Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation

描述（由申请人提供）：在所有真核mRNA的5'端添加一个帽是转录后加工的第一步，通常认为去除它会不可逆地导致mRNA衰变。在红细胞中，含无义的2-珠蛋白mRNA被细胞质内切酶切割，产生稳定和有帽盖的衰变中间体。虽然大多数盖帽酶是核的，但我们发现了一种140 kDa的细胞质盖帽酶复合物，它含有一个5‘-单磷酸激酶，能够将去盖RNA的5’端转化为二磷酸盖帽底物。尽管胞质盖顶酶与P小体或应激颗粒均无关联，但表达该蛋白显性阴性形式的细胞从应激中恢复的减少证明了其生物学作用。细胞质盖帽的必然结果是一个无盖帽的转录组，我们的实验室最近在哺乳动物细胞和其他拟南芥细胞中发现了这一证据。这些mrna与细胞质盖帽有关，因为在盖帽酶的显性阴性形式表达后，它们在未盖帽池中的代表性增加。目的1将使用生化方法来识别和表征细胞质盖帽酶复合物的成分，特别强调新型的5'-单磷酸激酶。这些发现将指导分子和遗传工具的发展，以表征细胞质盖顶的生物学功能。Aim 2中的实验将表征选定数量的已确定的重盖底物的5'端，并研究干扰细胞质盖盖后其盖状态的动态变化。这些rna的3'端也将被检测，以确定死蛋白化和/或寡尿苷化是否会导致无帽mrna的积累。Aim 2的最后一部分将结合深度测序与Aim 1中开发的工具，以生成未封顶转录组及其与细胞质封顶关系的全面图像。目标3将讨论细胞质封顶的生物学相关性，因为它与mrna在翻译状态和非翻译状态之间的循环有关。这些实验将研究改变P小体的大小和数量以及干扰导致脱帽和P小体组装的不同步骤的影响，并研究microRNA沉默与未封盖mrna的积累和/或其恢复到翻译池的关系。最后，iTRAQ质谱法将用于确定改变细胞质封顶是否会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与转录后控制相关的正常和疾病过程的理解，包括干细胞、胚胎发育、癌症和神经科学。

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.