The goal of this project is to define the mechanism that determines Xist mRNA stability. Based on previous work by the investigator, the structure of the murine Xist gene has been revised to include new information on the 3' end of the gene. The investigator has shown that this area of the gene is significantly larger than previously reported. Sequence comparison between mouse and human revealed sequence similarity in the new 3' ends. Using both Northern analysis and RNAse protection experiments, the investigator has confirmed that both Xist mRNA isoforms are produced by a mechanism involving differential polyadenylation. These polyadenylation sites are located in the new 3' sequences identified. Additional expression studies have shown that Xist mRNA isoforms are developmentally regulated, and therefore show different stabilities. This pattern of expression suggests that regulatory elements may interact differentially with each mRNA isoform, which in turn may influences Xist expression early in development. The data suggest that Xist mRNA isoforms change independently of Tsix and that the developmental regulation of the Xist isoforms is influenced by mRNA stabilization during the period in which upregulation of the chosen X chromosome is observed. The specific mechanism responsible for Xist stability remains poorly characterized. Although early in development Xist is unstable, after development Xist is exceptionally stable (T1/2 = >5 hr). Recent data have placed in doubt the importance of the 5' end of Xist in this regulation. The investigator plans to explore the role of the new 3' end of Xist in gene stability. He also proposes to pursue the mechanism of developmental regulation of Xist stability from two directions. First, a complete a mutational analysis of Xist and the regions immediately adjacent to Xist will be done. Second, the investigator plans to evaluate genes in the methylation pathway and define their role in determining Xist mRNA stability.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061079-05
Application #
6698082
Study Section
Mammalian Genetics Study Section (MGN)
Program Officer
Carter, Anthony D
Project Start
2001-02-01
Project End
2006-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
5
Fiscal Year
2004
Total Cost
$275,823
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Strauss, William M; Chen, Caifu; Lee, Chung-Tien et al. (2006) Nonrestrictive developmental regulation of microRNA gene expression. Mamm Genome 17:833-40
Ma, Mingchao; Strauss, William M (2005) Analysis of the Xist RNA isoforms suggests two distinctly different forms of regulation. Mamm Genome 16:391-404
Beletskii, A; Hong, Y K; Pehrson, J et al. (2001) PNA interference mapping demonstrates functional domains in the noncoding RNA Xist. Proc Natl Acad Sci U S A 98:9215-20