In erythroid cells a premature termination codon (PTC) in exons 1 or 2 of the beta-globin gene activates a cytoplasmic endonuclease that degrades beta-globin mRNA. The resulting loss of beta-globin expression is the mechanism underlying Cooley's anemia (beta-thalassemia), an often fatal disorder of hemoglobin production. Using cultured erythroid cells we showed that a PTC in exon 2 activates endonuclease cleavage by mPMR1, the mammalian ortholog of the Xenopus mRNA endonuclease PMR1. Biochemical differences in the decay of the PTC-containing beta-globin mRNA in erythroid versus non-erythorid cells indicate that a distinct process targets the decay of this mRNA in its native cell context.
Aim 1 will characterize the details of the PTC- stimulated endonuclease decay in erythroid cells and determine the role of 3'-UTR elements in stabililzing the decay intermediates. A sensitive FRET-based assay developed to study the polarity of mRNA decay will be used to quantify the selective loss of exon 1 by endonuclease cleavage.
Aim 2 will examine the relationship between the PTC-stimulated degradation of beta-globin mRNA and key proteins involved in mRNA surveillance (NMD) using RNAi and dominant negative proteins to interfere with key steps in the detection of a PTC and subsequent activation of endonuclease decay.
Aim 3 will characterize the role of mPMR1 in the degradation of PTC-containing beta-globin mRNA and characterize tyrosine kinase activation of this process.
Aim 4 will determine how the signal for recognition of a PTC is transduced to activate endonuclease cleavage using directed protein interaction analysis, tethering and RNAi to examine interactions between mPMR1 and key proteins involved in mRNA surveillance. The long-term goal of this research is to develop new treatments for beta-thalassemia by understanding the novel mechanisms involved in beta-globin mRNA decay in erythroid cells.

Public Health Relevance

Cooley's anemia is common inherited disease of that produces life-theatening anemia, particularly in young children. It is often caused by mutations in the hemoglobin beta- chain that activate the destruction of the mRNA made from the defective gene. This research seeks to understand how these defective gene products are destroyed and how this information might be used to develop new treatments for this debilitating disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079707-04
Application #
8208188
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
2009-01-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2013-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$301,381
Indirect Cost
$100,460
Name
Ohio State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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Mascarenhas, Roshan; Dougherty, Julie A; Schoenberg, Daniel R (2013) SMG6 cleavage generates metastable decay intermediates from nonsense-containing *-globin mRNA. PLoS One 8:e74791
Schoenberg, Daniel R (2011) Mechanisms of endonuclease-mediated mRNA decay. Wiley Interdiscip Rev RNA 2:582-600
Schoenberg, Daniel R; Maquat, Lynne E (2009) Re-capping the message. Trends Biochem Sci 34:435-42