Identifying the sequences and factors that govern the fate of elongating RNAPII

项目总结： 在发育过程中对基因表达的精确控制和对信号的响应对生物体来说是必不可少的 生长和动态平衡。因此，基因表达在多个步骤中受到调节，并严格控制 RNA聚合酶II(RNAPII)的转录延伸与信使RNA的协同加工 (M RNA)。我们实验室和其他实验室最近的研究表明，延长RNAPII可以针对早产儿 终止。耐人寻味的是，在真核生物中，哺乳动物表现出更高的早产率 和mRNAs内的“转录损耗”。这种现象在参与信号传递的基因中得到了丰富， DNA损伤反应、发育和组织特异性功能。尽管这样做的原因仍然是 根据定义，这些类别中的基因通常很长，并且含有广泛的第一内含子，这导致了人们认为 进化过程中内含子的扩展使得获得神秘的终止促进序列成为可能。显然， 全长信使核糖核酸的合成对于蛋白质的正常生产是必不可少的。因此，内含子终止具有 成为许多疾病的始作俑者，包括免疫功能障碍、神经退行性变和癌症。 在这里，我们建议系统地定义顺式作用序列和反式作用蛋白因子 决定RNAPII伸长复合体和新生RNA的命运。我们将定义RNAPII延伸率如何 在信使核糖核酸基因座被调节，以防止不适当的3‘末端形成和异常转录本的产生，以及 相反，增强子和反义RNA的转录是如何快速终止以防止产生 不受欢迎的非编码RNA(NcRNA)物种。为了实现这些目标，我们开发了体内协同作用和 在体外系统中。在目标1中，我们将使用一种强大的小鼠胚胎干细胞筛选策略来确定 影响RNAPII延伸特性和RNA命运的序列和蛋白质。为了补充这些内容 基于细胞的方法，Aim 2将利用一种新的无细胞转录系统来剖析生化 控制RNAPII延长以及与RNA加工和终止复合体相互作用的机制。 目标3将以我们的初步数据为基础，证明通过第一个外显子-内含子连接进行转录 刺激RNAPII延伸率，详细研究序列和蛋白质因子是如何参与 剪接影响RNAPII活性。这项工作将回答有关促进终止的性质的核心问题 序列和控制其识别的因素，并将描述伸长、 拼接和端接复合体。这些研究将确定对功能的伸长的要求 MRNA和用于防止转录磨损的机制。同时，我们将揭开这些序列 以及促进在增强子和其他调控位点提前终止以防止聚合酶的因素 碰撞、双链RNA的形成和基因组的不稳定。通过阐明RNAPII的这些机制 控制我们的目标是揭示疾病的原因，并提出可能的治疗方法，这些疾病包括 转录延伸、剪接和3‘末端选择的中断。

PROJECT SUMMARY: Precise control of gene expression during development and in response to signals is essential for organismal growth and homeostasis. Accordingly, gene expression is regulated at multiple steps, with tight control over transcription elongation by RNA polymerase II (RNAPII) and coordinated processing of messenger RNA (mRNA). Recent work by our lab and others has revealed that elongating RNAPII can be targeted for premature termination. Intriguingly, across eukaryotes, mammals display an increased prevalence of premature termination and “transcription attrition” within mRNAs. This phenomenon is enriched among genes involved in signaling, the DNA damage response, development and tissue-specific functions. Although the reasons for this remain to be defined, genes in these classes are often long and harbor extensive first introns, leading to suggestions that intron expansion during evolution enabled the acquisition of cryptic termination-promoting sequences. Clearly, full-length mRNA synthesis is essential for proper protein production. Accordingly, intronic termination has emerged as a contributor to many diseases, including immune dysfunction, neurodegeneration and cancer. Here, we propose to systematically define the cis-acting sequences and trans-acting protein factors that determine the fate of the RNAPII elongation complex and nascent RNA. We will define how RNAPII elongation is regulated at mRNA loci to prevent inappropriate 3’ end formation and production of aberrant transcripts, and conversely, how transcription of enhancer and antisense RNAs is rapidly terminated to prevent generation of unwanted non-coding RNA (ncRNA) species. To accomplish these goals, we developed synergistic in vivo and in vitro systems. In Aim 1, we will use a powerful screening strategy in mouse embryonic stem cells to define the sequences and proteins that influence RNAPII elongation properties and RNA fate. To complement these cell-based approaches, Aim 2 will make use of a novel cell-free transcription system to dissect the biochemical mechanisms that control RNAPII elongation and the interplay with RNA processing and termination complexes. Aim 3 will build on our preliminary data demonstrating that transcription through the first exon-intron junction stimulates RNAPII elongation rate, to investigate in detail how the sequences and protein factors involved in splicing impact RNAPII activity. This work will answer central questions about the nature of termination-promoting sequences and the factors that govern their recognition, and will describe the interactions between elongation, splicing and termination complexes. These studies will identify the requirements for elongation of a functional mRNA and the mechanisms employed to prevent transcription attrition. In parallel, we will uncover the sequences and factors that promote early termination at enhancers and other regulatory loci to prevent polymerase collisions, double-stranded RNA formation and genome instability. By elucidating these mechanisms of RNAPII control we aim to reveal the causes of, and suggest potential treatments for, the growing list of diseases involving disruption of transcription elongation, splicing and 3’ end choice.