The broad long-term goal of this project is to understand the role of mRNA degradation in regulating T lymphocyte gene expression. Steady state mRNA levels are determined by the balance between transcription and degradation. This proposal is based on the hypothesis that specific RNA regulatory sequences and specific trans-acting proteins that recognize these sequences function to regulate gene expression at the level of mRNA decay. We recently used microarray technology to measure mRNA decay rates of approximately 6,000 transcripts expressed in T cells under conditions of rest or activation, and we identified hundreds of transcripts that were regulated at the level of mRNA decay. We used computational methods to search for conserved sequences found in transcripts that were co-regulated at the level of mRNA decay and found that approximately 100 short-lived transcripts expressed in T cells contained the conserved 11-mer RNA sequence UGUUUGUUUGU in their 3' untranslated region. We have termed this sequence the GU-rich element (GRE) and have shown using a reporter system that this RNA element mediates mRNA decay in HeLa cells. In preliminary experiments, we have also shown that the CUG-binding protein 1 binds specifically to the GRE and mediates GRE-dependent mRNA decay. The goals of this grant are to characterize these novel regulators and to identify additional novel regulators of mRNA decay.
Specific aim 1 is to characterize the role of the GU-rich element (GRE) and the CUG-binding protein 1 (CUGBP1) in regulating T cell mRNA decay.
Specific aim 2 is to characterize the role of novel conserved RNA sequence elements in regulating T cell mRNA decay, and specific aim 3 is to characterize the role of novel RNA-binding proteins in regulating T cell mRNA decay. We will use biochemical and molecular biological methods to test the function of novel regulatory elements and RNA-binding proteins in the regulation of T cell mRNA decay. These experiments will provide insight into the mechanisms by which mRNA decay regulates the expression of important growth regulatory genes, including cytokine genes and proto-oncogenes. Understanding the mechanisms by which mRNA decay regulates the expression of these genes will provide insight into human disease states such as autoimmunity, immunodeficiency, cancer, and infectious diseases and will lead to the development of new anti-cancer and immunomodulatory drugs that act by targeting specific mRNA decay pathways. In order for human cells to develop and function normally, genes need to be turned on at precise times and they need to be turned off at precise times. An important method that cells use to turn genes off is to eliminate messenger RNA, which then stops the production of proteins. The elimination of messenger RNA occurs in the cell through a process known as messenger RNA decay. The goal of the experiments proposed in this grant application is to define the proteins that are responsible for mRNA decay and to understand how the process of mRNA decay is regulated. Many human diseases such as cancer or autoimmune diseases occur because genes are turned on but then are not turned off in a normal manner. Understanding how genes are turned off will lead to the development of new drugs that will be used to turn off specific genes in patients with diseases such as cancer or autoimmunity. ? ? ?
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