Role of nucleolin in regulating mRNA stability during DNA damage response (DDR)

描述（申请人提供）：Nucleolin是一种丰富的核仁磷酸化蛋白，具有RNA和p53结合特性，通过调节mRNA稳定性和p53信号通路来控制基因表达。本研究旨在研究核蛋白介导的DNA损伤反应（DDR）过程中mRNA稳定性调控的基本分子机制及其与细胞存活或死亡的生理相关性。具体的假设是，核仁与mRNA 3'加工因子相互作用，调节DNA损伤时参与DNA修复或凋亡反应的基因的mRNA稳定性。我们的初步数据表明，核仁蛋白与PARN deadenylase和p53相关，两者在DDR过程中调控许多基因的mRNA 3'加工，核仁蛋白磷酸化状态调节PARN活性。在Specific Aim 1中，我们将阐明核蛋白与mRNA 3'加工因子的功能相互作用及其对DDR期间mRNA稳定性的影响。我们将使用质分光光度法分析来鉴定与核蛋白磷酸化变体不同的蛋白质。在Specific Aim 2中，我们将在紫外线处理下表征p53介导的DDR中核仁蛋白/PARN mRNA靶点。我们将测量p53信号通路中靶mrna的稳态水平，分析rna与核仁蛋白/PARN复合物的直接结合，并在凋亡或细胞活力测定中评估这些相互作用的功能后果。核仁蛋白/PARN相互作用对不同途径中其他潜在靶点的影响将通过市售PCR阵列进行评估。本研究旨在了解核仁蛋白/PARN/p53相互作用如何调节mRNA稳定性，从而在细胞对DNA损伤的反应中调节p53信号通路、凋亡和DNA修复途径相关基因的表达。阐明细胞存活或死亡途径中这些相互作用对DNA损伤的功能影响，将推动基于核蛋白的小分子抑制剂领域的发展，为治疗提供新的选择。这笔拨款将为纽约市立大学少数族裔服务机构的两名女科学家（一名大三学生和另一名少数族裔教员）提供研究支持，以进行基本的分子癌症研究。这笔资金还将帮助在社会和教育方面处于不利地位的纽约市立大学本科生和研究生，并使他们接触与健康有关的研究。

DESCRIPTION (provided by applicant): 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.