these variants are enriched in hypoxic and NMD repressed erythroid cells. 3) Determine the mechanism of hypoxia-induced inhibition of nonsense mediated RNA decay by pursuing our working model, based on preliminary data, that eIF2a phosphorylation sequesters NMD targeted mRNAs to cytoplasmic stress granules, where these mRNAs cannot be degraded. Using confocal microscopy we will determine the localization of enzymes important for NMD, as well as NMD degraded mRNAs in stressed cells where eIF2a is phosphorylated.

Public Health Relevance

The normal growth and development of red blood cells is vital to sustain health. Impairment of red cell development, in diseases such as thalassemia, may lead to a severe anemia and a dependence on transfusions. We have identified a novel mechanism that controls the stability of messenger RNA, the carrier of genetic information from DNA to proteins in a cell. Because this mechanism is regulated by cellular stresses found in the normal, and particularly thalassemic, erythroid cells, we will determine if erythroid cell survival is affected by this novel form of gene regulation, and identify the genes that are regulated by this mechanism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK081641-03
Application #
7897664
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Bishop, Terry Rogers
Project Start
2008-09-01
Project End
2013-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
3
Fiscal Year
2010
Total Cost
$338,153
Indirect Cost
Name
New York University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Wengrod, Jordan; Wang, Ding; Weiss, Sarah et al. (2015) Phosphorylation of eIF2? triggered by mTORC1 inhibition and PP6C activation is required for autophagy and is aberrant in PP6C-mutated melanoma. Sci Signal 8:ra27
Martin, L; Gardner, L B (2015) Stress-induced inhibition of nonsense-mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11. Oncogene 34:4211-8
Gold, Heidi L; Wengrod, Jordan; de Miera, Eleazar Vega-Saenz et al. (2014) PP6C hotspot mutations in melanoma display sensitivity to Aurora kinase inhibition. Mol Cancer Res 12:433-9
Martin, Leenus; Grigoryan, Arsen; Wang, Ding et al. (2014) Identification and characterization of small molecules that inhibit nonsense-mediated RNA decay and suppress nonsense p53 mutations. Cancer Res 74:3104-13
Wengrod, Jordan; Martin, Leenus; Wang, Ding et al. (2013) Inhibition of nonsense-mediated RNA decay activates autophagy. Mol Cell Biol 33:2128-35
Karam, Rachid; Wengrod, Jordan; Gardner, Lawrence B et al. (2013) Regulation of nonsense-mediated mRNA decay: implications for physiology and disease. Biochim Biophys Acta 1829:624-33
Martin, L; Rainey, M; Santocanale, C et al. (2012) Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation. Oncogene 31:4076-84
Parathath, Sajesh; Mick, Stephanie L; Feig, Jonathan E et al. (2011) Hypoxia is present in murine atherosclerotic plaques and has multiple adverse effects on macrophage lipid metabolism. Circ Res 109:1141-52
Wang, Ding; Zavadil, Jiri; Martin, Leenus et al. (2011) Inhibition of nonsense-mediated RNA decay by the tumor microenvironment promotes tumorigenesis. Mol Cell Biol 31:3670-80
Chin, King-Tung; Kang, Guoxin; Qu, Jiaxiang et al. (2011) The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load. FASEB J 25:2583-91

Showing the most recent 10 out of 13 publications