TUT ase mediated degradation of histone mRNA

描述（由申请人提供）：复制依赖组蛋白主要在s期表达。通过羟基脲或紫外线诱导的检查点途径抑制DNA复制导致组蛋白转录减少和细胞质组蛋白mrna的快速降解。最近，Marzluff实验室已经证明组蛋白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)尾部。寡核苷酸(U)尾部的长度在8-15纳米之间，是组蛋白mRNA降解所必需的。此外，我们的研究结果表明，非典型聚(a)聚合酶。末端尿苷基转移酶（TUTase），负责组蛋白mrna的低聚尿苷化。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低尿嘧啶化的TUTase亚型，并定义其酶活性的生化要求。其次，我将阐明在抑制DNA复制后TUTase被招募到组蛋白mRNA的机制，我将确定先前报道的参与组蛋白mRNA降解的蛋白质是否与组蛋白mRNA上的TUTase活性相关并影响其活性。我还将确定组蛋白mRNA的初始低尿嘧啶化是否发生在翻译活性多体上，并研究TUTase在mRNA被降解时是否仍与mRNA相关。先前的研究表明，在酵母和哺乳动物中，s期组蛋白的过高或过低表达分别会导致染色体丢失和DNA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mRNA为我们提供了一个独特的机会来识别和理解细胞周期如何调节mRNA降解，无论是作为一组正常的过程，还是作为对基因组稳定性重要的检查点通路的反应的一部分。

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.