Pre-mRNA editing is a recently discovered process that leads to the production of a variety of protein isoforms from a single gene. The A-to-I RNA editing, a distinct mechanism of mRNA editing in mammalian cells has been previously identified by our laboratory. Specifically, using HeLa cells we have shown that glutamate receptor subunit B (gluR-B) mRNA can undergo site specific deamination at an adenosine residue which converts adenosine to inosine (A-to-I), a critical change required to maintain receptor function. Two RNA-dependent adenosine deaminases (A-to-I RNA editase) have been identified in HeLa as well as a variety of other mammalian cells. In agreement with these observations, numerous inosine-containing mRNA (I-mRNA) were detected in different tissues as well. The presence of A- to-I RNA editase and I-mRNA in mammalian cells suggests that A- to-I RNA editing play an important role in gene expression and function. The broad, long-term objective of this proposal is to understand the mechanism and function of A-to-I RNA editing.
Three specific aims are proposed as a basis for this research plan. First, to characterize I-mRNA in mammalian cells using the modified affinity purification approach to be established. Specifically, I-mRNA from HeLa cells will be enriched and identified. The cDNA of I-mRNA will be analyzed to determine the A-to-I editing site. The effect of the identified I-mRNA on protein identity, mRNA stability and structure will be studied in vitro and in vivo. Second, we will identify the site-specific interactions in the editase-RNA complex. Specifically, the conditions required for editase-RNA complex formation will be determined and intra-complex interactions between the editase and substrate and the catalytic core will be studied by site-specific UV cross-linking and base modifications. The possibility that the catalytic and RNA binding domains are functionally independent will be examined. Also, A-to-I RNA editase with a different RNA binding domain will be characterized, and protein factors associated with A-to-I RNA editase will be studied. Third, we will study the functions of A-to-I RNA editase in HeLa cells. Specifically, dominate-negative mutant and neutralizing antibody will be produced to establish a HeLa cell line in which editase activity can be quantitatively controlled. By using this cell line, the hypothesis that the dsRNA-dependent protein kinase (PKR) is mediated by the dsRNA editing activity of A-to-I RNA editase will be examined. The overall cellular effects of gene expression by A-to-I RNA editase and the I-mRNA decay will be studied. The proposed experiments will provide fundamental information regarding the role of A-to-I pre-mRNA editing in mammalian gene expression and regulation that could be used to combat disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060426-05
Application #
6689587
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
2000-01-01
Project End
2006-10-31
Budget Start
2004-01-01
Budget End
2006-10-31
Support Year
5
Fiscal Year
2004
Total Cost
$209,114
Indirect Cost
Name
Yale University
Department
Surgery
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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