TUT ase mediated degradation of histone mRNA

描述（由申请人提供）：复制依赖性组蛋白主要在S期表达。通过羟基脲或UV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降解需要转录后修饰，其导致在组蛋白mRNA的3'末端添加寡聚（U）尾。寡核苷酸（U）尾的长度范围为8-15 nt，并且其添加是组蛋白mRNA降解所需的。此外，我们的研究结果表明，一个非典型的聚（A）聚合酶。末端尿苷转移酶（TUTase）负责组蛋白mRNA的寡尿苷化。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寡核苷酸化的TUTase亚型，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mRNA的机制，并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相关并影响TUTase对组蛋白mRNA的活性。我还将确定组蛋白mRNA的初始寡核苷酸化是否发生在具有免疫活性的多聚核糖体上，并研究TUTase在降解时是否仍与mRNA相关。先前的研究已经证明，在S期期间，组蛋白的过度表达或表达不足分别导致酵母和哺乳动物的染色体丢失和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，既作为一组正常的过程，也作为对基因组稳定性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.