Molecular Regulation of Translational Regulation

描述(由申请人提供)：这个项目的长期目标是在翻译水平上表征基因表达的调节。作为我们的模型系统，我们将研究二氢叶酸还原酶(DHFR)的表达调控，DHFR是癌症化疗中的关键靶点。这种酶催化NADPH依赖的还原反应，产生还原的叶酸，四氢叶酸，一碳转移反应的关键中间体。因此，dhfr是从头合成嘌呤和嘧啶以及合成某些氨基酸所必需的。因此，DHFR在维持细胞的新陈代谢需求方面起着核心作用。该实验室以前的研究表明，DHFR除了在催化和细胞代谢中发挥作用外，还作为一种RNA结合蛋白发挥作用。这种蛋白与其自身的DHFR mRNA具有高亲和力(5-6 nM)结合，这种相互作用导致DHFR mRNA的翻译抑制，从而抑制新的DHFR蛋白的合成。这些研究表明，DHFR的表达至少在一定程度上受翻译自动调节反馈机制的控制。这种DHFR翻译自动调节模型具有生物学意义，因为它为严格控制给定细胞内DHFR的表达提供了一种合理的机制。然而，用抗叶酸类似物甲氨蝶呤(MTX)等抑制剂处理DHFR蛋白，改变了正常的DHFR蛋白与DHFR mRNA的相互作用，导致DHFR mRNA的翻译效率提高，新的DHFR蛋白的合成增加。这一正常调控过程的中断为恶性细胞提供了一种有效的机制来保护自己，以应对细胞毒应激，并发展细胞耐药性。为了进一步了解DHFR翻译调控的分子基础，本项目提出了三个具体目标：(1)鉴定DHFR mRNA上的关键顺式作用元件，这些元件是这种RNA-蛋白质相互作用所必需的。为此，我们建议确定蛋白质结合所需的关键核苷酸序列和/或二级结构；(2)表征DHFR蛋白质上识别RNA所必需的关键反式作用元件。作为这一目标的一部分，我们计划确定DHFR蛋白上的一个或多个结构域以及介导RNA结合过程的关键氨基酸接触点；以及(3)确定与DHFR蛋白相互作用的细胞RNA。为此，我们建议除dhfr mRNA外，还鉴定其表达和/或功能可能受人dhfr蛋白调控的细胞mRNAs。

DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize the regulation of gene expression at the translational level. As our model system, we will investigate the regulation of expression of dihydrofolate reductase (DHFR), a critical target in cancer chemotherapy. This enzyme catalyzes the NADPH-dependent reductive reaction, which gives rise to the reduced folate, tetrahydrofolate, a key intermediate in one-carbon transfer reactions. DHFR is, therefore, required for the de novo synthesis of purines and pyrimidines as well as for the synthesis of certain amino acids. Thus, DHFR plays a central role in maintaining the metabolic requirements of the cell. Previous studies from this lab have shown that in addition to its role in catalysis and cellular metabolism, DHFR also functions as an RNA binding protein. This protein binds with high affinity (5-6 nM) to its own DHFR mRNA, an interaction that results in the translational repression of DHFR mRNA with subsequent inhibition of synthesis of new DHFR protein. These studies demonstrate that the expression of DHFR is controlled at least, in part, by a translational autoregulatory feedback mechanism. This model of DHFR translational autoregulation has biological relevance in that it offers a rational mechanism for the tight control of DHFR expression within a given cell. However, treatment of DHFR protein with inhibitor compounds such as the antifolate analog, methotrexate (MTX), alters the normal DHFR protein-DHFR mRNA interaction, resulting in an enhanced translational efficiency of DHFR mRNA with an increased synthesis of new DHFR protein. Disruption of this normal regulatory process provides an efficient mechanism for malignant cells to protect themselves in response to exposure to cytotoxic stress and develop cellular drug resistance. To further our understanding of the molecular elements underlying the translational regulation of DHFR, three specific aims are proposed in this project: (1) Characterize the critical cis-acting elements on DHFR mRNA that are required for this RNA-protein interaction. In this aim, we propose to determine the critical nucleotide sequences and/or secondary structures required for protein binding; (2) Characterize the critical trans-acting elements on the DHFR protein that are necessary for RNA recognition. As part of this aim, we plan to identify the domain or domains on the DHFR protein as well as the critical amino acid contact points that mediate the process of RNA binding; and (3) Identify the cellular RNAs that interact with DHFR protein. In this aim, we propose to identify the cellular mRNAs in addition to DHFR mRNA whose expression and/or function may be under the control of human DHFR protein.