Nucleolin, an abundant nucleolar phosphoprotein with RNA- and p53-binding properties, controls gene expression by regulating mRNA stability and the p53 signaling pathway. This proposal is aimed to study the basic molecular mechanism of nucleolin-mediated regulation of mRNA stability during the DNA damage response (DDR) and its physiological relevance in cell survival or cell death. The specific hypothesis is that nucleoln interacts with mRNA 3'processing factors and regulates the mRNA stability of genes involved in either the DNA repair or apoptotic response upon DNA damage. Our preliminary data indicate that nucleolin associates with PARN deadenylase and p53, both of which play a role in the regulation of mRNA 3'processing of many genes during DDR, and nucleolin phosphorylation state regulates PARN activity.
In Specific Aim 1, we will elucidate the functional interaction of nucleolin with mRNA 3'processing factors and their effect on the mRNA stability during DDR. We will use mass-spectrophotometry analyses to identify proteins that differentially associate with nucleolin phospho-variants.
In Specific Aim 2, we will characterize nucleolin/PARN mRNA targets in the p53-mediated DDR upon UV treatment. We will measure the steady state levels of the target- mRNAs in the p53-signaling pathway, analyze direct RNA-binding to the nucleolin/PARN complex and assess the functional consequences of these interactions in apoptotic or cell- viability assays. The effect of nucleolin/PARN interaction on other potential targets in different pathways will be assessed by commercially available PCR arrays. This study is aimed at understanding how nucleolin/PARN/p53 interaction regulate mRNA stability and hence the expression of genes involved in the p53 signaling, apoptosis and DNA repair pathways during cellular response to DNA damage. Elucidating the functional consequences of these interactions in cell survival or death pathways upon DNA damage will advance the field of nucleolin-based small molecule inhibitors, offering new alternatives for therapeutics. This grant will provide research support to two women scientists at CUNY minority-serving institutions (one junior and the other minority faculty) to pursue basic molecular cancer research. This funding will also help to outreach socially and educationally disadvantaged CUNY undergraduate and graduate students, and expose them to health-related research.
Control of gene expression by regulating mRNA stability is critical in determining ultimate fate of the cells following DNA damage: DNA repair, death, aging or proliferation. This proposal is aimed to determine the role(s) of the RNA-binding phosphoprotein nucleolin, the tumor suppressor p53 and the mRNA 3'processing machinery in regulating mRNA stability of genes involved in the DNA damage response. Understanding the functional consequences of these interactions will provide new insights on how control of gene expression upon DNA damage decides cellular fate, offering new opportunities for therapeutics.
