Eukaryotic protein synthesis initiation is an intricate, highly stress-sensitive event that requires formation of a ternary complex composed of initiation factor 2 (eIF2), GTP, and methionine-charged initiator tRNA (met-tRNAi). During studies to reconstruct this process, we produced unique evidence for a potent ternary complex inhibitor (TCI) in the low molecular mass pool of eukaryotic RNA (sRNA). TCI readily isolates from the bulk of sRNA and tRNAs by ion exchange chromatography. In cell lysates, TCI suppresses the rate of protein synthesis, and disrupts assembly of heavy polymeric ribosomes. Moreover, another component in ribosome depleted cytoplasmic lysates significantly antagonizes TCI (?TCI) activity. In this proposal we intend three focused goals. First, we will purify and determin the primary nucleotide sequence of TCI, using standard extraction, co-immunoprecipitation, chromatography, and sequencing techniques. Second, we will measure TCI and ?TCI activity levels in primary proliferating, differentiating, and mineralizing osteoblast cultures. We will quantify TCI and ?TCI activity by rate and dosage assays, by relation to positive cell and tissue sources and to co-isolated primary fibroblasts using highly purified assay substrates and reagents. Third, we will examine the effect of two well known physiological stressors, amino acid deprivation and hypoxia, which each control protein synthesis through the eIF2 pathway, on TCI and ?TCI activity in osteoblasts, using the methods and results developed in the previous goals. Results from the first goal will permit quantitative assessment of TCI and secondary structure predictions. Results from goal two will provide evidence for TCI and ?TCI in cells within the physiological context with application to bone growth. Results from goal three will provide support for the importance of these factors by relation to stress induced bone pathology. Since assembly of the met- tRNAi/eIF2/GTP ternary complex is integral to all aspects of protein synthesis, and by extension to cellular physiology, our results will establish proof of principal fr future studies to purify ?TCI to homogeneity, to expand stress studies in normal and transgenic animals, and develop a better definition of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.

Public Health Relevance

Eukaryotic protein synthesis is a complex, stress-sensitive process that requires a protein factor termed eIF2. We recently reported a low molecular mass RNA inhibitor (TCI) of eIF2 activity as well as a cytoplasmic antagonist of TCI (?TCI) in eukaryotic cell extracts. We will next purify and determine the primary sequence of TCI, measure TCI and ?TCI activity in primary proliferating, differentiating, and mineralizing osteoblast cultures, and examine the effect of two potent stressors, nutrient deprivation and hypoxia, which both control protein synthesis through the eIF2 pathway, on TCI and ?TCI activity in bone forming osteoblasts. Our results will begin to define new effects by physiologic and pathologic situations, factors, and events that control osteoblast dependent bone formation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AR062764-01
Application #
8285345
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Chen, Faye H
Project Start
2012-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$165,875
Indirect Cost
$65,875
Name
Yale University
Department
Surgery
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
McCarthy, Thomas L; Yun, Zhong; Madri, Joseph A et al. (2014) Stratified control of IGF-I expression by hypoxia and stress hormones in osteoblasts. Gene 539:141-51