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

基本信息

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
项目类别：
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

项目摘要

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.

在所有真核生物 mRNA 的 5' 末端添加帽子是转录后加工的第一步，人们普遍认为它的去除会导致红细胞中的 mRNA 不可逆转地衰变。含有 ¿ -球蛋白 mRNA 被细胞质核酸内切酶切割，产生衰变中间体，虽然大多数加帽酶是核的，但我们鉴定出 140 kDa 的细胞质。加帽酶复合物，含有 5'-单磷酸激酶，能够转化 5' 末端尽管细胞质加帽酶不相关，但将 RNA 脱帽到二磷酸加帽底物中。与 P 体或应激颗粒有关其生物学作用的证据通过减少表达该蛋白质的显性失活形式的细胞从应激中恢复。细胞质的推论。 capping 是一种无帽转录组，我们的实验室最近在哺乳动物细胞中发现了其证据以及拟南芥中的其他人发现，这些 mRNA 通过其增加而与细胞质加帽相关。表达显性阴性形式的加帽酶后，未加帽池中的代表。 1 将使用生化方法来识别和表征细胞质加帽的成分酶复合物，特别是新型 5'-单磷酸激酶这些发现将指导。开发用于表征细胞质加帽的生物学功能的分子和遗传工具。目标 2 中的实验将表征选定数量的已识别重加帽底物的 5' 末端并研究干扰细胞质加帽后其帽子状态的动态变化。 3' 端。还将检查 RNA 以确定脱腺苷化和/或寡尿苷酰化是否会导致积累 Aim 2 的最后一部分将把深度测序与 Aim 1 中开发的工具结合起来。生成无帽转录组及其与细胞质的关系的全面图片目标 3 将解决细胞质加帽的生物学相关性，因为它与细胞的循环有关。这些实验将检查翻译状态和非翻译状态之间的 mRNA 改变的影响。 P体的大小和数量以及导致脱盖和P体的不同步骤的干扰组装，并检查 microRNA 沉默与未加帽 mRNA 积累的关系和/或将它们恢复到翻译库中，最后，将使用 iTRAQ 质谱来确定是否。改变细胞质加帽有可能改变蛋白质组的复杂性。广泛影响我们对与转录后相关的正常和疾病过程的理解控制，包括干细胞、胚胎发育、癌症和神经科学。