Determining the Role of the Stem-loop Binding Protein in Histone mRNA Decay.

描述(申请人提供)：组蛋白在后生动物中的表达是一个高度调控的过程，与细胞周期中的DNA合成有关。在细胞生命的大部分时间里，组蛋白基因水平一直保持在较低的基础水平，但在S期开始时，DNA合成开始时，组蛋白基因的水平增加了约30倍。在S期结束时，由于转录物在细胞质中的快速降解，组蛋白mRNA的水平下降到基础水平；然而，很少有证据解释组蛋白mRNA的降解是如何被触发的。茎环结合蛋白(SLBP)是组蛋白mRNAs加工、输出和翻译所必需的。SLBP还被认为通过识别或导致异常翻译终止和/或招募衰变蛋白来降解转录物，在组蛋白mRNA的降解中发挥关键作用。然而，SLBP在组蛋白mRNA衰退中的作用还没有得到彻底的评价。我推测SLBP在识别或导致异常翻译终止和/或起始组蛋白mRNAs的mRNA降解中起关键作用。了解组蛋白mRNA基因表达如何与DNA合成相结合，将使我们更清楚地了解组蛋白蛋白在细胞中的浓度是如何控制的。此外，这些研究还将深入了解细胞在其生命的不同阶段(如DNA合成后)如何调节基因表达。因此，完成拟议的实验将更好地理解这种类型的基因表达中断如何导致细胞异常生长，并可能导致更好的癌症治疗方法。具体目的1：确定SLBP的组蛋白基因降解所需的区域(S)和特定氨基酸残基(S)。我将利用各种生化和分子技术来专门寻找SLBP的结构域和/或氨基酸残基，这些结构域和/或氨基酸残基是这种功能所必需的。我还将寻找可能通过翻译后修饰而发出组蛋白mRNA衰退信号的氨基酸残基。具体目的2：确定SLBP是否具有将衰变蛋白招募到组蛋白转录本和/或识别异常翻译终止的功能。一旦DNA合成终止，我将确定与SLBP相互作用的蛋白质。然后，我将中断这些相互作用，并评估这些突变的影响在我的研究生工作中，我分析了ELS元件，这些元件对翻译和确定酵母中mRNAs的稳定性非常重要。这些研究教会了我基本的分子生物学技术，这些技术对于研究真核生物的基因表达是必不可少的。作为Marzluff博士实验室的博士后，我计划研究人类细胞中组蛋白mRNAs的衰变规律。这些研究的结果将帮助我理解组蛋白mRNAs在细胞周期中的表达是如何调节的，并为深入了解在细胞核中产生的最终导致细胞质中组蛋白mRNA降解的信号提供见解。此外，这些研究将扩展我的知识，包括更复杂的真核生物和生物化学中的技术。我的最终职业目标是获得一个教员的职位，在那里我可以开发一个独立的研究项目。我计划研究真核生物的转录后基因调控及其在人类疾病中的作用。我相信，我作为研究生获得的经验，加上我作为博士后研究员将获得的培训和经验，将为我提供必要的工具，以启动我自己的独立计划，旨在成功地理解真核生物如何在转录后调控基因表达。

DESCRIPTION (provided by applicant): Histone expression in metazoans is a highly regulated process that is linked with DNA synthesis during the cell cycle. Histone mRNA levels remain at a low basal level throughout most of the life of a cell but increase about 30-fold at the beginning of S phase, when DNA synthesis begins. At the end of S-phase, histone mRNA levels decrease back to basal level due to rapid degradation of the transcripts in the cytoplasm; however, little evidence exists to explain how degradation of histone mRNAs is triggered. The stem-loop binding protein (SLBP) is required for processing, export and translation of histone mRNAs. SLBP is also hypothesized to play a critical role in histone mRNA degradation by recognizing, or causing, aberrant translation termination and/or recruiting decay proteins to degrade the transcript. However, the role of SLBP In histone mRNA decay has not been thoroughly evaluated. I hypothesize that SLBP plays a critical role In recognizing, or causing, aberrant translation termination and/or in initiating mRNA degradation of histone mRNAs. Understanding how histone mRNA gene expression is coupled with DNA synthesis will give a clearer picture of how the concentration of histone proteins is controlled in a cell. Also, these studies will give insight into how a cell regulates gene expression during different stages of its life (e.g. after DNA synthesis). Therefore, completion of the proposed experiments will provide a better understanding of how disruption of this type of gene expression can result in abnormal cell growth and could lead to better treatments for cancer. Specific Aim 1: To identify the region(s) and specific amino acid residue(s) of SLBP that Is required for histone mRNA degradation. I will utilize various biochemical and molecular techniques to specifically search for domains and/or amino acid residues of SLBP that are required for this function. I will also search for amino acid residues that could signal histone mRNA decay by being post-translationally modified. Specific Aim 2: To determine if SLBP functions to recruit decay proteins to a histone transcript and/or to recognize aberrant translation termination. I will Identify proteins that interact with SLBP once DNA ^ synthesis is terminated. I will then disrupt these interactions and evaluate the effect those mutant During my graduate work, I analyzed els elements that are important for translation and in determining the stability of mRNAs in the yeast Saccharomyces cerevisiae. These studies have taught me basic molecular biological techniques that are essential for studying gene expression In eukaryotes. As a postdoctoral fellow in Dr. Marzluff's lab, I plan to study the regulation of decay of histone mRNAs in human cells. The results from these studies will help me understand how expression of histone mRNAs is regulated during the cell cycle, and provide^insights into the signals that are generated in the nucleus which ultimately lead to histone mRNA degradation in the cytoplasm. Additionally, these studies will expand my knowledge to include techniques in more complex eukaryotes and in biochemistry. My ultimate career goal is to obtain a faculty position where I can develop.an independent research program. I plan to study post-transcriptional gene regulation in eukaryotes and its role in human disease. I believe that the experience that I have obtained as a graduate student, together with the training and experience I will obtain as a postdoctoral fellow, will provide me with the necessary tools to initiate my own independent program aimed at successfully understanding how eukaryotes regulate gene expression post-transcriptionally.