Probing the specificity and activity of the metazoan Integrator complex

探讨后生动物整合复合体的特异性和活性

基本信息

批准号：
10224260
负责人：
Karen L Adelman
金额：
$ 65.07万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10224260
关键词：
Address Amino Acids Attenuated Behavior Binding Biochemical Biological Bruck-de Lange syndrome C-terminal Cells Chromatin Code Complex Computer Analysis Cues Data Development Disease Drosophila genus Elongation Factor Endoribonucleases Enhancers Explosion Functional disorder Gene Expression Gene Expression Regulation Generations Genes Genetic Transcription Growth Hematological Disease Hematopoiesis Hematopoietic Neoplasms Homeostasis Human Development In Vitro Knowledge Malignant Neoplasms Malignant neoplasm of liver Maps Mass Spectrum Analysis Mediating Messenger RNA Modeling Modification Molecular Monitor Mutation Neurons Output Phosphoric Monoester Hydrolases Phosphorylation Physiological Plasmids Polymerase Property Protein Dephosphorylation Protein phosphatase Proteins RNA RNA Polymerase II RNA Splicing RNA analysis RNA chemical synthesis Regulation Regulator Genes Reporter Research Role Serine Signal Transduction Site Small Nuclear RNA Specificity Stimulus Structure Time Transcript Transcription Elongation Transcriptional Regulation Untranslated RNA Work attenuation base cilium biogenesis cohesin developmental disease endonuclease experience functional genomics gene repression human disease improved in vivo insight interest lipid biosynthesis malignant stomach neoplasm migration mutant neurogenesis promoter protein complex public health relevance recruit response synergism termination factor tool transcription factor transcription termination transcriptome sequencing

项目摘要

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.

项目概要：响应外部信号和线索精确控制基因表达对于生物学至关重要在后生动物细胞中，RNA 聚合酶 II (RNAPII) 的受控暂停。然而，早期转录本延伸对于定义基因输出至关重要。我们的工作表明，RNAPII 释放到生产伸长中的情况仍然不明确。整合复合物 (INT) 以暂停的 RNAPII 为目标，以减弱信使 RNA (mRNA) 的表达。我们发现 INT 是果蝇细胞基因表达的中央调节因子，通过选择性招募已知需要特定的基因启动子和随后的 INT 介导的转录终止。用于参与剪接 (snRNA) 的小核 RNA 的 3' 端形成，并参与切割相应地，Integrator 11 亚基 (IntS11) 被发现具有一种 RNA 核酸内切酶活性，最近显示 INT 也与蛋白质上的 RNAPII 相关。然而，我们对 mRNA 的 INT 功能的了解非常有限，如下所述。初步数据和拟议的研究将解决有关基因调控的基本的、尚未解答的问题，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。 mRNA 基因启动子，但在其他基因启动子上高度不稳定。暂停的 RNAPII 的行为具有多样性。引起了人们对其根本原因的极大兴趣，特别是快速周转的证据。暂停的 RNAPII 表明存在启动子近端终止因子，但是没有这样的因子。我们的工作已在后生动物细胞中进行了描述，表明 INT 是这种“缺失”的终止因子，并且表明 IntS11 介导的 RNA 裂解在转录终止中发挥作用。将蛋白磷酸酶 PP2A 募集至 RNAPII，导致 RNAPII C 末端结构域去磷酸化 (CTD)，这一发现非常重要，因为 RNAPII CTD 磷酸化参与了暂停的释放。 RNAPII 转化为生产性 mRNA 延伸。拟议的工作将在此初步数据的基础上进行扩展。确定 INT 如何针对特定基因启动子，特别寻找相互作用的证据 INT 和粘连蛋白复合物之间的关系值得注意的是，INT 已被证明与粘连蛋白相互作用。哺乳动物神经元，并与 Cornelia de Lange 综合征中的粘连蛋白一起，将探测 Aim 2。 IntS11 RNA 核酸内切酶活性对 RNAPII 延伸特性的影响将定义目标 3。 INT介导的PP2A磷酸酶募集到早期延伸的底物和后果重要的是，INT 突变与许多疾病有关，其中每一种疾病都与 INT 突变有关。 INT 复合体中的 14 个亚基与经过测试的病理生理状态有关，从而改善了我们的研究。对 INT 活动的了解将为了解血液、肝脏等疾病的机制提供宝贵的见解。和胃癌，以及发育障碍，包括异常的神经元迁移。