TUT ase mediated degradation of histone mRNA

描述(申请人提供)：复制依赖的组蛋白主要在S期表达。通过羟基脲或紫外线诱导的检查点通路抑制DNA复制导致组蛋白转录减少和细胞质中组蛋白mRNAs的快速降解。最近，Marzluff实验室证明了组蛋白mRNA的降解需要转录后修饰，导致组蛋白mRNA的3‘端增加一个寡核苷酸(U)尾。寡头(U)尾巴的长度范围从8-15个核苷酸，它的添加是组蛋白mRNA降解所必需的。此外，我们的发现表明，一个非规范的聚(A)聚合酶。Uridyl末端转移酶(TUTase)负责组蛋白mRNAs的寡核苷酸。该提案的总体目标有三个方面。首先，我将确定负责组蛋白mRNA寡核苷酸的TUTase亚型(S)，并确定其酶活性的生化要求。其次，我将阐明TUTase在抑制DNA复制后被招募到组蛋白mRNAs的机制(S)，并将确定以前报道的与组蛋白mRNA降解有关的蛋白质是否与组蛋白mRNAs上的TUTase活性相关并影响其活性。我还将确定组蛋白mRNA的初始寡核苷酸是否发生在翻译活性的多聚体上，并调查TUTase是否仍与正在降解的mRNA相关。先前的研究表明，在酵母和哺乳动物中，组蛋白在S期的过度或过低表达分别导致了染色体的丢失和DNA的损伤。更重要的是，越来越多的数据表明，转录后机制是基因表达的主要决定因素。作为一类受调控的信息，组蛋白mRNAs为我们提供了一个独特的机会来识别和理解细胞周期是如何调控mRNA降解的，无论是作为一系列正常的过程，还是作为对对基因组稳定性至关重要的检查点途径的反应的一部分。

DESCRIPTION (provided by applicant): The replication dependent histones are expressed predominantly during S-phase. Inhibition of DNA replication by hydroxyurea or UV induced checkpoint pathways result in a reduction in histone transcription and rapid degradation of cytoplasmic histone mRNAs. Recently, the Marzluff lab has demonstrated that histone mRNA degradation requires a post-transcriptional modification that results in the addition of a oligo(U) tail to the 3' end of the histone mRNA. The oligo(U) tail ranges in size from 8-15 nts in length and its addition is required for histone mRNA degradation. Furthermore, our findings indicate that a non-canonical poly(A) polymerase. Terminal Uridyl Transferase (TUTase), is responsible for the oligouridylation of the histone mRNAs. The overall goals of the proposal are three fold. First, I will identify the TUTase isoform(s) responsible for histone mRNA oligouridylation and define the biochemical requirements for its enzymatic activity. Second, I will elucidate the mechanism(s) by which TUTase is recruited to histone mRNAs following inhibition of DNA replication and I will determine if proteins previously reported to be involved in histone mRNA degradation associate with and affect TUTase activity on histone mRNAs. I will also determine if initial oligouridylation of histone mRNA occurs on translationally active polysomes and investigate if TUTase remains associated with the mRNA as it is being degraded. Prior research has demonstrated that over or under expression of histones during S-phase contributes to chromosome loss and DNA damage in yeast and mammals, respectively. Of further importance is the mounting data that post-transcriptional mechanisms are a major determinant of gene expression. As a regulated class of messages, histone mRNAs present us a unique opportunity to identify and understand how the cell cycle regulates mRNA degradation, both as a normal set of processes, and as part of a response to checkpoint pathways important for genome stability.