Regulation of RNA surveillance by the dynamic Exon Junction Complex

动态外显子连接复合物对 RNA 监视的调节

基本信息

批准号：
9384336
负责人：
Guramrit Singh
金额：
$ 31.27万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9384336
关键词：
Address Affinity Chromatography Binding Sites Biological Biological Process Biology Cell physiology Cells Communication Complex Coupled DNA DNA sequencing Data Defect Deposition Development Ensure Event Excision Exons Functional disorder Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Goals Health Human Immunoprecipitation Intellectual functioning disability Introns Kinetics Label Lead MLN51 Gene Malignant Neoplasms Mass Spectrum Analysis Mediating Mental Retardation Messenger RNA Metabolic Metabolism Methods Mutation Pathway interactions Play Positioning Attribute Process Proteins Quality Control RNA Recruitment Activity Regulation Regulator Genes Regulatory Pathway Reporter Rest Ribonucleoproteins Role Site Specific qualifier value Specificity Spliceosomes Stereotyping Terminator Codon Testing Transcript Transcription Process Translations Work base deep sequencing genetic information human disease in vivo insight interdisciplinary approach mRNA Decay messenger ribonucleoprotein neglect next generation novel premature protein complex protein purification tool transcriptome transcriptome sequencing unpublished works

项目摘要

7. Project Summary/Abstract The proposal is focused on understanding the functions of the extremely conserved multi-protein exon junction complex (EJC) in specifying parallel nonsense-mediated mRNA decay (NMD) pathways. NMD is as an essential post-transcriptional mechanism that regulates normal gene expression and also serves a quality control function. A detailed understanding of these processes is crucial for betterment of human health as mutations that disrupt the EJC and NMD proteins cause developmental defects, intellectual disability and mental retardation. The EJC is deposited upstream of mRNA exon-exon junctions by the spliceosome, and has major consequences on downstream mRNA metabolism. An EJC downstream of a termination codon is widely accepted as an absolute mark for premature translation termination and an NMD trigger. The current proposal is motivated by two unexpected discoveries that challenge the dogmatic view of EJC composition and function. First, we recently revealed in vivo EJC occupied sites by developing a novel high-throughput approach termed RIPiT-Seq that purifies RNPs containing a distinct pair of proteins and identifies their transcriptome-wide footprints. This demonstrated that the EJC is not detected at all predicted binding sites and, frequently, the complex is also detected at unexpected positions. We now show that the EJC composition also varies from position to position, and there exist in human cells at least two mutually exclusive or 'alternate' EJCs, that contain different protein factors. Notably, the two alternate EJC factors, RNPS1 and MLN51 were described previously as unique components of parallel NMD branches. We show that the two alternate EJC factors have distinct NMD targets, and they interact differentially with proteins of the parallel NMD branches. Our overall hypothesis is that the alternate EJCs control distinctly different biological activities in the cell. We propose to use a multi-disciplinary approach to discover how different EJC compositions are connected to parallel NMD branches and what biological activities are regulated by each unique complex. In Aim 1, we will use RNA-Seq and RIPiT-Seq approaches to define the alternate EJC-regulated NMD targets, their distinctive features, and hence the unique biological functions of alternate EJCs. In Aim 2, we will use a metabolic labeling approach to address a largely neglected question regarding the kinetics and order of events during EJC remodeling. Using well-established strategies to enhance and/or inhibit NMD at different steps, we will investigate when and where in the NMD pathway do alternate EJCs function. In Aim 3, we will use reporters from parallel NMD branches to test the relationship between alternate EJCs and parallel NMD pathways. We will also use RNA- Seq and RIPiT-Seq based global analyses to reveal specific mRNA and protein interactions of alternate EJC- Upf complexes in parallel NMD branches. Our goal is to reveal the target specificity, underlying mechanisms and cellular functions of parallel NMD branches.

7. 项目总结/摘要该提案的重点是了解极其保守的多蛋白外显子连接的功能复合体（EJC）指定平行的无义介导的mRNA衰减（NMD）途径。 NMD 是作为调节正常基因表达并提供质量的重要转录后机制控制功能。详细了解这些过程对于改善人类健康至关重要破坏 EJC 和 NMD 蛋白的突变会导致发育缺陷、智力障碍和智力低下。 EJC 通过剪接体沉积在 mRNA 外显子-外显子连接的上游，并且具有对下游 mRNA 代谢产生重大影响。终止密码子下游的 EJC 被广泛使用被接受为提前翻译终止和 NMD 触发器的绝对标记。目前的提案受到两个意想不到的发现的推动，这些发现挑战了 EJC 组成和功能的教条观点。首先，我们最近通过开发一种新的高通量方法揭示了体内 EJC 占据的位点 RIPiT-Seq 可纯化包含一对不同蛋白质的 RNP，并在全转录组范围内识别它们脚印。这表明 EJC 并未在所有预测的结合位点上被检测到，并且通常情况下，在意想不到的位置也检测到复合物。我们现在表明 EJC 的组成也有所不同位置到位置，并且在人类细胞中存在至少两个相互排斥或“替代”的 EJC，即含有不同的蛋白质因子。值得注意的是，描述了两个替代 EJC 因子 RNPS1 和 MLN51 以前作为并行 NMD 分支的独特组件。我们证明了两个替代 EJC 因子不同的 NMD 靶点，并且它们与平行 NMD 分支的蛋白质的相互作用存在差异。我们的整体假设是交替的 EJC 控制着细胞中明显不同的生物活性。我们建议使用多学科方法来发现不同的 EJC 组合如何与并行 NMD 连接分支以及每个独特的复合体调节哪些生物活性。在目标 1 中，我们将使用 RNA-Seq 和 RIPiT-Seq 方法来定义替代 EJC 监管的 NMD 目标、其独特特征以及因此，替代 EJC 具有独特的生物学功能。在目标 2 中，我们将使用代谢标记方法解决了一个很大程度上被忽视的问题，即 EJC 重塑过程中的动力学和事件顺序。使用在不同步骤中增强和/或抑制 NMD 的完善策略，我们将研究何时和 EJCs 在 NMD 通路中的哪个位置发挥替代功能。在目标 3 中，我们将使用来自并行 NMD 的报告器分支来测试替代 EJC 和平行 NMD 路径之间的关系。我们还将使用 RNA- 基于 Seq 和 RIPiT-Seq 的全局分析揭示了替代 EJC-的特定 mRNA 和蛋白质相互作用并行 NMD 分支中的 UPF 复合体。我们的目标是揭示目标特异性、潜在机制和平行 NMD 分支的细胞功能。