Relationship of Cytoplasmic Capping to Post-transcriptional Gene Regulation

在所有真核mRNAs的5‘端添加帽是转录后处理的第一步， 而它的去除通常被认为会不可逆转地使mrna腐烂。在红系细胞里胡说八道- 含有-珠蛋白的mRNA被细胞质核酸内切酶切割，产生腐烂的中间产物 都是稳定的和有上限的。尽管大多数封闭酶是核的，但我们发现了一个140 kDa的细胞质 封端酶复合体，含有5‘-单磷酸激酶，能够转化5’端的 将RNA解封到二磷酸封盖底物中。尽管细胞质封顶酶不相关 无论是P小体还是应激颗粒，其生物学作用的证据都通过减少 表达这种蛋白质的显性阴性形式的细胞从应激中恢复。细胞质的推论 Capping是一种无上限的转录组，最近我们的实验室在哺乳动物细胞中发现了它的证据 而在拟南芥中也有其他人这样做。这些mRNAs与细胞质封顶有关，因为它们增加了 在显性阴性形式的封闭酶表达后，在无封闭池中的表达。目标 1将使用生化方法来鉴定和表征细胞质封顶的成分 酶复合体，特别强调新的5‘-单磷酸激酶。这些发现将指导 用于表征细胞质封顶生物学功能的分子和遗传工具的发展。 目标2中的实验将表征选定数量的已确定的重新封盖底物的5‘端 并研究干扰细胞质封顶后其帽子状态的动态变化。它们的3‘端 还将检查RNA，以确定死烯基化和/或寡脲酰化是否会导致 不封顶的mRNA。AIM 2的最后一部分将结合深度测序和AIM 1中开发的工具 以生成未封顶的转录组及其与细胞质的关系的全面图像 封顶。目标3将讨论细胞质封顶的生物学相关性，因为它与细胞周期有关 翻译和非翻译状态之间的mRNAs。这些实验将检验改变的影响 P小体的大小和数量以及不同步骤的干扰导致去顶和P小体 组装，并研究microRNA沉默与无上限mRNAs积累的关系 和/或将它们恢复到翻译池。最后，将使用iTRAQ质谱仪来确定 改变细胞质的封顶改变了蛋白质组的复杂性。细胞质封顶有可能 广泛影响我们对正常和疾病过程的理解，这些过程与转录后 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.