mRNA Capping Enzyme

描述(由申请人提供)： 项目摘要。该项目的长期目标是了解RNA聚合酶II(RNAPII)的转录是如何与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。这些研究是在酿酒酵母模型系统中进行的，但这个过程在进化过程中是高度保守的。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”。为了更好地了解介导CTD磷酸化变化的两种酶(激酶、磷酸酶等)，该项目的继续是必要的。以及识别这些模式的蛋白质。在下一个筹资阶段，提出了四个具体目标。第一个是继续研究，以了解两种已知终止途径的机制以及RNAPII如何在它们之间进行选择：用于小的非编码转录本和许多“隐蔽”转录本的早期终止途径，或用于mRNAs的后来的多聚腺苷偶联终止途径。第二个相关的目的是研究由许多RNAPII启动子的发散转录产生的短而不稳定的转录本的生物发生，重点是它们能告诉我们什么关于起始、早期延伸和终止。第三个目标将使用一种新的高分辨率技术来定位新生转录本的3‘端，以进一步探讨CTD磷酸化在RNA延长、停顿和终止中的作用。第四个具体目标建立在一个令人惊讶的发现之上，即Rpb1 CTD可以转移到不同的RNAPII亚单位上并仍然发挥作用。这一发现形成了一系列实验的基础，这些实验询问不同的CTD修饰和功能是否需要在同一CTD上的“顺”中发生，或者是否可以在两个CTD之间分开，以及是否可以通过将它们的靶直接拴在RNAPII上来绕过这些磷酸化。

DESCRIPTION (provided by applicant): Project Summary. 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. These studies are done in the model system Saccharomyces cerevisiae, but this process is highly conserved over evolution. 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. Continuation of 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 next funding period, four specific aims are proposed. The first is a continuation of studies to understand the mechanisms of the two known termination pathways and how RNApII chooses between them: an early termination pathway used for small noncoding transcripts and many "cryptic" transcripts or the later polyadenylation-coupled termination pathway used for mRNAs. A second related aim is to study the biogenesis of short unstable transcripts produced by divergent transcription of many RNApII promoters, with a focus on what they can tell us about initiation, early elongation, and termination. The third aim will use a new, high- resolution technique for mapping 3' ends of nascent transcripts to further probe the role of CTD phosphorylation in RNA elongation, pausing, and termination. The fourth specific aim builds upon the surprising discovery that the Rpb1 CTD can be transferred onto a different RNApII subunit and still function. This finding forms the basis for a set of experiments asking whether the different CTD modifications and functions need to occur in "cis" on the same CTD or can be split between two CTDs, and whether these phosphorylations can be bypassed by tethering their targets directly to RNApII.