Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation

在所有真核生物mRNA的5'末端添加帽是转录后加工的第一步， 并且通常认为其去除会使mRNA不可逆地衰退。在红细胞中毫无意义- 含有β-珠蛋白的mRNA被细胞质核酸内切酶切割，产生衰变中间体， 既稳定又封顶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，其含有能够转化5'末端的5'-单磷酸激酶， 将RNA脱帽成二磷酸加帽底物。虽然细胞质加帽酶与 无论是P体或应力颗粒的证据，其生物学作用是证明了减少 表达该蛋白质的显性阴性形式的细胞从应激中恢复。细胞质的必然结果 加帽是一种未加帽的转录组，我们的实验室最近在哺乳动物细胞中发现了这一证据 和拟南芥中的其他物种。这些mRNA通过其增加的表达与细胞质帽化有关。 在加帽酶的显性阴性形式表达后，未加帽池中的表型。目的 1将使用生物化学方法来识别和表征细胞质帽的组分， 酶复合物，特别强调新的5 '-单磷酸激酶。这些发现将指导 开发分子和遗传工具，用于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帽后它们的帽状态的动态变化。这些的3'末端 还将检查RNA以确定去腺苷化和/或寡尿苷酰化是否导致RNA的积累。 无帽mRNA。Aim 2的最后一部分将联合收割机与Aim 1中开发的工具相结合 为了全面了解无帽转录组及其与细胞质的关系， 封顶目标3将解决细胞质加帽的生物学相关性，因为它涉及细胞质的循环。 翻译和非翻译状态之间的mRNA。这些实验将检验改变 P体的大小和数量以及干扰导致脱盖和P体的不同步骤 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池。最后，将使用iTRAQ质谱法确定是否 改变细胞质帽改变了蛋白质组的复杂性。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。

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.