mRNA Capping Enzyme

描述(申请人提供)：这个项目的长期目标是了解RNA聚合酶II(RNAPII)转录是如何与RNA处理和终止相耦合的。该项目的早期资助期产生了一个模型，在该模型中，RNAPII亚单位Rpb1的C-末端结构域(CTD)在转录周期的不同阶段显示出特征的磷酸化模式，以促进共转录RNA加工的适当因子的结合。该项目产生的基础知识为CTD磷酸化循环如何影响重要的医学过程提供了重要的洞察力，例如病毒Tat蛋白刺激HIV转录，以及RNAPII在胚胎干细胞中的发育调节基因上的“暂停”。这个项目对于更好地了解介导CTD磷酸化变化的两种酶(激酶、磷酸酶等)是必要的。以及识别这些模式的蛋白质。在家长赠款的当前资助期内，正在实现四个具体目标。首先是了解两条已知的终止途径(对于Polya和非Polya转录本)以及RNAPII如何在它们之间进行选择。第二个目的是研究在许多RNAPII启动子上发散转录产生的短的不稳定转录本的生物发生。第三个目的是使用Net-Seq来探讨CTD磷酸化在RNA延长、暂停和终止中的作用。第四个目的利用了我们的发现，即Rpb1 CTD可以转移到不同的RNAPII亚基上，询问不同的CTD修饰和功能是否需要在同一CTD的“顺”中发生，还是可以在两个CTD之间拆分。这一补充将增加一个新的具体目标，试图回答不同位置的CTD重复之间是否存在功能差异。在第一个子目标中，将设计一个CTD，它添加了几个赖氨酸和非共识重复，以便可以使用质谱学来区分近端和远端重复。这些CTD片段的体内磷酸化将在野生型细胞和各种CTD修饰酶的突变体中进行分析。在第二个子目标中，将构建一系列在体内加入非天然光反应氨基酸的CTD突变体。体内交联和相关蛋白的分析将显示CTD相关因子优先与近端或远端重复序列相关联。总之，这些实验的结果将表明CTD的磷酸化模式和相关蛋白质在CTD的不同区域是不同的还是相同的。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. Earlier funding periods of this project produced a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the appropriate factors for co-transcriptional RNA processing. The fundamental knowledge generated by this project provides significant insight into how the CTD phosphorylation cycle affects medically important processes such as the stimulation of HIV transcription by the viral Tat protein and the "pausing" of RNApII at developmentally regulated genes in embryonic stem cells. This project is necessary to better understand both the enzymes that mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that recognize these patterns. In the current funding period of the parent grant, four specific aims are being pursued. The first is to understand the two known termination pathways (for polyA and non-polyA transcripts) and how RNApII chooses between them. A second aim studies the biogenesis of short unstable transcripts produced by divergent transcription at many RNApII promoters. The third aim uses NET-Seq to probe the role of CTD phosphorylation in RNA elongation, pausing, and termination. The fourth aim exploits our discovery that the Rpb1 CTD can be transferred onto a different RNApII subunit, asking whether the different CTD modifications and functions need to occur in "cis" on the same CTD or can be split between two CTDs. This supplement will add one new specific aim attempting to answer whether there are functional differences between CTD repeats at different positions. In the first subaim, a CTD will be engineered that adds several lysines and non-consensus repeats so that mass spectrometry can be used to distinguish proximal and distal repeats. The in vivo phosphorylations on these CTD fragments will be analyzed in wild-type cells and mutants of various CTD modifying enzymes. In the second subaim, a series of CTD mutants will be constructed that incorporate a non-native, photoreactive amino acid in vivo. In vivo crosslinking and analysis of associated proteins will show whether CTD-associated factors preferentially associate with proximal or distal repeats. Together, the results of these experiments will show whether the CTD phosphorylation patterns and associated proteins are distinct or identical in different regions of the CTD.