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: 10667577
• 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/10667577
• 关键词: Address; Aging; Binding; Binding Proteins; Biochemical; Biogenesis; Biological; Biology; Cell Communication; Cell Culture System; Cell surface; Cells; Development; Disease; Endoplasmic Reticulum; Gene Expression; Genes; 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; posttranscriptional; 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.

内质网是分泌和膜蛋白合成的亚细胞场所 并在分泌/膜蛋白生物合成和细胞蛋白质稳定中发挥关键作用。此外 它在分泌/膜蛋白合成中的既定作用，最近的研究检查了mRNA 胞浆和ER结合核糖体的转录组显示，胞浆蛋白质转录本广泛地 在ER上代表，核糖体足迹分析表明细胞溶质蛋白的翻译 ER相关核糖体上的mRNA。这些发现鉴定了一种意想不到的mRNA转录组范围内的 ER在蛋白质组表达中的功能，并重新开启了有关其机制的基本问题 调节mRNA在ER上的定位和翻译。原则上，当前的模型忽略了mRNA ER的定位是共翻译和信号序列依赖性的， ER上胞浆蛋白mRNA的翻译表明， 介导mRNA定位到ER。同样，尽管SRP途径在蛋白质易位中的功能是 虽然已经建立了一个很好的机制，但SRP通路在mRNA定位于ER中的功能问题仍然存在很大程度上 未开发的同样重要的是，最近的研究发现，一些translocon相关蛋白，包括 Sec 61 α、β、TRAPα、ribophorin I和p180是mRNA结合蛋白（RBP）， ER常驻RBP在ER上的RNA定位和翻译生物学中的未被重视的作用。 该建议融合了我们实验室的三个主要研究主题：mRNA中的SRP通路功能 和核糖体定位到ER; ii）ER定位的翻译起始作为定位蛋白的机制 合成，和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mRNA和核糖体定位于ER的细胞机制的问题。立足过去 我们对ER的RNA定位和翻译调控的研究已有十五年，包括 发现证据表明ER在细胞蛋白质组表达中的mRNA转录组范围内的作用， 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相互作用组，以及翻译的亚细胞组织的生物化学分析。 机械，以获得新的见解细胞组织和蛋白质组表达的调控。这 研究有望推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的研究， RNA和核糖体运输动力学，这项研究是重要的，在其努力严格测试现有的 范例和先进的理解细胞机制的本地化蛋白质合成。