Mechanisms of RNA localization and translational regulation on the endoplasmic reticulum

内质网RNA定位和翻译调控机制

• 批准号: 10460908
• 负责人: Christopher V. Nicchitta
• 金额: $ 64.66万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-06 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10460908
• 关键词: Address; Aging; Binding Proteins; Biochemical; Biogenesis; Biological; Biology; Cell Communication; Cell Culture System; Cell surface; Cells; Code; Development; Disease; Endoplasmic Reticulum; Gene Expression; Genes; Genetic Transcription; Hormone secretion; Individual; Laboratories; Learning; Location; Mammalian Cell; Mediating; Membrane Proteins; Memory; Messenger RNA; Metabolism; Modeling; Neurotransmitters; Pathway interactions; Peptide Signal Sequences; Process; Protein Biosynthesis; Protein translocation; Proteins; Proteome; RNA; RNA-Binding Proteins; Regulation; Research; Ribosomes; Role; Signal Recognition Particle; Signal Transduction; Site; System; Testing; Transcript; Translation Initiation; Translational Regulation; Translations; crosslinking and immunoprecipitation sequencing; in vivo; insight; protein function; proteostasis; ribophorin I; ribosome profiling; secretory protein; signal sequence receptor; tissue/cell culture; trafficking; transcriptome; transcriptome sequencing

The endoplasmic reticulum (ER) is the subcellular site of secretory and membrane protein synthesis and performs critical functions in secretory/membrane protein biogenesis and cellular proteostasis. In addition its established role in secretory/membrane protein synthesis, recent studies examining the mRNA transcriptomes of cytosolic and ER-bound ribosomes reveal that cytosolic protein transcripts are broadly represented on the ER, with ribosome footprinting analyses demonstrating translation of cytosolic protein mRNAs on ER-associated ribosomes. These findings identify an unexpected mRNA transcriptome-wide function for the ER in proteome expression and reopen fundamental questions regarding the mechanisms regulating mRNA localization and translation on the ER. Principally, where current models posit that mRNA localization to the ER is co-translational and signal sequence-dependent, the abundant presence and translation of cytosolic protein mRNAs on the ER indicates that either alternative and/or multiple pathways mediate mRNA localization to the ER. As well, and although SRP pathway function in protein translocation is well established, the question of SRP pathway function in mRNA localization to the ER remains largely unexplored. Also of significance, the recent findings that a number of translocon-associated proteins, including Sec61α,β, TRAPα, ribophorin I, and p180, are mRNA binding proteins (RBPs) suggest previously unappreciated roles for ER resident RBPs in the biology of RNA localization and translation on the ER. This proposal merges three primary research themes of our laboratory; SRP pathway function in mRNA and ribosome localization to the ER; ii) ER-localized translation initiation as a mechanism of localized protein synthesis, and iii) RNA binding protein function in RNA localization and translational regulation, to address new questions regarding cellular mechanisms of mRNA and ribosome localization to the ER. Building on the past decade and a half of our research into RNA localization and translational regulation on the ER, including founding evidence identifying an mRNA transcriptome-wide role for the ER in cellular proteome expression, the proposed research will utilize mammalian tissue culture cell systems, gene editing and silencing approaches, RNA-seq and Ribo-seq transcriptome analyses, HITS-CLiP and PAR-CLIP studies of ER RNA binding proteins and their RNA interactomes, and biochemical analyses of the subcellular organization of the translation machinery, to obtain new insights into the cellular organization and regulation of proteome expression. This research is expected to advance understanding into the systems and pathways governing post-transcriptional gene expression in the cell. In emphasizing in vivo analyses and native biosynthetic approaches to the study of RNA and ribosome trafficking dynamics, this research is significant in its efforts to rigorously test existing paradigms and advance understanding of cellular mechanisms of localized protein synthesis.

内质网(ER)是分泌和膜蛋白合成的亚细胞场所 在分泌/膜蛋白生物发生和细胞蛋白平衡中发挥重要作用。此外 它在分泌/膜蛋白合成中的既定作用，最近的研究检测了它的mRNA 胞质核糖体和内质网结合核糖体的转录本显示胞质蛋白转录本广泛存在 以核糖体足迹分析显示胞浆蛋白的翻译 ER相关核糖体上的mRNAs。这些发现确定了一个意想不到的整个转录组的mrna。 内质网在蛋白质组表达中的作用及其机制的再认识 调节内质网上的信使核糖核酸定位和翻译。最主要的是，目前的模型假设 ER的定位是共翻译和信号序列依赖的，丰富的存在和 胞浆蛋白mRNAs在内质网上的翻译表明，要么是替代的和/或多个途径 将信使核糖核酸定位于内质网。同样，尽管SRP途径在蛋白质转位中的作用是 SRP通路在内质网信使核糖核酸定位中的作用在很大程度上仍是个问题 未被开发的。同样重要的是，最近的研究发现，一些转运子相关蛋白，包括 SEC61α，β、TRAPα、核转录因子I和P180是先前发现的基因 ER常驻限制性商业惯例在ER上的RNA本地化和翻译的生物学中所扮演的未被认可的角色。 这一建议融合了我们实验室的三个主要研究主题：SRP途径在mRNA中的作用 核糖体定位于内质网；II)内质网定位翻译起始作为定位蛋白的一种机制 合成，以及iii)RNA结合蛋白在RNA定位和翻译调节中的功能，以解决新的 关于内质网基因和核糖体定位的细胞机制的问题。在过去的基础上建设 十年半以来，我们对内质网的RNA本地化和翻译调控进行了研究，包括 有证据表明内质网在细胞蛋白质组表达中具有转录组学上的作用 拟议的研究将利用哺乳动物组织培养细胞系统、基因编辑和沉默方法， ER RNA结合蛋白的RNA-seq和ribo-seq转录组分析、HITS-CLIP和PAR-CLIP研究 和它们的RNA相互作用，以及翻译的亚细胞组织的生化分析 机械，以获得对蛋白质组表达的细胞组织和调节的新见解。这 研究有望促进对转录后调控系统和途径的理解 基因在细胞中的表达。在强调体内分析和天然生物合成方法研究中 RNA和核糖体运输动力学，这项研究对于严格测试现有的 对局部蛋白质合成的细胞机制的范例和深入理解。