EXCEED THE SPACE PROVIDED. An exquisitely site-specific C to U deamination event in the nuclear transcript encoding apolipoproteinB (apoB) mRNA results in the generation of a translational termination codon (UAA) and production of apoB48 within mammalian enterocytes. Considerable progress has been made in understanding the molecular basis for this post-transcriptional modification and in identifying the key genes involved. The overarching theme of this renewal application will address the broader biological role and functionally relevant targets of these genes. These obiectives will be pursued in two related Specific Aims.
Aim 1 will focus on apobec- , the catalytic deaminase;
Aim 2 will focus on ACF, the regulatory subunit of the mammalian C to U RNA editing enzyme.
Aim 1 (a). We will undertake fine mapping of the apobec-1 binding site in the 3' untranslated regions of Cox-2, VEGF, GM-CSF, TNFot, IL-8 mRNAs and examine the functional interactions between apobec-1 and HuR binding, both in-vitro and in-vivo.
Aim 1 (b). We will examine the mechanisms that regulate [Cox-2- and VEGF- mRNA-apobec- 1] interaction in-vivo, including the role of sequestration by ACF and other apobec-1 binding proteins (GRY-RBP, CUGBP2, HuR). We propose that post-transcriptional regulation of Cox-2 and VEGF gene expression, which are important prognostic markers in both genetic and sporadic colorectal cancer, are regulated through alterations in apobec-1 compartmentalization or abundance.
Aim 1 (c). We will examine the biochemical properties of the anti-retroviral cytidine deaminase, apobec-3G, including its endogenous enzymatic targets, RNA and DNA specificity and protein interacting partners.
Aim 2 (a). We will characterize the minimal binding site for ACF in apoB mRNA and deduce a consensus binding site that likely exists in other targets, beyond apoB.
Aim 2 (b). We will use microarray analysis to identify RNAs whose regulation is altered as a result of ACF deletion in both cell lines and primary isolated murine hepatocytes.
Aim 2 (c). We will undertake genetic ACF deletion following homologous recombination in ES cells. In addition, we have constructed a conditional targeting vector in order to create a conditional ACF knockout. 'Floxed', mice will be crossed into lines of tissue-specific Cre-deletors (liver, intestine) in order to determine the tissue-specific targets of ACF in adult mice. These studies will identify the range of endogenous targets of these multifunctional RNA binding proteins and may point to an unanticipated role in human disease. PERFORMANCE SITE ========================================Section End===========================================
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