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

确定茎环结合蛋白在组蛋白 mRNA 衰变中的作用。

• 批准号: 8106408
• 负责人: Stacie Ann Meaux
• 金额: $ 5.13万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8106408
• 关键词: Abnormal Cell; Amino Acids; Back; Beryllium; Binding Proteins; Biochemical; Biochemistry; Biological; Cell Cycle; Cell Nucleus; Cells; Chromosomes; Codon Nucleotides; Complex; Coupled; Cytoplasm; DNA biosynthesis; Educational process of instructing; Eukaryota; Faculty; Gene Expression; Gene Expression Regulation; Genomic Instability; Goals; Histones; Human; In Vitro; Knowledge; Lead; Life; Link; Malignant Neoplasms; Messenger RNA; Molecular; Pathway interactions; Play; Positioning Attribute; Postdoctoral Fellow; Process; Proteins; Recruitment Activity; Regulation; Research; Role; S Phase; Saccharomyces cerevisiae; Signal Transduction; Staging; Techniques; Testing; Training; Transcript; Translations; Work; Yeasts; cancer therapy; career; cell growth; experience; graduate student; human disease; in vivo; insight; mRNA Decay; mRNA Stability; mRNA Transcript Degradation; mutant; programs; protein function; research study; stem; tool

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.

描述（由申请人提供）：后生动物中的组蛋白表达是一个高度调节的过程，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mRNA水平保持在较低的基础水平，但在DNA合成开始的S期开始时增加约30倍。在S期结束时，由于细胞质中转录物的快速降解，组蛋白mRNA水平降低回到基础水平;然而，几乎没有证据解释组蛋白mRNA的降解是如何触发的。茎环结合蛋白（SLBP）是组蛋白mRNA加工、输出和翻译所必需的。SLBP还被假设通过识别或引起异常翻译终止和/或募集衰变蛋白以降解转录物而在组蛋白mRNA降解中起关键作用。然而，SLBP在组蛋白mRNA衰变中的作用尚未得到彻底评估。我推测SLBP在识别或引起异常翻译终止和/或启动组蛋白mRNA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，将更清楚地了解细胞中组蛋白的浓度是如何控制的。此外，这些研究将深入了解细胞在其生命的不同阶段（例如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了解这种类型的基因表达的破坏如何导致异常细胞生长，并可能导致更好的癌症治疗。 具体目的1：鉴定组蛋白mRNA降解所需的SLBP区域和特定氨基酸残基。我将利用各种生物化学和分子技术来专门搜索SLBP的结构域和/或氨基酸残基，这些结构域和/或氨基酸残基是该功能所需的。我还将寻找氨基酸残基，这些氨基酸残基可以通过后期修饰来发出组蛋白mRNA衰变的信号。 具体目标二：确定SLBP是否具有将衰变蛋白募集到组蛋白转录物和/或识别异常翻译终止的功能。一旦DNA合成终止，我将鉴定与SLBP相互作用的蛋白质。然后，我将破坏这些相互作用，并评估这些突变体的影响在我的研究生工作中，我分析了对翻译和确定酵母酿酒酵母中mRNA稳定性很重要的ELS元件。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中的基因表达至关重要。作为Marzluff博士实验室的博士后研究员，我计划研究人类细胞中组蛋白mRNA衰变的调控。这些研究的结果将帮助我了解组蛋白mRNA的表达在细胞周期中是如何调节的，并提供对细胞核中产生的最终导致细胞质中组蛋白mRNA降解的信号的见解。此外，这些研究将扩大我的知识，包括在更复杂的真核生物和生物化学技术。我的最终职业目标是获得一个教师职位，在那里我可以发展一个独立的研究项目。我计划研究真核生物中的转录后基因调控及其在人类疾病中的作用。我相信，我作为一名研究生获得的经验，以及我作为博士后获得的培训和经验，将为我提供必要的工具，以启动我自己的独立项目，旨在成功地了解真核生物如何调节基因表达转录后。