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'端添加帽是转录后加工的第一步，并且通常认为其去除会不可逆地使mRNA衰变。在红系细胞中，含无义2-珠蛋白的mRNA被胞质核酸内切酶切割，产生稳定且加帽的衰变中间体.虽然大多数加帽酶是核，我们确定了一个140 kDa的细胞质加帽酶复合物，其中含有一个5 '-单磷酸激酶，能够将5'端的decapped RNA转化为二磷酸加帽底物。虽然细胞质加帽酶是不相关的P机构或应力颗粒的证据表明，其生物学作用的细胞表达这种蛋白质的显性负性形式的应力的恢复减少。细胞质加帽的必然结果是一个未加帽的转录组，我们的实验室最近在哺乳动物细胞和拟南芥中发现了这一证据。这些mRNA通过在加帽酶的显性阴性形式表达后在未加帽池中增加的代表性与细胞质加帽相关联。目的1将使用生物化学方法来鉴定和表征细胞质加帽酶复合物的组分，特别强调新的5 '-单磷酸激酶。这些发现将指导分子和遗传工具的发展，以表征细胞质帽的生物学功能。目标2中的实验将表征选定数量的鉴定的再加帽底物的5'端，并研究在干扰细胞质加帽后其帽状态的动态变化。还将检查这些RNA的3'末端以确定去腺苷化和/或寡尿苷基化是否导致未加帽的mRNA的积累。目标2的最后一部分将联合收割机与目标1中开发的工具相结合，以生成未加帽转录组及其与细胞质加帽的关系的全面图片。目标3将解决细胞质加帽的生物学相关性，因为它涉及mRNA在翻译和非翻译状态之间的循环。这些实验将检查改变P体的大小和数量以及干扰导致去帽和P体组装的不同步骤的影响，并检查microRNA沉默与未加帽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加帽是否会改变蛋白质组的复杂性。细胞质加帽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。 公共卫生相关性：大多数基因表达的终点是蛋白质产物的产生，mRNA翻译的调节对于发育、学习和记忆、细胞对压力的反应以及癌症的发展和生长等多种过程至关重要。非翻译的mRNA被储存起来供以后使用或降解，关于这些储存的mRNA的状态或它们如何被重新激活的知之甚少。该建议将细胞质加帽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.