Probing the specificity and activity of the metazoan Integrator complex

项目总结： 对于生物体来说，精确控制基因表达以响应外界信号和提示是必不可少的 发展、增长和动态平衡。在后生动物细胞中，RNA聚合酶II(RNAPII)的调节暂停 在早期的转录本中，伸长是决定基因输出的关键。然而，管理停顿的机制 以及RNAPII释放到生产性伸长中仍然没有明确的定义。我们的工作表明， 整合子复合体(INT)靶标暂停RNAPII以减弱信使RNA(MRNA)的表达。此外， 我们发现INT是果蝇细胞中基因表达的中心调节因子，它通过选择性地募集到 特定基因启动子和随后INT介导的转录终止。已知需要INT 参与剪接的小核RNA(SnRNAs)的3‘端形成，并与切割有关 以及其他非编码RNA的处理。因此，整合者11亚基(IntS11)被发现具有 核糖核酸内切酶活性。有趣的是，INT最近被证明在蛋白质上也与RNAPII有关- 然而，对于编码基因，我们对mRNAs上的int功能的了解非常有限。如下所述，我们的 初步数据和拟议的研究将解决关于基因调控的基本、尚未回答的问题， 旨在阐明INT在人类疾病中的作用。例如，暂停的RNAPII在某些情况下非常稳定 信使核糖核酸基因启动子，但在其他高度不稳定。暂停的RNAPII的这种行为多样性具有 引起了人们对其根本原因的相当大的兴趣。特别是，人员流动迅速的证据 暂停的RNAPII提示存在启动子-近端终止因子。然而，没有这样的因素 已经在后生动物细胞中被描述过。我们的工作证明了INT是这个‘缺失’的终止因子 提示了IntS11介导的RNA切割在转录终止中的作用。此外，我们发现int 向RNAPII招募一种蛋白磷酸酶PP2A，导致RNAPII C末端结构域的去磷酸化 (CTD)。这一发现非常有意义，因为RNAPII CTD磷酸化参与了暂停的释放 RNAPII转化为生产性的信使核糖核酸。拟议的工作是在这一初步数据的基础上展开的。目标1将 确定INT如何针对特定的基因启动子，特别是寻找相互作用的证据 在INT和粘附素复合体之间。值得注意的是，int已经被证明与粘附素在 哺乳动物神经元，并与粘附素一起与科尼莉亚-德朗格综合征有关。目标2将探测 IntS11 RNA内切酶活性对RNAPII延伸特性的影响。目标3将定义 INT介导的PP2A磷酸酶在早期伸长中的底物和后果 很复杂。关键的是，int基因的突变与许多疾病有关，每一种 INT复合体中的14个亚基涉及到一种病理生理状态(S)。因此，改善我们的 对int活性的了解将对血液、肝脏等疾病的机制提供宝贵的见解。 和胃癌，以及发育障碍，包括异常神经元迁移。

PROJECT SUMMARY: Precise control of gene expression in response to external signals and cues is essential for organismal development, growth and homeostasis. In metazoan cells, the regulated pausing of RNA polymerase II (RNAPII) in early transcript elongation is pivotal for defining gene output. However, the mechanisms that govern pausing and release of RNAPII into productive elongation remain poorly defined. Our work demonstrates that the Integrator complex (INT) targets paused RNAPII to attenuate messenger RNA (mRNA) expression. Moreover, we find that INT is a central regulator of gene expression in Drosophila cells, through its selective recruitment to specific gene promoters and subsequent INT-mediated termination of transcription. INT is known to be required for 3'-end formation of small nuclear RNAs involved in splicing (snRNAs), and has been implicated in cleavage and processing of other non-coding RNAs. Accordingly, the Integrator 11 subunit (IntS11) was found to possess an RNA endonuclease activity. Interestingly, INT was recently shown to also associate with RNAPII at protein- coding genes, however, our understanding of INT function on mRNAs is very limited. As described below, our preliminary data and proposed research will address fundamental, unanswered questions about gene regulation, and aim to elucidate the roles of INT in human disease. For example, paused RNAPII is very stable at some mRNA gene promoters, yet highly unstable at others. This diversity of behaviors for paused RNAPII has generated considerable interest concerning its underlying causes. In particular, the evidence for rapid turnover of paused RNAPII suggests the presence of a promoter-proximal termination factor. However, no such factor has been described in metazoan cells. Our work demonstrates that INT is this ‘missing’ termination factor and suggests a role for IntS11-mediated RNA cleavage in transcript termination. In addition, we discovered that INT recruits a protein phosphatase, PP2A, to RNAPII leading to de-phosphorylation of the RNAPII C-terminal domain (CTD). This finding is highly significant, since RNAPII CTD phosphorylation is involved in the release of paused RNAPII into productive mRNA elongation. The proposed work expands on this preliminary data. Aim 1 will determine how INT is targeted to specific gene promoters, pursuing in particular evidence for an interaction between INT and the cohesin complex. Notably, INT has already been shown to interact with cohesin in mammalian neurons, and implicated together with cohesin in Cornelia de Lange Syndrome. Aim 2 will probe the impact of the IntS11 RNA endonuclease activity on RNAPII elongation properties. Aim 3 will define the substrates and consequences of INT-mediated recruitment of the PP2A phosphatase to the early elongation complex. Critically, mutations in INT have been associated with a large number of diseases, with every one of the 14 subunits in the INT complex being implicated a pathophysiological state(s). Consequently, improving our understanding of INT activity will provide invaluable insights into mechanisms of diseases such as blood, hepatic and gastric cancers, and developmental disorders, including aberrant neuronal migration.