Regulation of RNA surveillance by the dynamic Exon Junction Complex

7.项目概要/摘要 该提案的重点是了解极端保守的多蛋白外显子连接的功能 复合物（EJC）在指定平行的无义介导的mRNA衰变（NMD）途径。国家导弹防御系统是一个 重要的转录后机制，调节正常的基因表达，也提供了一个质量 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、智力残疾和 智力迟钝。EJC通过剪接体沉积在mRNA外显子-外显子连接的上游， 对下游mRNA代谢的主要影响。终止密码子下游的EJC被广泛地 被认为是过早终止翻译和触发国家导弹防御系统的绝对标志。现时的建议 是由两个意想不到的发现，挑战教条的观点EJC组成和功能的动机。 首先，我们最近通过开发一种新的高通量方法， RIPiT-Seq，纯化含有不同蛋白质对的RNP并鉴定其转录组范围 脚印这表明EJC在所有预测的结合位点都没有被检测到，并且通常， 在非预期位置也检测到复合物。我们现在表明，EJC组成也不同， 在人细胞中存在至少两个相互排斥或“交替”的EJC， 含有不同的蛋白质因子。值得注意的是，两个替代EJC因子RNPS 1和MLN 51被描述为 以前作为并行NMD分支的独特组件。我们表明，这两个交替EJC因素有 不同的NMD目标，它们与平行的NMD分支的蛋白质有差异地相互作用。我们的整体 这一假设是交替的EJC控制细胞中明显不同的生物活性。我们建议 采用多学科方法，研究不同的EJC组成与并行的NMD之间的联系 分支以及每个独特的复合物调节哪些生物活动。在目标1中，我们将使用RNA测序 和RIPiT-Seq方法来定义替代EJC调节的NMD靶点，它们的独特特征， 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来 解决了一个很大程度上被忽视的问题，关于动力学和顺序的事件在EJC重塑。使用 在不同阶段加强和/或抑制国家导弹防御系统的既定战略中，我们将研究何时以及 在NMD通路中，替代EJC发挥作用。在目标3中，我们将使用来自并行NMD的报告器， 分支来测试交替EJC和平行NMD途径之间的关系。我们还将使用RNA- 基于Seq和RIPiT-Seq的全球分析，以揭示替代EJC-1的特异性mRNA和蛋白质相互作用。 平行NMD分支中的UPF复合物。我们的目标是揭示靶点特异性，潜在机制 和并行NMD分支的细胞功能。

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.