Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation

描述(由申请人提供)：在所有真核细胞mRNAs的5‘端增加一个帽是转录后处理的第一步，它的去除通常被认为是不可逆转地使mRNAs腐烂。在红系细胞中，含有无义的2-珠蛋白的信使核糖核酸被细胞质内切酶切割，以产生稳定的、有上限的腐烂中间体。虽然大多数封闭酶是核的，但我们发现了一个140 kDa的细胞质封闭酶复合体，它包含一个5‘-单磷酸激酶，能够将去掉的RNA的5’端转化为二磷酸的封盖底物。尽管细胞质封顶酶与P小体或应激颗粒无关，但该蛋白的显性负性表达细胞从应激中恢复的能力降低，证明了它的生物学作用。细胞质封顶的必然结果是一个未封顶的转录组，最近我们的实验室在哺乳动物细胞中发现了这一证据，其他人在拟南芥中也发现了这一证据。这些mRNAs与细胞质封顶有关，因为它们在未封顶的池中的表达增加，随后表达了一种显性的负型封顶酶。目的1将使用生化方法来鉴定和表征细胞质封端酶复合体的成分，特别是新的5‘-单磷酸激酶。这些发现将指导分子和遗传工具的发展，以表征细胞质封顶的生物学功能。目标2中的实验将表征选定数量的已确定的重新封盖底物的5‘端，并研究干扰细胞质封盖后其封盖状态的动态变化。还将检查这些RNA的3‘端，以确定死烯化和/或寡酰化是否导致未封顶的mRNAs的积累。AIM 2的最后部分将把深度测序与AIM 1中开发的工具结合起来，以生成未封顶转录组及其与细胞质封顶的关系的全面图像。目标3将讨论细胞质封顶的生物学相关性，因为它与翻译和非翻译状态之间的mRNAs循环有关。这些实验将研究改变P小体的大小和数量以及干扰导致解帽和P小体组装的不同步骤的影响，并研究microRNA沉默与无上限mRNAs的积累和/或它们恢复到翻译池的关系。最后，iTRAQ质谱仪将被用来确定改变细胞质封顶是否改变蛋白质组的复杂性。细胞质封顶有可能广泛影响我们对正常和疾病过程的理解，这些过程与转录后控制有关，包括干细胞、胚胎发育、癌症和神经科学。 与公共健康相关：大多数基因表达的终点是蛋白质产物的产生，而mRNA翻译的调控对于发育、学习和记忆、细胞对压力的反应以及癌症的发展和生长等各种过程都是必不可少的。非翻译的mRNAs被储存起来供以后使用或降解，人们对这些储存的mRNAs的状态或它们如何被重新激活知之甚少。这项建议研究了细胞质封顶作为一种新的调控过程，对转录后基因调控、RNA沉默和翻译控制具有广泛的意义。

DESCRIPTION (provided by applicant): The addition of a cap to the 5' end of all eukaryotic mRNAs is the first step in post-transcriptional processing, and its removal is generally thought to irreversibly commit mRNA to decay. In erythroid cells nonsense- containing 2-globin mRNA is cleaved by a cytoplasmic endonuclease to generate decay intermediates that are both stable and capped. Although most capping enzyme is nuclear, we identified a 140 kDa cytoplasmic capping enzyme complex that contains a 5'-monophosphate kinase capable of transforming the 5' end of decapped RNA into a diphosphate capping substrate. Although cytoplasmic capping enzyme is not associated with either P bodies or stress granules evidence for its biological role was demonstrated by the reduced recovery from stress of cells expressing a dominant negative form of this protein. The corollary to cytoplasmic capping is an uncapped transcriptome, evidence of which was recently identified by our lab in mammalian cells and by others in Arabidopsis. These mRNAs were linked to cytoplasmic capping by their increased representation in the uncapped pool following expression of a dominant negative form of capping enzyme. Aim 1 will use biochemical approaches to identify and characterize the components of the cytoplasmic capping enzyme complex, with particular emphasis on the novel 5'-monophosphate kinase. These findings will guide development of molecular and genetic tools for characterizing the biological function of cytoplasmic capping. Experiments in Aim 2 will characterize the 5' ends of a selected number of the identified re-capping substrates and study dynamic changes in their cap status after interfering with cytoplasmic capping. The 3' ends of these RNAs will also be examined to determine if deadenylation and/or oligouridylylation lead to the accumulation of uncapped mRNAs. The last portion of Aim 2 will combine deep sequencing with the tools developed in Aim 1 to generate a comprehensive picture of the uncapped transcriptome and its relationship to cytoplasmic capping. Aim 3 will address the biological relevance of cytoplasmic capping as it relates to the cycling of mRNAs between translating and non-translating states. These experiments will examine the impact of altering the size and number of P bodies and interfering with different steps leading to decapping and P body assembly, and examine the relationship of microRNA silencing to the accumulation of uncapped mRNAs and/or their restoration to the translating pool. Lastly, iTRAQ mass spectrometry will be used to determine if altering cytoplasmic capping changes the complexity of the proteome. Cytoplasmic capping has the potential to broadly impact our understanding of normal and disease processes that are linked to post-transcriptional control, including stem cells, embryonic development, cancer and neuroscience. PUBLIC HEALTH RELEVANCE: The endpoint of most gene expression is the production of a protein product, and the regulation of mRNA translation is essential for such diverse processes as development, learning and memory, the cellular response to stress and the development and growth of cancers. Non-translating mRNAs are stored for later use or degraded, and little is known about the state of these stored mRNAs or how they are re-activated. This proposal examines cytoplasmic capping as a new regulatory process with broad implications for post- transcriptional gene regulation, RNA silencing and translational control.