Regulation of RNA surveillance by the dynamic Exon Junction Complex

7.项目摘要/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。 复杂(EJC)在指定平行的无义介导的mRNA衰变(NMD)途径。NMD是作为一种 基本的转录后机制，调节正常的基因表达，也是一种质量 控制功能。对这些过程的详细了解对于改善人类健康至关重要，因为 破坏EJC和NMD蛋白的突变会导致发育缺陷、智力残疾和 精神发育迟滞。EJC由剪接体沉积在mrna外显子-外显子连接的上游，并具有 对下游信使核糖核酸代谢的主要影响。终止密码子下游的EJC广泛存在于 被接受为过早终止翻译和触发国家导弹防御系统的绝对标志。目前的提案 是由两个意想不到的发现推动的，这两个发现挑战了对EJC组成和功能的教条主义观点。 首先，我们最近通过开发一种名为 RIPiT-Seq，纯化包含一对不同蛋白质的RNPs并鉴定其整个转录组 脚印。这表明并不是在所有预测的结合位点上都检测到EJC，而且经常是 在意想不到的位置也检测到复合体。我们现在展示的是，EJC组成也不同于 位置对位置，并且在人类细胞中存在至少两个相互排斥或“交替”的ECCs， 含有不同的蛋白质因子。值得注意的是，描述了两个替代的EJC因子，RNPS1和MLN51 以前作为平行的NMD分支的独特组成部分。我们证明了两个交替的EJC因素具有 不同的NMD靶点，它们与平行NMD分支的蛋白质有不同的相互作用。我们的整体 假说是交替的EjCs控制着细胞中明显不同的生物学活动。我们建议 使用多学科方法来发现不同的EJC组合如何连接到并行NMD 以及哪些生物活动受每个独特的复合体的调节。在目标1中，我们将使用RNA-Seq 和RIPiT-Seq方法，以定义替代的EJC调节的NMD目标，它们的独特特征，以及 因此，替代的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 回答了一个很大程度上被忽视的问题，即在EJC重塑过程中事件的动力学和顺序。vbl.使用 在不同步骤加强和/或抑制NMD的成熟战略，我们将调查何时和 在NMD途径中，Ejcs的功能有哪些变化。在目标3中，我们将使用来自平行NMD的记者 分支，以测试备用EGC和并行NMD路径之间的关系。我们还将使用RNA- 基于SEQ和RIPiT-Seq的全球分析，以揭示交替的EJC-2基因的特异性mRNA和蛋白质相互作用 NMD平行分支中的UPF复合体。我们的目标是揭示靶标的特异性，潜在的机制 和平行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.