Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation

细胞质加帽与转录后基因调控的关系

• 批准号: 8242018
• 负责人: DANIEL R. SCHOENBERG
• 金额: $ 30.2万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242018
• 关键词: Abbreviations; Active Sites; Address; Arabidopsis; Biochemical; Biological; Biological Process; Cell Extracts; Cells; Cellular Stress; Cleaved cell; Commit; Complex; Cytoplasm; Cytoplasmic Granules; Development; Diphosphates; Disease; Dominant-Negative Mutation; Embryonic Development; Enzymes; Erythroid Cells; Excision; Family; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic; Genetic Translation; Globin; Glycerol; Growth and Development function; Lead; Learning; Link; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Memory; Messenger RNA; MicroRNAs; Molecular Genetics; Multienzyme Complexes; Mutation; Neurons; Neurosciences; Nuclear; Phosphotransferases; Play; Polyadenylation; Population; Post-Transcriptional Regulation; Process; Production; Protein Binding; Proteins; Proteome; Publishing; RNA; RNA Interference; RNA Splicing; RNA, Messenger, Stored; Recovery; Regulation; Role; Site; Small RNA; Stem cells; Stress; Structure; System; Thinking; Translating; Translation Initiation; Work; endonuclease; mRNA Decay; mRNA Surveillance; novel; research study; response; restoration; tool

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 ¿-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.

在所有真核mRNA的5'端增加一个盖是第一个步骤转录处理， 并去除通常会在异性细胞中腐烂的mRNA。 包含� - 珠蛋白mRNA被细胞质内核酸酶切割，以产生衰减中间体 稳定和上限。 限制含有5'单磷酸激酶的含量，能够转化的5'端 dece rNA进入双磷酸盐限制底物。 用p身体或压力颗粒的证据是生物角色角色角色角色角色角色角色角色角色角色角色 从表达该蛋白质的显性负形式的细胞中恢复。 封盖是一个未盖上的转录组，我们的实验室最近在哺乳动物细胞中鉴定出其证据 在拟南芥中，这些mRNA与细胞质上限有关 在封盖酶的主要负面形式的未盖池表达中的抑制。 1将使用生化方法来识别和表征细胞质上限的成分 酶复合物，特别着重于新型5'-Onophathate激酶。 开发分子和遗传工具，以表征细胞质封盖的生物学功能。 AIM 2中的经验将表征所选数量的重新封码基板的5'端 并研究其帽状态的动态变化在细胞质上限之后。 还将检查RNA，以确定deadenylation和/寡烯基化是否导致积累 毫无疑问的mRNA。 为了产生无盖转录组的全面图片，是与细胞质的关系 封盖3。 翻译和非翻译状态之间的mRNA将检查改变的影响 p身体的大小和数量，干扰不同的步骤，导致decapping和p身体 组装，并检查microRNA沉默与未封闭mRNA的积累的关系 和/或他们的翻译池。 改变细胞质上限会改变蛋白质组的复杂性。 广泛影响我们对与转录后有关的正常和疾病过程的理解 控制，包含干细胞，胚胎发育，癌症和神经科学。