Mechanisms of RNA localization and translational regulation on the endoplasmic reticulum

内质网 (ER) 是分泌和膜蛋白合成的亚细胞位点 并在分泌/膜蛋白生物发生和细胞蛋白质稳态中发挥关键功能。另外 它在分泌/膜蛋白合成中的既定作用，最近的研究检查了 mRNA 胞质和内质网结合核糖体的转录组揭示胞质蛋白转录物广泛存在 代表内质网，核糖体足迹分析证明胞浆蛋白的翻译 ER 相关核糖体上的 mRNA。这些发现发现了一个意想不到的 mRNA 转录组范围 ER 在蛋白质组表达中的功能并重新讨论有关机制的基本问题 调节 ER 上 mRNA 的定位和翻译。原则上，当前模型假定 mRNA 内质网的定位是共翻译和信号序列依赖性的，丰富的存在和 ER 上胞浆蛋白 mRNA 的翻译表明替代途径和/或多途径 介导 mRNA 定位至 ER。同样，尽管 SRP 通路在蛋白质易位中的功能是 已明确，SRP 通路在 mRNA 定位到 ER 中的功能问题在很大程度上仍然存在 未经探索。同样重要的是，最近的发现表明许多易位子相关蛋白，包括 Sec61α、β、TRAPα、核糖蛋白 I 和 p180 是先前建议的 mRNA 结合蛋白 (RBP) ER 驻留 RBP 在 ER 上 RNA 定位和翻译生物学中的作用未被重视。 该提案合并了我们实验室的三个主要研究主题； SRP 通路在 mRNA 中的功能 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机制 合成，以及 iii) RNA 结合蛋白在 RNA 定位和翻译调控中的功能，以解决新的问题 有关 mRNA 和核糖体定位到 ER 的细胞机制的问题。立足于过去 我们对 ER 的 RNA 定位和翻译调控进行了十五年的研究，包括 发现了 ER 在细胞蛋白质组表达中在 mRNA 转录组范围内的作用的证据， 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， RNA-seq 和 Ribo-seq 转录组分析、ER RNA 结合蛋白的 HITS-CLiP 和 PAR-CLIP 研究 及其 RNA 相互作用组，以及翻译亚细胞组织的生化分析 机械，以获得对细胞组织和蛋白质组表达调节的新见解。这个 研究预计将增进对转录后调控系统和途径的理解 细胞中的基因表达。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，这项研究对于严格测试现有的 范例并促进对局部蛋白质合成的细胞机制的理解。

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.