Probing the specificity and activity of the metazoan Integrator complex

项目概要： 精确控制基因表达以响应外部信号和提示对于生物体的发育至关重要。 发育生长和体内平衡在后生动物细胞中，RNA聚合酶II（RNAPII）的调节性暂停 在早期转录物延长是决定基因输出关键。然而，控制暂停的机制 以及RNAPII释放到生产性伸长中的情况仍然不清楚。我们的工作表明， 整合子复合物（INT）靶向暂停RNAPII以减弱信使RNA（mRNA）表达。此外，委员会认为， 我们发现INT是果蝇细胞中基因表达的中心调节因子，通过它的选择性募集， 特异性基因启动子和随后INT介导的转录终止。已知需要INT 参与剪接的小核RNA（snRNA）的3 '端形成，并与切割有关 和其他非编码RNA的加工。因此，发现整合子11亚基（IntS 11）具有 RNA内切核酸酶活性。有趣的是，INT最近被证明也与RNAPII在蛋白质- 然而，我们对INT在mRNA上的功能的理解非常有限。如下所述，我们的 初步数据和拟议的研究将解决有关基因调控的基本的、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在一些温度下非常稳定， mRNA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 引起了人们对其根本原因的极大兴趣。特别是，快速更替的证据 暂停RNAPII表明启动子近端终止因子的存在。然而，没有这样的因素 已经在后生动物细胞中被描述过。我们的工作表明，INT是这种“缺失”的终止因子， 提示IntS 11介导的RNA切割在转录终止中的作用。此外，我们还发现， 募集蛋白磷酸酶PP 2A至RNAPII，导致RNAPII C末端结构域去磷酸化 （CTD）。这一发现是非常重要的，因为RNAPII CTD磷酸化参与了暂停的 RNAPII转化为生产性mRNA延伸。拟议的工作扩展了这一初步数据。目标1将 确定INT如何靶向特定的基因启动子，特别是寻求相互作用的证据 INT和cohesin复合物之间的联系值得注意的是，INT已经被证明与粘附素相互作用， 哺乳动物神经元，并与科尔内利亚de Lange综合征的cohesin。目标2将探测 IntS 11 RNA内切酶活性对RNAPII延伸特性的影响。目标3将定义 底物和INT介导的PP 2A磷酸酶募集到早期伸长的结果 复杂.重要的是，INT的突变与大量疾病有关， INT复合物中的14个亚基与病理生理状态有关。因此，改善我们的 对INT活性的了解将为研究血液、肝脏和其他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.